WO2012090479A1 - Mcm7 as a target gene for cancer therapy and diagnosis - Google Patents

Mcm7 as a target gene for cancer therapy and diagnosis Download PDF

Info

Publication number
WO2012090479A1
WO2012090479A1 PCT/JP2011/007285 JP2011007285W WO2012090479A1 WO 2012090479 A1 WO2012090479 A1 WO 2012090479A1 JP 2011007285 W JP2011007285 W JP 2011007285W WO 2012090479 A1 WO2012090479 A1 WO 2012090479A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
mcm7
gene
double
expression level
Prior art date
Application number
PCT/JP2011/007285
Other languages
French (fr)
Inventor
Ryuji Hamamoto
Yusuke Nakamura
Takuya Tsunoda
Original Assignee
Oncotherapy Science, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oncotherapy Science, Inc. filed Critical Oncotherapy Science, Inc.
Publication of WO2012090479A1 publication Critical patent/WO2012090479A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to methods of detecting and diagnosing cancer or a predisposition for developing cancer in a subject, methods of assessing or determining the prognosis of a subject so diagnosed, as well as methods of treating and preventing cancer, particularly cancers associated with the overexpression of MCM7 such as lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • the present invention also relates to methods of screening for a candidate substance for treating and preventing a MCM7-associated cancer.
  • the present invention relates to double-stranded molecules that reduce MCM7 gene expression and uses thereof.
  • Lung cancer is the leading cause of cancer-related mortality in many countries, accounting for 1.3 million deaths worldwide annually, as of 2004 (World Health Organization, 2006). In Japan, more than 60,000 patients with lung cancer died in 2005. Despite improvements in surgical techniques and disease prognosis and the development of anticancer therapeutics, the survival of patients 5 years after diagnosis with NSCLC, the most common form of lung cancer, remains low, at 46.8%, according to a new report published by the Japanese Joint Committee for Lung Cancer Registration (NPL1). Therefore, it is vitally important for scientists to develop an understanding of the molecular mechanism of lung carcinogenesis so as to develop novel therapeutic methods for lung cancer that maximize efficiency while minimizing negative side-effects.
  • NPL1 Japanese Joint Committee for Lung Cancer Registration
  • NPL2 therapeutic monoclonal antibodies and small-molecule agents
  • a number of targeted therapies such as bevacizumab, cetuximab, erlotinib, gefitinib, sorafenib, and sunitinib have been investigated in phase II and phase III trials for the treatment of advanced non-small-cell lung cancer (NSCLC; NPLs 3-6).
  • NPLs 2-3 proangiogenic protein vascular endothelial growth factor
  • cetuximab epidermal growth factor receptor
  • erlotinib and gefitinib Two small-molecule epidermal growth factor receptor tyrosine kinase inhibitors, erlotinib and gefitinib, have been shown to be effective for a subset of advanced NSCLC patients (NPLs 4-5).
  • sorafenib inhibitor for c-RAF, b-RAF, vascular endothelial growth factor receptors 2 and 3, platelet-derived growth factor receptor h, and KIT
  • sunitinib inhibitor for platelet-derived growth factor receptor, KIT, FLT3, and vascular endothelial growth factor receptor
  • DNA replication in eukaryotic cells is a highly regulated process that ensures the accurate duplication of genetic information while preserving genome stability.
  • a large number of molecular players, including mini-chromosome maintenance (MCM) proteins, are involved in DNA replication (NPL 7-9).
  • MCM proteins are essential replication initiation and elongation factors originally found in Saccharomyces cerevisiae that exist in a functional complex composed of MCM2-7. They are evolutionally conserved in all eukaryotes (NPL 10).
  • the MCM protein complex belongs to the AAA+ family (ATPases associated with various cellular activities), and operates to ensure that DNA undergoes a single round of replication per cell cycle through use of a licensing mechanism (NPL 10-14).
  • the MCM4, 6 and 7 subcomplexes possess DNA helicase activity that promotes unwinding of double strand DNA at the replication forks (NPL 15-17).
  • NPL 1 Sawabata N, et al. Nihon Kokyuki Gakkai Zasshi 2010;48: 333-44.
  • NPL 2 Thatcher N. Lung Cancer 2007;57 Suppl 2:S18-23
  • NPL 3 Sandler A, et al. N Engl J Med 2006;355:2542-50
  • NPL 4 Shepherd FA, et al. N Engl J Med 2005;353:123-32
  • NPL 5 Thatcher N, et al. Lancet 2005;366:1527-37
  • NPL 6 Cesare G, et al. Oncologist 2007;12:191-200
  • NPL 7 Branzei D, et al.
  • NPL 8 Blow JJ, et al.Nat Rev Cancer 2008;8: 799-806.
  • NPL 9 Ilves I, et al. Mol Cell 2010;37: 247-58.
  • NPL 10 Tye BK. et al. Annu Rev Biochem 1999;68: 649-86.
  • NPL 11 Chong JP,et al. Nature 1995;375: 418-21.
  • NPL 12 Blow JJ, et al. Nature 1988;332: 546-8.
  • NPL 13 Tada S, et al. Biol Chem 1998;379: 941-9.
  • NPL 14 Costa A, et al.
  • the present invention relates to the MCM proteins and the roles they play in carcinogenesis,. Central to the present invention is the discovery of a specific expression pattern of the MCM7 gene in cancerous cells. The present invention further relates to the discovery that an inhibitor of the MCM 4, 6 and 7 complex which has DNA helicase activity effectively suppress the growth of cancer cells.
  • the MCM7 gene was revealed to be frequently up-regulated in human tumors, in particular, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor
  • small interfering RNA small interfering RNA
  • the MCM7 gene finds diagnostic utility as a marker for lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor and as an oncogene target, the expression and/or activity of which may be altered to treat or alleviate a symptom of cancer.
  • various agents for treating or preventing cancer can be identified.
  • test gene an increase in the level of expression of the test gene as compared to a normal control level indicates that the subject suffers from or is at risk of developing cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
  • cancer particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
  • the present invention further relates to the discovery that a high expression level of MCM7 correlates to poor survival rate in a cancer patient, particularly in a lung cancer patient such as an NSCLC patient. Accordingly, it is an objective of the present invention to provide a method for monitoring, assessing or determining the prognosis of a patient diagnosed with cancer, for example, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor cancer, particularly lung cancer such as NSCLC. Such a method includes the steps of detecting the expression level of MCM7 gene, comparing it to a pre-determined reference expression level and determining the prognosis of the patient from the difference between them.
  • Such methods involve the steps of: contacting the MCM7 protein with a test substance and detecting the binding between the MCM7 protein and the test substance.
  • Test substances that bind the MCM7 protein may prove effective in the treatment and/or the prophylaxis of cancer, e.g., by providing a clinical benefit such as improving prognosis, decreasing mortality levels, reducing tumor marker levels, and/or alleviating detectable symptoms of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
  • the biological activity of the MCM7 protein to be detected is preferably cell proliferative activity (cell proliferation enhancing activity).
  • a decrease in the biological activity of the MCM7 protein as compared to a control level in the absence of the test substance indicates that the test substance may prove effective in the treatment and/or the prophylaxis of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
  • cancer particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
  • the test cell may be an epithelial cell, such as cancerous epithelial cell.
  • a decrease in the expression level of the gene as compared to a control level in the absence of the test substance indicates that the test substance may prove effective in the treatment and/or the prophylaxis of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • cancer particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • a test substance e.g., a test substance
  • detecting the biological activity of the MCM protein complex e.g., a trimer composed of MCM4, MCM6 and MCM7 proteins and a hexamer composed of MCM 2-7 proteins.
  • the biological activity of the MCM protein complex to be detected is preferably DNA helicase activity.
  • a decrease in the biological activity of the MCM protein complex as compared to a control level in the absence of the test substance indicates that the test substance may prove effective in the treatment and/or the prophylaxis of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • cancer particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • siRNAs for the MCM7 gene were confirmed.
  • the data presented in the Examples section demonstrates the inhibition of cell proliferation of cancer cells by siRNAs.
  • the data herein support the utility of the MCM7 gene as a preferred therapeutic target for cancer, particularly lung and bladder cancer and the use of double-stranded molecules (e.g., siRNA) against MCM7 genes in the context of cancer therapy.
  • double-stranded molecules e.g., siRNA
  • the double-stranded molecules of the present invention are composed of a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13 and 15 as a target sequence, and the antisense strand comprises a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule.
  • Such double-stranded molecules are shown herein to inhibit expression of the MCM7 gene when introduced into a cell expressing an MCM7 gene and thus may prove effective in the treatment and/or the prophylaxis of cancer.
  • the present methods contemplate administration of an siRNA or double-stranded molecule composition to a subject in need thereof with the aim of reducing the expression of the MCM7 gene and thereby prove effective in the treatment and/or the prophylaxis of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, more particularly lung cancer and bladder cancer.
  • cancer particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, more particularly lung cancer and bladder cancer.
  • It is yet another objective of the present invention is to provide a pharmaceutical composition formulated for the treatment and/or the prophylaxis of cancer, or the inhibition of cancer cell growth.
  • the pharmaceutical composition of the present invention preferably comprises an antisense nucleotide or double-stranded molecule (e.g., siRNA) against the MCM7 gene, which inhibit the expression of the MCM7 gene.
  • the disease may be identified prior to detection of overt clinical symptoms of lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
  • Fig. 1 depicts the dysregulation of MCM7 in lung cancer tissues, and the relevance of MCM7 expression to a negative outcome for NSCLC patients after surgical operation.
  • Part B includes representative cases for positive MCM7 expression in lung ADC, SCC tissues and normal lung tissues. Original magnification, x100 and x200.
  • Part C depicts the results of immunohistochemical analysis of MCM7 in various normal tissues. No significant staining was observed.
  • Fig. 2 depicts the elevated MCM7 expression in bladder cancer :
  • Part A depicts the quantitative real-time PCR results for mRNA expression levels of MCM7 gene in 124 bladder TCCs, 12 upper tract TCCs and 23 normal bladder. Statistical analysis was done using Kruskal-Wallis test:*, P ⁇ 0.0001; Mann Whitney U-test: **, P ⁇ 0.0001.
  • Part B depicts the correlation between MCM7 gene expression and pathological tumor stages. Statistical analysis was done using Kruskal-Wallis test:*, P ⁇ 0.0001; Mann Whitney U-test: **, P ⁇ 0.0001.
  • Part C depicts the results of immunohistochemical analysis of MCM7 in bladder cancer tissues. Original magnification, x200.
  • Part D depicts the results of immunohistochemical analysis of MCM7 in liver cancer tissues. Original magnification, x200.
  • Fig. 3 depicts the involvement of MCM7 in the proliferation of lung cancer cells.
  • Part A depicts the results of Western blot analysis of MCM7 in A549 and SBC5 cells after treatment with two MCM7 siRNAs (siMCM7#1 and siMCM7#2) and two control siRNAs (siEGFP and siNC).
  • Part B depicts the results of immunocytochemical analysis of MCM7 in A549 and SBC5 cells after treatment with siRNAs. The nucleus was stained with DAPI, and the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating DNA was used to label proliferating cells.
  • Part C depicts the effects of MCM7 knockdown on the viability of lung cancer cell lines (A549 and SBC5). Statistical analysis was done using Student's t-test. *, P ⁇ 0.05.
  • Fig. 4 depicts mRNA expression levels of MCM7 gene in 15 lung-cancer cell lines, 2 bladder-cancer cell lines, 2 liver-cancer cell lines and 1 normal human cell line examined by quantitative real-time PCR.
  • Fig. 5 depicts involvement of MCM7 in the growth of bladder cancer.
  • Part A depicts the results of Western blot analysis of MCM7 in SW780 cells after treatment with two MCM7 siRNAs and control siRNAs (siEGFP and siNC).
  • Part B depicts the results of immunocytochemical analysis of MCM7 in SW780 cells after treatment with siRNAs. The nucleus was stained with DAPI, and the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating DNA was used to label proliferating cells.
  • Part C depicts the effects of MCM7 knockdown on the viability of a bladder cancer cell line (SW780). Statistical analysis was done using Student's t-test. *, P ⁇ 0.05.
  • Fig. 6 depicts effects of a DNA helicase inhibitor on the growth of cancer cells.
  • Heliquinomycin reduced growth rate in two lung cancer cell lines (A549 and SBC5) and one bladder cancer cell line (SW780) in a dose-dependent manner. Cell viability was measured by Cell Counting Kit-8 at 72 hours after treatment with indicated concentration of heliquinomycin.
  • a method for detecting or diagnosing cancer or a predisposition for developing cancer in a subject comprising the step of determining an expression level of an MCM7 gene in a subject-derived biological sample, wherein an increase in said expression level as compared to a normal control level of said gene indicates that said subject suffers from or is at a risk of developing cancer, wherein said expression level is determined by a method selected from a group consisting of: (a) detecting mRNA of an MCM7 gene; (b) detecting a protein encoded by an MCM7 gene; and (c) detecting a biological activity of a protein encoded by an MCM7 gene, [2] The method of [1], wherein said MCM7 gene expression level is at least 10% greater than the normal control level, [3] The method of [1] or [2], wherein the subject-derived biological sample is a biopsy, saliva, sputum, blood, serum, plasma, pleural effusion or
  • an isolated or purified antibody refers to an antibody that is substantially free of cellular material such as carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • heterologous protein also referred to herein as a "contaminating protein”
  • the polypeptide is recombinantly produced, it is also preferably substantially free of culture medium, which includes preparations of polypeptide with culture medium less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • polypeptide When the polypeptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, which includes preparations of polypeptide with chemical precursors or other chemicals involved in the synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation. That a particular protein preparation contains an isolated or purified polypeptide can be shown, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining or the like of the gel.
  • SDS sodium dodecyl sulfate
  • antibodies and polypeptides of the present invention are isolated or purified.
  • biological sample refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • body fluids including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen.
  • Biological sample further refers to a homogenate, lysate, extract, cell culture or tissue culture prepared from a whole organism or a subset of its cells, tissues or component parts, or a fraction or portion thereof.
  • biological sample refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or polynucleotides.
  • polypeptide polypeptide
  • peptide protein
  • protein polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly functions to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine).
  • amino acid analog refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium).
  • modified R group or modified backbones e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium.
  • amino acid mimetic refers to chemical compounds that have different structures but similar functions to general amino acids. Amino acids may be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • nucleic acid and nucleic acid molecule
  • gene refers to the amino acids referred to by their commonly accepted single-letter codes. Similar to the amino acids, they encompass both naturally-occurring and non-naturally occurring nucleic acid polymers.
  • the polynucleotide, oligonucleotide, nucleic acid, or nucleic acid molecule may be composed of DNA, RNA or a combination thereof.
  • an "isolated nucleic acid” is a nucleic acid removed from its original environment (e.g., the natural environment if naturally occurring) and thus, synthetically altered from its natural state.
  • isolated nucleic acid include DNA, RNA, and derivatives thereof, for example, a cDNA molecule, substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • MCM7 gene encompasses the human MCM7 gene as well as equivalents from other animals including, but not limited to, non-human primate, mouse, rat, dog, cat, horse, and cow, and further includes allelic mutants and genes found in other animals as corresponding to the MCM7 gene.
  • the typical nucleotide sequences of the MCM7 gene are shown in SEQ ID NOs 17 and 19. This sequence data is also available via GenBank accession numbers: NM_005916 and NM_182776, respectively.
  • MCM7 amino acid sequences encoded the human MCM7 gene are shown in SEQ ID NO: 18 and SEQ ID NO: 20 and available via GenBank accession numbers: MCM7: NP_005907 and NP_877577.
  • MCM7 amino acid sequences encoded the human MCM7 gene are shown in SEQ ID NO: 18 and SEQ ID NO: 20 and available via GenBank accession numbers: MCM7: NP_005907 and NP_877577.
  • the MCM7 genes and proteins can be obtained from nature as naturally occurring proteins via conventional cloning methods or through chemical synthesis based on the selected nucleotide or amino acid sequence. Methods for cloning genes and proteins using cDNA libraries and such are well known in the art.
  • cancer refers to cancer over-expressing the MCM7 gene, in particular, lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia (AML), osteosarcoma and soft tissue tumor.
  • lung cancer includes non-small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC).
  • non-small cell lung cancer (NSCLC) includes adenocarcinoma (ADC), squamous-cell carcinoma (SCC) and large-cell carcinoma (LCC).
  • prevention and prophylaxis can occur “at primary, secondary and tertiary prevention levels”. While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g. reducing the proliferation and metastasis of tumors.
  • certain embodiments of the present invention encompass the treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence
  • such methods may include any of the following steps: the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis.
  • Effective treatment and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer.
  • a treatment may also deem “efficacious” if it leads to clinical benefit such as, reduction in expression of the gene, or a decrease in size, prevalence, or metastatic potential of the cancer in the subject.
  • "efficacious” means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer. Efficaciousness is determined in association with any known method for diagnosing or treating the particular tumor type.
  • double-stranded molecule refers to a nucleic acid molecule that inhibits expression of a target gene, including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)).
  • siRNA short interfering RNA
  • dsRNA double-stranded ribonucleic acid
  • shRNA small hairpin RNA
  • siD/R-NA short interfering DNA/RNA
  • sense strand of a target sequence is a nucleotide sequence within mRNA or cDNA sequence of a gene, which will result in suppress of translation of the whole mRNA if a double-stranded nucleic acid molecule of the invention was introduced within a cell expressing the gene.
  • a nucleotide sequence within mRNA or cDNA sequence of a gene can be determined to be a target sequence when a double-stranded polynucleotide comprising a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene.
  • the double stranded polynucleotide by which suppresses the gene expression may be composed of the target sequence and 3'overhang having 2 to 5 nucleotides in length (e.g., uu).
  • double-stranded molecule is also referred to as “double-stranded nucleic acid ", “ double-stranded nucleic acid molecule”, “double-stranded polynucleotide”, “double-stranded polynucleotide molecule”, “double-stranded oligonucleotide” and “double-stranded oligonucleotide molecule”.
  • target sequence refers to a nucleotide sequence within mRNA or cDNA sequence of a target gene, which will result in suppression of translation of the whole mRNA of the target gene if a double-stranded molecule targeting the sequence is introduced into a cell expressing the target gene.
  • a nucleotide sequence within mRNA or cDNA sequence of a gene can be determined to be a target sequence when a double-stranded molecule including a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene.
  • a target sequence is shown by cDNA sequence
  • a sense strand sequence of a double-stranded cDNA i.e., a sequence that mRNA sequence is converted into DNA sequence, is used for defining a target sequence.
  • a double-stranded molecule is composed of a sense strand that has a sequence corresponding to a target sequence and an antisense strand that has a complementary sequence to the target sequence, and the antisense strand hybridizes with the sense strand at the complementary sequence to form a double-stranded molecule.
  • the phrase "corresponding to” means converting a target sequence according to the kind of nucleic acid that constitutes a sense strand of a double-stranded molecule.
  • base "t"s within the RNA region is replaced with base "u”s.
  • base "u"s within the DNA region is replaced with "t”s.
  • a sequence corresponding to a target sequence is "5'-GGCUAAUGGAGATGTCAA -3'" (for SEQ ID NO: 13) or "5'-GAAAGAAGATGTGAATGA-3'” (for SEQ ID NO: 15).
  • a complementary sequence to a target sequence for an antisense strand of a double-stranded molecule can be defined according to the kind of nucleic acid that constitutes the antisense strand.
  • a complementary sequence to a target sequence is 3'- CCGAUUACCTCTACAGTT-5'" (for SEQ ID NO: 13) or "3'-CUUUCUUCTACACTTACT -5'" (for SEQ ID NO: 15).
  • the sequence corresponding to a target sequence shown in SEQ ID NO: 13 or 15 is the RNA sequence of SEQ ID NO: 13 or 15, and the complementary sequence corresponding to a target sequence shown in SEQ ID NO: 13 or 15 is the RNA sequence of "3'- CCGAUUACCUCUACAGUU-5'" (for SEQ ID NO:13) or "3'- CUUUCUUCUACACUUACU-5'" (for SEQ ID NO:15).
  • a double-stranded molecule may have one or two 3' overhangs having 2 to 10, preferably 2 to 5 nucleotides in length (and/or a loop sequence that links a sense strand and an antisense strand to form hairpin structure, in addition to a sequence corresponding to a target sequence and complementary sequence thereto.
  • the added nucleotides form a single strand at the 3'-end of the sense strand and/or antisense strand of the double-stranded molecule and are preferably composed of "t" and "u” nucleotides, though are not necessarily limited thereto.
  • siRNA refers to a double-stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed.
  • the siRNA includes a sense nucleic acid sequence (also referred to as “sense strand"), an antisense nucleic acid sequence (also referred to as “antisense strand”) or both.
  • the siRNA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, e.g., a hairpin.
  • the siRNA may either be a dsRNA or shRNA.
  • dsRNA refers to a construct of two RNA molecules including complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded RNA molecule.
  • the nucleotide sequence of two strands may include not only the "sense” or “antisense” RNAs selected from a protein coding sequence of target gene sequence, but also RNA molecule having a nucleotide sequence selected from non-coding region of the target gene.
  • shRNA refers to an siRNA having a stem-loop structure, including the first and second regions complementary to one another, i.e., sense and antisense strands.
  • the degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
  • siD/R-NA refers to a double-stranded polynucleotide molecule which is composed of both RNA and DNA, and includes hybrids and chimeras of RNA and DNA and prevents translation of a target mRNA.
  • a hybrid indicates a molecule wherein a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize to each other to form the double-stranded molecule; whereas a chimera indicates that one or both of the strands composing the double stranded molecule may contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used.
  • the siD/R-NA includes a sense nucleic acid sequence (also referred to as "sense strand”), an antisense nucleic acid sequence (also referred to as “antisense strand”) or both.
  • the siD/R-NA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences from the target gene, e.g., a hairpin.
  • the siD/R-NA may either be a dsD/R-NA or shD/R-NA.
  • the term "dsD/R-NA” refers to a construct of two molecules including complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded polynucleotide molecule.
  • the nucleotide sequence of two strands may include not only the "sense” or "antisense” polynucleotides sequence selected from a protein coding sequence of target gene sequence, but also polynucleotide having a nucleotide sequence selected from non-coding region of the target gene.
  • One or both of the two molecules constructing the dsD/R-NA are composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules is composed of RNA and the other is composed of DNA (hybrid double-strand).
  • shD/R-NA refers to an siD/R-NA having a stem-loop structure, including the first and second regions complementary to one another, i.e., sense and antisense strands.
  • the degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the loop region of an shD/R-NA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
  • the present invention is based in part on the discovery that the expression of the MCM7 gene is elevated in cancerous cells, particularly cancer cells obtained from patients of lung cancer and various other cancers.
  • the protein encoded by the MCM7 gene is one of the highly conserved mini-chromosome maintenance proteins (MCM) that are essential for the initiation of eukaryotic genome replication.
  • MCM mini-chromosome maintenance proteins
  • the hexameric protein complex formed by the MCM proteins is a key component of the pre-replication complex and may be involved in the formation of replication forks and in the recruitment of other DNA replication related proteins.
  • Both the MCM2-MCM7 complex and the complex composed of MCM4, MCM6 and MCM7 have helicase activity and ATPase activity.
  • the typical nucleotide sequences of the MCM7 gene are shown in SEQ ID NOs 17 and 19 and available via GenBank accession numbers: NM_005916 and NM_182776, respectively.
  • the typical amino acid sequences encoded the human MCM7 gene are shown in SEQ ID NO: 18 and SEQ ID NO: 20 and available via GenBank accession numbers: MCM7: NP_005907 and NP_877577.
  • the present invention is not limited to these disclosed sequences.
  • MCM7 polypeptide encoded by the MCM7 gene
  • MCM7 polypeptide or “MCM7 protein”.
  • MCM7 sequences need not be limited to the sequences mentioned herein and that variants (e.g., functional equivalents and allelic variants) can be used in the present invention as described below.
  • a “functional equivalent” of a protein is a polypeptide that has a biological activity equivalent to that of the original reference protein. Namely, any polypeptides that retain the biological ability of the MCM7 protein may be used as such functional equivalents of the MCM7 protein in the present invention. Relevant biological activities of the MCM7 protein include, for example, cancer cell proliferation activity (cancer cell proliferation enhancing activity) and activity of forming a complex with MCM4 and MCM6 proteins.
  • functional equivalents include those polypeptides in which one or more amino acids are substituted, deleted, added, and/or inserted to the natural occurring amino acid sequence of the MCM7 protein.
  • the polypeptide may be one that includes an amino acid sequence having at least about 80% homology (also referred to as sequence identity) to the sequence of the MCM7 protein (e.g., SEQ ID NO: 18 or 20), more preferably at least about 90% to 95% homology, even more preferably 96%, 97%, 98% or 99% homology.
  • a polypeptide of the present invention may have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has a functional equivalent to that of the human protein of the present invention, it is within the scope of the present invention.
  • MCM7 polypeptide examples include those wherein one or more amino acids, e.g., 1-5 amino acids, e.g., up to 5% of amino acids, are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the MCM7 protein.
  • the polypeptide can be encoded by a polynucleotide that hybridizes under stringent conditions to the natural occurring nucleotide sequence of the gene.
  • the polypeptide is encoded by a polynucleotide that shares at least about 90%, 93%, 95%, 97%, 99% sequence identity to a reference sequence of MCM7, e.g., SEQ ID NO: 17 or 19, as determined using a known sequence comparison algorithm.
  • stringent (hybridization) conditions refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10 degrees C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times of background, preferably 10 times of background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 degrees C, or, 5x SSC, 1% SDS, incubating at 65 degrees C, with wash in 0.2x SSC, and 0.1% SDS at 50 degrees C.
  • hybridization may be performed by conducting pre-hybridization at 68 degrees C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 degrees C for 1 hour or longer.
  • the following washing step can be conducted, for example, in a low stringent condition.
  • An exemplary low stringent condition may include 42 degrees C, 2x SSC, 0.1% SDS, preferably 50 degrees C, 2x SSC, 0.1% SDS. High stringency conditions are often preferably used.
  • An exemplary high stringency condition may include washing 3 times in 2x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37 degrees C for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50 degrees C for 20 min.
  • factors such as temperature and salt concentration, can influence the stringency of hybridization and one skilled in the art can routinely adjust these and other factors to arrive at the desired stringency.
  • mutated or modified proteins i.e., peptides composed of an amino acid sequence in which one, two, or several amino acid residues have been modified through substitution, deletion, insertion and/or addition
  • mutated or modified proteins have been known to retain the original biological activity (Mark et al., Proc Natl Acad Sci USA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10:6487-500 (1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13 (1982)).
  • the peptides of the present invention may have an amino acid sequence wherein one, two or even more amino acids are added, inserted, deleted, and/or substituted in an originally disclosed reference sequence, such as the human MCM7 sequence.
  • the number of amino acid mutations or modifications is not particularly limited. However, it is generally preferred to alter 5% or less of the amino acid sequence, more preferably less than 3%, even more preferably less than 1%. Accordingly, in a preferred embodiment, the number of amino acids to be mutated in such a mutant is generally 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, more preferably 5 or 6 amino acids or less, and even more preferably 3 or 4 amino acids or less.
  • An amino acid residue to be mutated is preferably mutated into a different amino acid in which the properties of the amino acid side-chain are conserved (a process known as conservative amino acid substitution).
  • properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W).
  • A, I, L, M, F, P, W, Y, V hydrophilic
  • Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Aspargine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).
  • Such conservatively modified polypeptides are included in the present MCM7 protein.
  • the present invention is not restricted thereto and the MCM7 protein includes non-conservative modifications so long as the resulting modified peptide retains at least one biological activity of the original MCM7 protein.
  • the modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.
  • Mutations and/or modifications may be introduced at the N- and C-terminals as well as intermediate sites.
  • An example of a protein modified by addition of one or more amino acids residues is a fusion protein of the MCM7 protein. Fusion proteins can be made by techniques well known to a person skilled in the art, for example, by linking the DNA encoding the MCM7 gene with a DNA encoding another peptide or protein, so that the frames match, inserting the fusion DNA into an expression vector and expressing it in a host.
  • the "other" component of the fusion protein is typically a small epitope composed of several to a dozen amino acids.
  • fusion proteins contemplated by the instant invention include fusions of the MCM7 protein and other small peptides or proteins such as FLAG (Hopp TP, et al., Biotechnology 6: 1204-10 (1988)), a polyhistidine (His-tag) such as 6xHis containing six His (histidine) residues or 10xHis containing 10 His residues, Influenza aggregate or agglutinin (HA), human c-myc fragment, Vesicular stomatitis virus glycoprotein (VSV-GP), p18HIV fragment, T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage), SV40T antigen fragment, lck tag, alpha-tubulin fragment,
  • FLAG Hopp TP, et al., Biotechnology 6: 1204-10 (1988)
  • His-tag polyhistidine
  • proteins that can be fused to a protein of the invention include GST (glutathione-S-transferase), Influenza agglutinin (HA), immunoglobulin constant region, beta-galactosidase, MBP (maltose-binding protein), and such.
  • modified proteins contemplated by the present invention include polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.
  • the present invention further contemplates and thus encompasses polynucleotides that encode such functional equivalents of the MCM7 protein and deems such to fall within the scope of "MCM genes".
  • a gene amplification method for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a polynucleotide encoding a polypeptide functionally equivalent to the protein, using a primer synthesized based on the sequence above information.
  • PCR polymerase chain reaction
  • High homology typically refers to a homology of 40% or higher, preferably 60% or higher, more preferably 80% or higher, even more preferably 90% to 95% or higher.
  • the homology of a particular polynucleotide or polypeptide can be determined by following the algorithm in "Wilbur and Lipman, Proc Natl Acad Sci USA 80: 726-30 (1983)".
  • Percent sequence identity and sequence similarity can be readily determined using conventional techniques such as the BLAST and BLAST 2.0 algorithms, which are described (Altschul SF, et al., J Mol Biol. 1990 Oct 5; 215 (3):403-10; Nucleic Acids Res. 1997 Sep 1; 25(17):3389-402).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the worldwide web at ncbi.nlm.nih.gov/).
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra).
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff S & Henikoff JG. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10915-9).
  • a partial peptide having an amino acid sequence specific to the MCM7 protein is preferably composed of less than about 400 amino acids, usually less than about 200 and often less than about 100 amino acids, and at least about 7 amino acids, for example, about 8 amino acids or more, for example, about 9 amino acids or more.
  • the expression of the MCM7 gene is significantly and specifically elevated in cancer tissues, more particularly, in tissue samples derived from lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • the MCM7 gene finds utility as a diagnostic marker for a cancer over-expressing the MCM7 gene such as lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • a cancer over-expressing the MCM7 gene such as lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • subject-derived biological sample such as a cell sample and a tissue sample
  • Such diagnosis or detection may be performed by comparing the expression level of the MCM7 gene between a subject-derived sample and a normal sample. More particularly, the present invention provides a method for detecting or diagnosing cancer and/or a predisposition for developing cancer in a subject by determining the expression level of the MCM7 gene in the subject-derived biological sample.
  • the MCM7 gene finds further utility in determining the prognosis of a cancer patient as well as in assessing and/or monitoring the efficacy or applicability of a cancer immunotherapy.
  • Preferred cancers to be diagnosed, detected or assessed by the present method include lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • Lung cancer includes small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).
  • NSCLC includes adenocarcinoma, squamous cell carcinoma (SCC) and large-cell carcinoma.
  • the present invention also provides a method for detecting or identifying cancer cells in a subject-derived biological sample such as a tissue sample, the method including the step of determining the expression level of the MCM7 gene in a subject-derived biological sample, wherein an increase in said expression level as compared to a normal control level indicates the presence or suspicion of cancer cells in the tissue.
  • the present invention may provide a doctor with useful information to diagnose that the subject suffers from the disease. For example, according to the present invention, when the suspicion or doubt of the presence of cancer cells in the tissue obtained from a subject is indicated, clinical decisions would be made by a doctor with consideration of this observation and another aspect including the pathological finding of the tissue, levels of known tumor marker(s) in blood, or clinical course of the subject, etc.
  • some well-known diagnostic lung cancer markers in blood include ACT, BFP, CA19-9, CA50, CA72-4, CA130, CA602, CEA, IAP, KMO-1, SCC, SLX, SP1, Span-1, STN, TPA, and cytokeratin 19 fragment.
  • Some well-known bladder cancer markers in blood include NMP22, BFP and TPA.
  • diagnostic esophageal tumor markers in blood such as CEA, DUPAN-2, IAP, NSE, SCC, SLX and Span-1 are also well known.
  • Some well-known diagnostic colorectal cancer markers in blood include CA72-4,STN,CA19-9,CEA and NCC-ST-439, liver cancer markers in blood include AFP and PIVKA-2, pancreatic cancer marker in blood include CA19-9, Span-1,SLX and CEA, testicular cancer markers in include AFP and BFP, acute myeloid leukemia markers in blood include ACT and SOD, and osteosarcoma markers in blood include ICTP, NTx, DPD and BAP.
  • an intermediate result for examining the condition of a subject may also be provided.
  • the present invention provides a method for detecting a diagnostic marker of cancer, the method including the step of detecting the expression level of the MCM7 gene in a subject-derived biological sample as a diagnostic marker of cancer.
  • Preferable cancers to be diagnosed by the present method include lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • the term "diagnosing" is intended to encompass predictions and likelihood analysis.
  • the present method is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria such as disease stages, and disease monitoring and surveillance for cancer.
  • an intermediate result for examining the condition of a subject may also be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to determine that a subject suffers from the disease.
  • the present invention may be used to detect cancerous cells in a subject-derived tissue, and provide a doctor with useful information to diagnose that the subject suffers from the disease.
  • a subject to be diagnosed by the present method is preferably a mammal. Exemplary mammals include, but are not limited to, human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • a biological sample from a subject to be diagnosed.
  • Any biological material can be used as the biological sample for the determination so long as it can include the objective transcription or translation product of the MCM7 gene due to cancer.
  • suitable biological samples include, but are not limited to, bodily tissues such as biopsy specimen and fluids such as saliva, sputum, blood, serum, plasma, pleural effusion and urine.
  • the biological sample contains a cell population including an epithelial cell, more preferably a epithelial cell derived from tissue suspected to be cancerous.
  • the cells may be purified from the obtained bodily tissues and fluids, and then used as the biological sample.
  • biological sample may be a tissue sample collected from an area suspected to be cancerous.
  • the tissue sample may be a lung tissue sample for lung cancer, bladder tissue sample for bladder cancer, esophageal tissue sample for esophageal cancer, colorectal tissue sample for colorectal cancer, liver tissue sample for liver cancer, pancreatic tissue sample pancreatic cancer, testicular tissue sample for testicular cancer, myeloid tissue sample for acute myeloid leukemia, osseous tissue sample for osteosarcoma or soft tissue sample for soft tissue tumor.
  • the expression level of the MCM7 gene in a subject-derived biological sample is determined and then correlated to a particular healthy or disease state by comparison to a control sample.
  • the expression level can be determined at the transcription product (i.e., mRNA) level, using methods known in the art.
  • the mRNA of the MCM7 gene may be quantified using probes by hybridization methods (e.g., Northern hybridization).
  • the detection may be carried out on a filter, a chip or an array.
  • the use of an array is preferable for detecting the expression level of a plurality of genes (e.g., various cancer specific genes) including the MCM7 gene.
  • Those skilled in the art can prepare such probes utilizing the known sequence information for the MCM7 gene.
  • the cDNA of the MCM7 gene may be used as the probes.
  • the probe may be labeled with a suitable label, such as dyes and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.
  • the transcription product of the MCM7 gene may be quantified using primers by amplification-based detection methods (e.g., RT-PCR).
  • primers can also be prepared based on the available sequence information of the gene.
  • the primers used in the Example (SEQ ID NOs: 3 and 4) may be employed for the detection by RT-PCR, but the present invention is not restricted thereto.
  • a probe or primer suitable for use in the context of the present method will hybridize under stringent, moderately stringent, or low stringent conditions to the mRNA of the MCM7 gene.
  • stringent (hybridization) conditions refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degrees C lower than the thermal melting point (T m ) for a specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees C for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degrees C for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • a probe or primer of the present invention is typically a substantially purified oligonucleotide.
  • the oligonucleotide typically includes a region of nucleotide sequence that hybridizes under stringent conditions to at least about 2000, 1000, 500, 400, 350, 300, 250, 200, 150, 100, 50, or 25 bases, consecutive sense strand nucleotide sequence of a nucleic acid including a MCM7 sequence, or an anti sense strand nucleotide sequence of a nucleic acid including a MCM7 sequence, or of a naturally occurring mutant of these sequences.
  • an oligonucleotide having 5-50 bases in length can be used as a primer for amplifying the genes, to be detected.
  • mRNA or cDNA of a MCM7 gene can be detected with oligonucleotide probe or primer of a specific size, generally 15- 30 bases in length.
  • length of the oligonucleotide probe or primer can be selected from 15-25 bases.
  • Assay procedures, devices, or reagents for the detection of gene by using such oligonucleotide probe or primer are well known (e.g. oligonucleotide microarray or PCR).
  • probes or primers can also include tag or linker sequences. Further, probes or primers can be modified with detectable label or affinity ligand to be captured.
  • a polynucleotide having a few hundreds (e.g., about 100-200) bases to a few kilo (e.g., about 1000-2000) bases in length can also be used for a probe (e.g., northern blotting assay or cDNA microarray analysis).
  • diagnosis may involve detection of a translation product (i.e., protein) of the MCM7 gene.
  • a translation product i.e., protein
  • the quantity of the MCM7 protein may be determined and correlated to a disease or normal state.
  • the quantity of the translation products/proteins may be determined using, for example, immunoassay methods that use an antibody specifically recognizing the protein.
  • the antibody may be monoclonal or polyclonal.
  • any antibody fragments or modified antibodies e.g., chimeric antibody, scFv, Fab, F(ab') 2 , Fv, etc.
  • chimeric antibody e.g., scFv, Fab, F(ab') 2 , Fv, etc.
  • the intensity of staining may be observed via immunohistochemical analysis using an antibody against the MCM7 protein. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of the MCM7 gene.
  • the translation product may also be detected based on its biological activity.
  • the MCM7 protein was demonstrated herein to be involved in the growth of cancer cells.
  • the cancer cell growth promoting ability of the MCM7 protein may be used as an index of the MCM7 protein existing in the biological sample.
  • cell growth promoting ability is also referred to as "cell proliferative activity” or "cell proliferation enhancing activity”.
  • the expression level of other cancer-associated genes for example, genes known to be differentially expressed in lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor, may also be determined. Furthermore, in the case where the expression levels of multiple cancer-related genes are compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
  • methods for detecting or identifying cancer in a subject or cancer cells in a subject-derived sample begin with a determination of MCM7 gene expression level.
  • the expression level may be determined by any of the aforementioned techniques. Once determined, then, this level may be compared to a control level.
  • control level refers to the expression level of the MCM7 gene detected in a control sample and encompasses both a normal control level and a cancer control level.
  • normal control level refers to a level of the MCM7 gene expression detected in a normal healthy individual or in a population of individuals known not to be suffering from cancer. A normal individual is one with no clinical symptom of cancer. A normal control level can be determined using a normal cell obtained from a non-cancerous tissue.
  • a "normal control level” may also be the expression level of the MCM7 gene detected in a normal healthy tissue or cell of an individual or population known not to be suffering from cancer.
  • cancer control level refers to an expression level of the MCM7 gene detected in the cancerous tissue or cell of an individual or population suffering from cancer.
  • An increase in the expression level of the MCM7 gene detected in a sample as compared to a normal control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing cancer such as lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • the subject-derived sample may include epithelial cells, more preferably epithelial cells suspected to be cancerous.
  • the expression level of the MCM7 gene in a sample can be compared to a cancer control level of the MCM7 gene.
  • a similarity between the expression level of a sample and the cancer control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing cancer.
  • the expression levels of other cancer-related genes are also measured and compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing cancer.
  • gene expression levels are deemed to be "altered” or “increased” when the gene expression changes or increases by, for example, 10%, 25%, or 50% from, or at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more compared to a control level.
  • the expression level of cancer marker genes including the MCM7 gene in a biological sample can be considered to be increased if it increases from the normal control level of the corresponding cancer marker gene by, for example, 10% or more, 25% or more, or 50% or more; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
  • the control level may be determined at the same time with a test biological sample by using a sample(s) previously collected and stored from a subject/subjects whose disease state (cancerous or non-cancerous) is/are known.
  • the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of the MCM7 gene in samples from subjects whose disease state are known.
  • the control level can be a database of expression patterns from previously tested cells.
  • the expression level of the MCM7 gene in a biological sample may be compared to multiple control levels, which control levels are determined from multiple reference samples.
  • control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample.
  • standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as standard value.
  • the subject When the expression level of the MCM7 gene is increased compared to the normal control level or is similar to the cancerous control level, the subject may be diagnosed to be suffering from or at a risk of developing cancer. Furthermore, in case where the expression levels of multiple cancer-related genes are compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing cancer.
  • Difference between the expression levels of a test biological sample and the control level can be normalized to the expression level of control nucleic acids, e.g., housekeeping genes. Genes whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell. Exemplary control genes include, but are not limited to, beta actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.
  • the present invention provides the use of the MCM7 gene as cancerous markers.
  • the MCM7 gene are particularly useful for lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor as cancerous markers.
  • it can be determined whether a biological sample contains cancerous cells, especially lung cancerous cells, bladder cancerous cells, esophageal cancerous cells, colorectal cancerous cells, liver cancerous cells, pancreatic cancerous cells ,testicular cancerous cells, myeloid tissue tumor cells, osteosarcoma cells or soft tissue tumor cells , by detecting the expression level of the MCM7 gene as cancerous markers.
  • the expression level of the MCM7 gene in a biological sample as compared to a normal control level indicates that the biological sample contains cancerous cells.
  • the expression level of the MCM7 gene can be determined by detecting the transcription or translation products of the gene as described above.
  • cancer treatment targeting MCM7 can be achieved by the present invention.
  • the cancer treatment targeting MCM7 refers to suppression or inhibition of MCM7 activity and/or expression in the cancer cells. Any anti-MCM7 agents may be used for the cancer treatment targeting MCM7.
  • a cancer overexpressing MCM7 can be treated with at least one active ingredient selected from the group consisting of: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof.
  • the present invention provides a method of (i) diagnosing whether a subject has the cancer to be treated, and/or (ii) selecting a subject for cancer treatment, which method includes the steps of: a) determining the expression level of MCM7 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated; b) comparing the expression level of MCM7 with a normal control level; c) diagnosing the subject as having the cancer to be treated, if the expression level of MCM7 is increased as compared to the normal control level; and d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
  • such a method includes the steps of: a) determining the expression level of MCM7 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated; b) comparing the expression level of MCM7 with a cancerous control level; c) diagnosing the subject as having the cancer to be treated, if the expression level of MCM7 is similar or equivalent to the cancerous control level; and d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
  • the cancer includes, but is not limited to, lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor. Accordingly, prior to the administration of the double-stranded molecule of the present invention as active ingredient, it is preferable to confirm whether the expression level of MCM7 in the cancer cells or tissues to be treated is enhanced as compared with normal cells of the same organ.
  • the present invention provides a method for treating a cancer (over)expressing MCM7, which method may include the steps of: i) determining the expression level of MCM7 in cancer cells or tissue(s) obtained from a subject with the cancer to be treated; ii) comparing the expression level of MCM7 with normal control; and iii) administrating at least one component selected from the group consisting of (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof, to a subject with a cancer overexpressing MCM7 compared with normal control.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one component selected from the group consisting of: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof, for use in administrating to a subject having a cancer overexpressing MCM7.
  • the present invention further provides a method for identifying a subject to be treated with: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, or (c) a vector encoding thereof, which method may include the step of determining an expression level of MCM7 in subject-derived cancer cells or tissue(s), wherein an increase of the level compared to a normal control level of the gene indicates that the subject has cancer which may be treated with a double-stranded molecule of the present invention.
  • a subject to be treated by the present method is preferably a mammal.
  • exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • the expression level of MCM7 in cancer cells or tissues obtained from a subject is determined.
  • the expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art.
  • the translation product of the MCM7 gene may be detected for the treatment of the present invention, using methods known in the art. Those methods for determining the expression level of the MCM7 gene were described above.
  • the present invention relates, in part, to the discovery that MCM7 expression is significantly associated with poorer prognosis of subjects with lung cancer, e.g. NSCLC.
  • the present invention provides a method for determining, predicting, monitoring or assessing the prognosis of a subject with a cancer caused or promoted in part by the over-expression of MCM7, by detecting the expression level of the MCM7 in a biological sample collected from a subject with cancer; comparing the detected expression level to a control level, wherein an increased expression level of MCM7 in comparison to the normal control level as indicative of poor prognosis (poor survival).
  • Cancers to be assessing or determining the prognosis in the method of the present invention are preferably lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, more preferably lung cancer such as NSCLC.
  • NSCLC includes adenocarcinoma, squamous cell carcinoma (SCC) and large-cell carcinoma.
  • determining a similar or increased expression level of MCM7 gene in comparison to a cancerous control level is indicative of a poor prognosis.
  • prognosis refers to a forecast as to the probable outcome of the disease as well as the prospect of recovery from the disease as indicated by the nature and symptoms of the case. Accordingly, a less favorable, negative, or poor prognosis is defined by a lower post-treatment survival term or survival rate. Conversely, a positive, favorable, or good prognosis is defined by an elevated post-treatment survival term or survival rate.
  • assessing the prognosis refer to the ability of predicting, forecasting or correlating a given detection or measurement with a future outcome of cancer of the subject (e.g., malignancy, likelihood of curing cancer, survival, and the like). For example, a determination of the expression level of MCM7 gene over time enables a predicting of an outcome for the subject (e.g., increase or decrease in malignancy, increase or decrease in grade of a cancer, likelihood of curing cancer, survival, and the like).
  • the phrase "assessing (or determining or predicting) the prognosis” is intended to encompass predictions and likelihood analysis of cancer progression, particularly cancer recurrence, metastatic spread and disease relapse.
  • the present method for determining or assessing prognosis is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria such as disease staging, and disease monitoring and surveillance for metastasis or recurrence of neoplastic disease.
  • the subject-derived biological sample used for the method may be any sample derived from the subject to be assessed so long as the expression level of the MCM7 gene can be detected in the sample.
  • the subject-derived biological sample may be any sample derived from a subject, e.g., a subject known to have cancer.
  • the biological sample is a cancerous lung tissue sample for lung cancer, a cancerous esophageal tissue sample for esophageal cancer, a cancerous colorectal tissue sample for colorectal cancer, a cancerous liver tissue sample for liver cancer, a cancerous pancreatic tissue sample for pancreatic cancer, a cancerous bladder tissue sample for bladder cancer, a cancerous testicular tissue sample for testicular cancer, a cancerous myeloid tissue sample for acute myelocytic leukemia, a cancerous osterous tissue sample for osteosarcoma or a cancerous soft tissue sample for soft tissue tumor.
  • the biological sample may include bodily fluids such as saliva, sputum, blood, serum, plasma, pleural effusion or urine.
  • the sample may be cells purified from a tissue.
  • the biological samples may be obtained from a subject at various time points, including before, during, and/or after a treatment.
  • control level used for comparison may be, for example, the expression level of the MCM7 gene detected before any kind of treatment in an individual, or a population of individuals who showed good or positive prognosis of cancer after the treatment, which herein is referred to as "good prognosis control level".
  • control level may be the expression level of the MCM7 gene detected before any kind of treatment in an individual, or a population of individuals who showed poor or negative prognosis of cancer after the treatment, which herein will be referred to as "poor prognosis control level".
  • the "control level” may be a single expression pattern derived from a single reference population or from a plurality of expression patterns.
  • the control level may be determined based on the expression level of the MCM7 gene detected before any kind of treatment of cancer in a subject, or a population of the subjects whose disease state (good or poor prognosis) are known.
  • cancer is lung cancer, in particular NSCLC.
  • the standard value of the expression levels of the MCM7 gene in a patient group with a known disease state.
  • the standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as standard value.
  • control level may be determined at the same time with the test biological sample by using a sample(s) previously collected and stored before any kind of treatment from cancer subject(s) (control or control group) whose disease state (good prognosis or poor prognosis) are known.
  • control level may be determined by a statistical method based on the results obtained by analyzing the expression level of the MCM7 gene in samples previously collected and stored from a control group.
  • control level can be a database of expression patterns from previously tested cells.
  • the expression level of the MCM7 gene in a biological sample may be compared to multiple control levels, which control levels are determined from multiple reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample.
  • a similarity between a measured or calculated the expression level of the MCM7 gene and a level corresponding to a good or positive prognosis control level indicates a more favorable patient prognosis.
  • an increase in the expression level as compared to the good or positive prognosis control level indicates a less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome.
  • a decrease in the expression level of the MCM7 as compared to a poor or negative prognosis control level indicates a more favorable prognosis of the patient, with a similarity between the two indicating a less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome.
  • a following cell and/or tissue obtained from a subject who showed good, or poor prognosis of cancer after treatment is a preferable biological sample for good, or poor prognosis control level, respectively: - a lung cancer cell and/or cancerous lung tissue sample for lung cancer, - a esophageal cancer cell and/or cancerous esophageal tissue sample for esophageal cancer, - a colorectal cancer cell and/or cancerous colorectal tissue sample for colorectal cancer, - a liver cancer cell and/or cancerous liver tissue sample for liver cancer, - a pancreatic cancer cell and/or cancerous pancreatic tissue sample for pancreatic cancer, - a bladder cancer cell and/or cancerous bladder tissue sample for bladder cancer, - a testicular cancer cell and/or cancerous testicular tissue sample for testicular cancer, - an acute myelocytic leukemia cell and/or cancerous myeloid tissue sample for acute myelocytic
  • An expression level of the MCM7 gene in a subject-derived biological sample is considered altered (i.e., increased or decreased) when the expression level differs from the control level by more than 1.0, 1.5, 2.0, 5.0, 10.0, or more fold.
  • the difference in the expression level between the test biological sample and the control level can be normalized to a control, e.g., housekeeping gene.
  • a control e.g., housekeeping gene.
  • polynucleotides whose expression levels are known not to differ between the cancerous and non-cancerous cells including those coding for beta-actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1 may be used to normalize the expression level of the MCM7 gene.
  • the expression level may be determined by detecting the gene transcript in the patient-derived biological sample using techniques well known in the art, including those described previously, in section II-1. Method for diagnosing cancer or a predisposition for developing cancer, for instance using hybridization, immunoassay, immunostaining, and cell proliferation assays.
  • the gene transcripts of interest detected by the present method include both the transcription and translation products, such as mRNA and protein.
  • the transcription product of the MCM7 gene can be detected by hybridization, e.g., Northern blot hybridization analyses, that use a MCM7 gene probe to the gene transcript.
  • the detection may be carried out on a chip or an array. The use of an array is preferable for detecting the expression level of a plurality of genes including the MCM7 gene.
  • amplification-based detection methods such as reverse-transcription based polymerase chain reaction (RT-PCR) which use primers specific to the MCM7 gene may be employed for the detection (see “EXAMPLES").
  • RT-PCR reverse-transcription based polymerase chain reaction
  • the MCM7 gene-specific probe or primers may be designed and prepared using conventional techniques by referring to the whole sequence of the MCM7 gene (e.g., SEQ ID NO: 17 or 19).
  • the primers (SEQ ID NOs: 3 and 4) used in the "EXAMPLES” may be employed for the detection by RT-PCR, but the present invention is not restricted thereto.
  • a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of the MCM7 gene.
  • stringent (hybridization) conditions refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences.
  • the temperature of a stringent condition is selected to be about 5 degree Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degrees Centigrade for longer probes or primers.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • the translation product may be detected for the assessment of the present invention.
  • the quantity of the MCM7 protein may be determined.
  • a method for determining the quantity of the protein as the translation product includes immunoassay methods that use an antibody specifically recognizing the MCM7 protein.
  • the antibody may be monoclonal or polyclonal.
  • any fragment or modification e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.
  • Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the intensity of staining may be observed via immunohistochemical analysis using an antibody against MCM7 protein. Namely, the observation of strong staining indicates increased presence of the MCM7 protein and at the same time high expression level of the MCM7 gene. Furthermore, herein, the MCM7 protein has been demonstrated to have a cell proliferating activity. Therefore, the expression level of the MCM7 gene can be determined using such cell proliferating activity as an index.
  • cells which express MCM7 are prepared and cultured in the presence of a biological sample, and then by detecting the extent of proliferation in a predetermined time period, or by measuring the cell cycle or the colony forming ability the cell proliferating activity of the biological sample can be determined.
  • the expression level of other cancer-associated genes for example, genes known to be differentially expressed in lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and/or soft tissue tumor, especially lung cancer, e.g., NSCLC, may also be determined to improve the accuracy of the assessment.
  • lung cancer e.g., NSCLC
  • lung cancer e.g., NSCLC
  • an intermediate result may also be provided in addition to other test results for assessing the prognosis of a subject.
  • Such intermediate result may assist a doctor, nurse, or other practitioner to assess, determine, or estimate the prognosis of a subject.
  • Additional information that may be considered, in combination with the intermediate result obtained by the present invention, to assess prognosis includes clinical symptoms and physical conditions of a subject.
  • the subject to be assessed for the prognosis of cancer according to the method is preferably a mammal and includes human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • the expression level of the MCM7 gene is useful prognostic marker for assessing, predicting or determining the prognosis of a subject suffering from lung cancer (e.g. NSCLC), bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and/or soft tissue tumor. Therefore, the present invention also provides a method for detecting a prognostic marker for assessing, predicting or determining the prognosis of a subject suffering from lung cancer (e.g.
  • NSCLC NSCLC
  • bladder cancer esophageal cancer
  • colorectal cancer liver cancer
  • pancreatic cancer testicular cancer
  • acute myeloid leukemia osteosarcoma
  • soft tissue tumor which comprises steps of: a) detecting or determining an expression level of a MCM7 gene in a subject-derived biological sample, wherein the subject-derived biological is collected from a cancerous area, and b) correlating the expression level detected or determined in step a) with the prognosis of the subject.
  • an increased expression level to the good or positive prognosis control level is indicative of potential or suspicion of poor prognosis (poor survival).
  • kits and reagents In addition to diagnosing cancer, assessing the prognosis of cancer, and/or monitoring the efficacy of a cancer therapy, the present invention provides kits for detecting or diagnosing cancer or determining or assessing the prognosis of cancer.
  • the cancer to be detected or diagnosing by the present kit is selected from the group consisting of lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • the kits of the present invention preferably include reagents described bellow.
  • the present invention also provides reagents for detecting or diagnosing cancer or a predisposition for developing cancer, determining or assessing the prognosis of cancer, or monitoring the efficacy of a cancer therapy i.e., reagents that can detect the transcription or translation product of the MCM7 gene.
  • reagents include those capable of: (a) detecting mRNA of the MCM7 gene; (b) detecting the MCM7 protein; or (c) detecting the biological activity of the MCM7 protein, in a subject-derived biological sample.
  • Suitable reagents include nucleic acids that specifically bind to or identify a transcription product of the MCM7 gene.
  • a nucleic acid that specifically binds to or identifies a transcription product of the MCM7 gene includes, for example, oligonucleotides (e.g., probes and primers) having a sequence that is complementary to a portion of the MCM7 gene transcription product.
  • oligonucleotides exemplified by primers and probes that are specific to the mRNA of the gene of interest and may be prepared based on methods well known in the art.
  • antibodies can be exemplified as reagents for detecting the translation product of the gene.
  • reagents may be used for the above-described diagnosis or detection of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor. Also, above-mentioned reagents may be used for the determining or assessing the prognosis of cancer.
  • the reagents may be preferably used for lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, more preferably lung cancer such as NSCLC.
  • the assay format for using the reagents may be Northern hybridization or sandwich ELISA, both of which are well-known in the art.
  • the detection reagents may be packaged together in the form of a kit.
  • the detection reagents may be packaged in separate containers.
  • the detection reagents may be packaged with other reagents necessary for the detection.
  • a kit may include a nucleic acid or antibody (either bound to a solid matrix or packaged separately with reagents for binding them to the matrix) as the detection reagent, a control reagent (positive and/or negative), and/or a detectable label.
  • a kit of the present invention may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, and syringes. These reagents and such may be retained in a container with a label.
  • Suitable containers include bottles, vials, and test tubes.
  • the containers may be formed from a variety of materials, such as glass or plastic.
  • Instructions e.g., written, tape, VCR, CD-ROM, etc.
  • for carrying out the assay may also be included in the kit.
  • the present kit is suited for the detection and diagnosis of cancer, for example, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor, it may also be useful in assessing the prognosis of cancer and/or monitoring the efficacy of a cancer therapy, for example, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
  • lung cancer such as NSCLC.
  • the kit of the present invention for diagnosing cancer may further include either of positive or negative controls sample, or both.
  • the positive control sample of the present invention may be established a lung cancer cell line(s) (e.g., an NSCLC cell line(s)), esophageal cancer cell line(s), colorectal cancer cell line(s), liver cancer cell line(s), pancreatic cancer cell line(s), bladder cancer cell line(s), testicular cancer cell line(s), acute myeloid leukemia cell line(s), osteosarcoma cell line(s) and soft tissue tumor cell line(s).
  • lung cancer cell line(s) e.g., an NSCLC cell line(s)
  • esophageal cancer cell line(s) e.g., colorectal cancer cell line(s)
  • liver cancer cell line(s) e.g., pancreatic cancer cell line(s), bladder cancer cell line(s)
  • testicular cancer cell line(s) e.g., acute myeloid le
  • the MCM7 positive control samples may also be a tissue(s) selected from the group consisting of: - a lung cancer tissue(s) obtained from a lung cancer patient(s), - an esophageal cancer tissue(s) obtained from an esophageal cancer patient(s), - a colorectal cancer tissue(s) obtained from a colorectal cancer patient(s), - a liver cancer tissue(s) obtained from a liver cancer patient(s), - a pancreatic cancer tissue(s) obtained from a pancreatic cancer patient(s), - a bladder cancer tissue(s) obtained from a bladder cancer patient(s), - a testicular cancer tissue(s) obtained from a testicular cancer patient(s), - an acute myeloid leukemia tissue(s) obtained from an acute myeloid leukemia patient(s), - an osteosarcoma tissue(s) obtained from an osteosarcoma patient(s), and - a soft tissue tumor tissue(s) obtained from the group
  • such lung cancer tissue may be an NSCLC tissue(s) obtained from a NSCLC patient(s).
  • such NSCLC tissue(s) may be a clinical lung adenocarcinoma tissue(s) obtained from a lung adenocarcinoma patient(s), a lung squamous cell carcinoma tissue(s) obtained from a lung squamous cell carcinoma patient(s), and/or a lung squamous cell carcinoma tissue(s) obtained from a large cell carcinoma patient(s).
  • positive control samples may be prepared by determined a cut-off value and preparing a sample containing an amount of an MCM7 mRNA or protein more than the cut-off value.
  • cut-off value refers to the value dividing between a normal range and a cancerous range.
  • ROC receiver operating characteristic
  • the present kit may include an MCM7 standard sample providing a cut-off value amount of an MCM7 mRNA or polypeptide.
  • negative control samples may be prepared from non-cancerous cell lines or non-cancerous tissues such as normal lung tissues, esophageal tissues, colorectal tissues, liver tissues, pancreatic tissues, bladder tissues, testicular tissues, bone marrow, bone tissues or soft tissue tissues, or may be prepared by preparing a sample containing an MCM7 mRNA or protein less than cut-off value.
  • non-cancerous cell lines or non-cancerous tissues such as normal lung tissues, esophageal tissues, colorectal tissues, liver tissues, pancreatic tissues, bladder tissues, testicular tissues, bone marrow, bone tissues or soft tissue tissues, or may be prepared by preparing a sample containing an MCM7 mRNA or protein less than cut-off value.
  • the kit of the present invention for assessing the prognosis of cancer may further include either of a good or positive prognosis control sample or a poor or negative prognosis control sample, or both.
  • a good or positive control sample may be a cancerous tissue sample obtained from an individual or a population of individuals who showed good or positive prognosis of cancer, after the treatment.
  • a poor or negative control sample may be a cancerous tissue sample obtained from an individual or a population of individuals who showed poor or negative prognosis of cancer, after the treatment.
  • a good or positive prognosis control sample may also be a clinical lung cancer tissue(s) obtained from a lung cancer patient(s) who showed good or positive prognosis of lung cancer, after treatment.
  • an esophageal cancer tissue(s), colorectal cancer tissue(s), liver cancer tissue(s), pancreatic cancer tissue(s), bladder cancer tissue(s), testicular cancer tissue(s), acute myeloid leukemia tissue(s), osteosarcoma tissue(s), or soft tissue tumor tissue(s) obtained from a respective cancer patient(s) who showed good or positive prognosis of cancer, after the treatment may also be a preferable good or positive prognosis control sample for respective cancer.
  • such lung cancer tissue may be an NSCLC tissue(s) obtained from an NSCLC cancer patient(s).
  • NSCLC tissue may be a lung adenocarcinoma (ADC) tissue(s), a lung squamous cell carcinoma (SCC) tissue(s), and/or a large cell carcinoma tissue(s).
  • ADC lung adenocarcinoma
  • SCC lung squamous cell carcinoma
  • a good or positive prognosis control sample may be prepared by determined a cut-off value and preparing a sample containing an amount of an MCM7 mRNA or protein less than the cut-off value.
  • cut-off value refers to the value dividing between a good prognosis range and a poor prognosis range.
  • ROC receiver operating characteristic
  • the present kit may include an MCM7 standard sample providing a cut-off value amount of an MCM7 mRNA or polypeptide.
  • a poor or negative prognosis control sample may be a clinical lung cancer tissue(s) obtained from a lung cancer patient(s) who showed poor or negative prognosis of lung cancer, after the treatment.
  • an esophageal cancer tissue(s), colorectal cancer tissue(s), liver cancer tissue(s), pancreatic cancer tissue(s), bladder cancer tissue(s), testicular cancer tissue(s), acute myeloid leukemia tissue(s), osteosarcoma tissue(s), or soft tissue tumor tissue(s) obtained from a respective cancer patient(s) who showed poor or negative prognosis of cancer, after the treatment may also be a preferable poor or negative prognosis control sample for respective cancer.
  • such lung cancer tissue may be an NSCLC tissue(s) obtained from an NSCLC cancer patient(s).
  • NSCLC tissue may be a lung adenocarcinoma (ADC) tissue(s), a lung squamous cell carcinoma (SCC) tissue(s), and/or a large cell carcinoma tissue(s).
  • ADC lung adenocarcinoma
  • SCC lung squamous cell carcinoma
  • a poor or negative prognosis control sample may be prepared by determined a cut-off value and preparing a sample containing an amount of an MCM7 mRNA or protein more than the cut-off value.
  • the reagents for diagnosing or detecting cancer, or determining or assessing the prognosis of cancer may be immobilized on a solid matrix, such as a porous strip, to form at least one site for detecting cancer.
  • the measurement or detection region of the porous strip may include a plurality of sites, each containing a detection reagent (e.g., nucleic acid).
  • a test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a separate strip from the test strip.
  • the different detection sites may contain different amounts of immobilized detection reagents (e.g., nucleic acid), i.e., a higher amount in the first detection site and lesser amounts in subsequent sites.
  • the number of sites displaying a detectable signal provides a quantitative indication of the expression level of the MCM7 gene in the sample.
  • the detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
  • MCM7 is involved in cancer cell growth. Accordingly, substances that suppress an expression level of the MCM7 gene and/or a biological activity of the MCM7 polypeptide are expected to be useful for the treatment or prevention of cancer.
  • a polypeptide encoded by the gene or fragment thereof, or a transcriptional regulatory region of the gene it is possible to screen substances that alter the expression of the gene or the biological activity of a polypeptide encoded by the gene.
  • Such substances may be used as pharmaceuticals for treating or preventing cancer, in particular, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor,.
  • the present invention provides methods of screening for candidate substances for treating or preventing cancer using the MCM7 gene, a polypeptide encoded by the gene or fragments thereof, or a transcriptional regulatory region of the gene.
  • a substance isolated and identified by the screening method of the present invention is expected to inhibit the expression of the MCM7 gene, or the activity of the translation product of the gene, and thus, is a candidate for the treatment or prevention of diseases attributed to MCM7, for example, cell proliferative diseases, such as cancer (in particular, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor).
  • cancer in particular, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • the substances identified through the screening methods of the present invention are deemed to have a clinical benefit and can be further tested for the ability to prevent cancer cell growth in animal models or test subjects.
  • substances identified through the present screening methods may be any substance or composition and may in fact be a combination of several substances.
  • the substances may be contacted sequentially or simultaneously.
  • Any test substances for example, cell extracts, cell culture supernatants, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds (including nucleic acid constructs, such as antisense RNA, siRNA, Ribozymes, etc.) and natural compounds can be used in the screening methods of the present invention.
  • Test substances useful in the screenings described herein can also be antibodies that specifically bind to a protein of interest or a partial peptide thereof that lacks the biological activity of the original proteins in vivo.
  • Test substances of the present invention can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including: (1) biological libraries, (2) spatially addressable parallel solid phase or solution phase libraries, (3) synthetic library methods requiring deconvolution, (4) the "one-bead one-compound” library method and (5) synthetic library methods using affinity chromatography selection.
  • test substance libraries are well known in the art, herein below, additional guidance in identifying test substances and construction libraries of such substances for the present screening methods are provided.
  • test substance libraries are facilitated by knowledge of the molecular structure of compounds known to have the properties sought, and/or the molecular structure of MCM7 protein.
  • One approach to preliminary screening of test substances suitable for further evaluation utilizes computer modeling of the interaction between the test substance and its target.
  • Computer modeling technology allows for the visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule.
  • the three-dimensional construct typically depends on data from x-ray crystallographic analysis or NMR imaging of the selected molecule.
  • the molecular dynamics require force field data.
  • the computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.
  • CHARMM performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
  • a putative inhibitor has been identified, combinatorial chemistry techniques can be employed to construct any number of variants based on the chemical structure of the identified putative inhibitor, as detailed below.
  • the resulting library of putative inhibitors may be screened using the methods of the present invention to identify test substances suited to the treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • Combinatorial Chemical Synthesis Combinatorial libraries of test substances may be produced as part of a rational drug design program involving knowledge of core structures existing in known inhibitors. This approach allows the library to be maintained at a reasonable size, facilitating high throughput screening.
  • simple, particularly short, polymeric molecular libraries may be constructed by simply synthesizing all permutations of the molecular family making up the library.
  • An example of this latter approach would be a library of all peptides six amino acids in length. Such a peptide library could include every 6 amino acid sequence permutation. This type of library is termed a linear combinatorial chemical library.
  • Combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6).
  • peptide libraries see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptides (e.g., PCT Publication No.
  • WO 91/19735 encoded peptides (e.g., WO 93/20242), random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g., US Patent 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90:6909-13), vinylogous polypeptides (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J Amer Chem Soc 1992, 114: 9217-8), analogous organic syntheses of small compound libraries (Chen et al., J.
  • Another approach uses recombinant bacteriophage to produce libraries. Using the "phage method" (Scott & Smith, Science 1990, 249: 386-90; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Devlin et al., Science 1990, 249: 404-6), very large libraries can be constructed (e.g., 10 6 -10 8 chemical entities).
  • a second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 1986, 23: 709-15; Geysen et al., J Immunologic Method 1987, 102: 259-74); and the method of Fodor et al.
  • Aptamers are macromolecules composed of nucleic acid that bind tightly to a specific molecular target.
  • Tuerk and Gold disclose SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method for selection of aptamers.
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • a large library of nucleic acid molecules e.g., 10 15 different molecules
  • the screened test substance is a protein
  • for obtaining a DNA encoding the protein either the whole amino acid sequence of the protein may be determined to deduce the nucleic acid sequence coding for the protein, or partial amino acid sequence of the obtained protein may be analyzed to prepare an oligo DNA as a probe based on the sequence, and screen cDNA libraries with the probe to obtain a DNA encoding the protein.
  • the obtained DNA finds use in preparing the test substance which is a candidate for treating or preventing cancer.
  • the expression of the MCM7 gene is crucial for the growth and/or survival of cancer cells, in particular lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor cells. Accordingly, substances that suppress the function of the MCM7 polypeptide are presumed to inhibit the growth and/or survival of cancer cells, and therefore find utility in either or both of the treatment and prevention of cancer.
  • the present invention provides methods of screening a candidate substance for treating or preventing cancer, using the MCM7 polypeptide. Further, the present invention also provides methods of screening a candidate substance capable of inhibiting the growth and/or survival of cancer cells, using the MCM7 polypeptide.
  • fragments of the polypeptides may be used for the present screening, so long as it retains at least one biological activity of the naturally occurring MCM7 polypeptide.
  • the polypeptides or fragments thereof may be further linked to other substances, so long as the polypeptides and fragments retain at least one of their biological activities.
  • Usable substances include: peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc. These kinds of modifications may be performed to confer additional functions or to stabilize the polypeptide and fragments.
  • the polypeptides or fragments used for the present method may be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on the selected amino acid sequence.
  • conventional peptide synthesis methods that can be adopted for the synthesis include: 1) Peptide Synthesis, Interscience, New York, 1966; 2) The Proteins, Vol. 2, Academic Press, New York, 1976; 3) Peptide Synthesis (in Japanese), Maruzen Co., 1975; 4) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985; 5) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991; 6) WO99/67288; and 7) Barany G. & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.
  • the proteins may be obtained through any known genetic engineering methods for producing polypeptides (e.g., Morrison J., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62).
  • a suitable vector including a polynucleotide encoding the objective protein in an expressible form e.g., downstream of a regulatory sequence including a promoter
  • the host cell is cultured to produce the protein.
  • a gene encoding the MCM7 polypeptide is expressed in host (e.g., animal) cells and such by inserting the gene into a vector for expressing foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8.
  • a promoter may be used for the expression. Any commonly used promoters may be employed including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3.
  • the EF-alpha promoter (Kim et al., Gene 1990, 91:217-23), the CAG promoter (Niwa et al., Gene 1991, 108:193), the RSV LTR promoter (Cullen, Methods in Enzymology 1987, 152:684-704), the SR alpha promoter (Takebe et al., Mol Cell Biol 1988, 8:466), the CMV immediate early promoter (Seed et al., Proc Natl Acad Sci USA 1987, 84:3365-9), the SV40 late promoter (Gheysen et al., J Mol Appl Genet 1982, 1:385-94), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 1989, 9:946), the HSV TK promoter, and such.
  • the introduction of the vector into host cells to express the MCM7 gene can be performed according to any methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 1987, 15:1311-26), the calcium phosphate method (Chen et al., Mol Cell Biol 1987, 7:2745-52), the DEAE dextran method (Lopata et al., Nucleic Acids Res 1984, 12:5707-17; Sussman et al., Mol Cell Biol 1985, 4:1641-3), the Lipofectin method (Derijard B, Cell 1994, 7:1025-37; Lamb et al., Nature Genetics 1993, 5:22-30; Rabindran et al., Science 1993, 259:230-4), and such.
  • the polypeptides may be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C- terminus of the polypeptide.
  • a commercially available epitope-antibody system may be used (Experimental Medicine 13: 85-90 (1995)).
  • Vectors which are capable of expressing a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP), and so on, by the use of its multiple cloning sites are commercially available.
  • a fusion protein prepared by introducing only small epitopes composed of several to a dozen amino acids so as not to change the property of the original polypeptide by the fusion, is also provided herein.
  • Epitopes such as polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such, and antibodies recognizing them may be used as the epitope-antibody system for detecting the binding activity between the polypeptides (Experimental Medicine 13: 85-90 (1995)).
  • the MCM7 protein may also be produced in vitro adopting an in vitro translation system.
  • the present invention provides a method of screening for a candidate substance for either or both of treating and preventing cancer, which includes steps of: a) contacting a test substance with an MCM7 polypeptide or a fragment thereof; b) detecting binding (or binding activity) between the polypeptide or fragment and the test substance; and c) selecting the test substance that binds to the polypeptide as a candidate substance for either or both of treating and preventing cancer.
  • Identified substances by the screening method of the present invention can be applied to any cancer that over-expresses the MCM7 gene, such as lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor cells.
  • the potential therapeutic effect of the test substance in inhibiting the cell growth or treating or preventing MCM7 associated disease may be evaluated or estimated using methods described herein.
  • the present invention also provides a method of screening for a candidate substance capable of inhibiting the cell growth or a candidate substance for treating or preventing MCM7 associating disease (e.g., cancer), using the MCM7 polypeptide or fragments thereof including the steps as follows: a) contacting a test substance with an MCM7 polypeptide or a fragment thereof; b) detecting the binding (or binding activity) between the polypeptide or fragment and the test substance; and c) correlating the binding of b) with the therapeutic effect of the test substance.
  • the therapeutic effect may be correlated with the binding level to MCM7 polypeptide or a functional fragment thereof.
  • the test substance when the test substance binds to MCM7 polypeptide or a functional fragment thereof, the test substance may identified or selected as the candidate substance having the requisite therapeutic effect.
  • the test substance when the test substance does not bind to an MCM7 polypeptide or a functional fragment thereof, the test substance may identified as the substance having no significant therapeutic effect.
  • the binding of a test substance to the MCM7 polypeptide may be, for example, detected by immunoprecipitation using an antibody against the polypeptide. Therefore, for the purpose for such detection, it is preferred that the MCM7 polypeptide or fragments thereof used for the screening contains an antibody recognition site.
  • the antibody used for the screening may be one that recognizes an antigenic region (e.g., epitope) of the MCM7 polypeptide. Preparation methods for such antibodies are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the MCM7 polypeptide or a fragment thereof may be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody at its N- or C-terminus. The specificity of the antibody has been revealed, to the N- or C- terminus of the polypeptide.
  • a commercially available epitope-antibody system can be used (Experimental Medicine 1995, 13:85-90).
  • Vectors which can express a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP), and such by the use of its multiple cloning sites are commercially available and can be used for the present invention.
  • fusion proteins containing much smaller epitopes to be detected by immunoprecipitation with an antibody against the epitopes are also known in the art (Experimental Medicine 1995, 13:85-90).
  • epitopes composed of several to a dozen amino acids so as not to change the property of the MCM7 polypeptide or fragments thereof, can also be used in the present invention.
  • Examples include polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage), and such and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the MCM7 polypeptide (Experimental Medicine 13: 85-90 (1995)).
  • His-tag polyhistidine
  • influenza aggregate HA human c-myc
  • FLAG Vesicular stomatitis virus glycoprotein
  • VSV-GP Vesicular stomatitis virus glycoprotein
  • T7-tag T7 gene 10 protein
  • HSV-tag human simple herpes virus glycoprotein
  • E-tag an epitope on monoclonal phage
  • Glutathione S-transferase is also well-known as the counterpart of the fusion protein to be detected by immunoprecipitation.
  • GST is used as the protein to be fused with the MCM7 polypeptide or fragment thereof to form a fusion protein
  • the fusion protein can be detected either with an antibody against GST or a substance specifically binding to GST, i.e., such as glutathione (e.g., glutathione-Sepharose 4B).
  • an immune complex is formed by adding an antibody (recognizing the MCM7 polypeptide or a fragment thereof itself, or an epitope tagged to the polypeptide or fragment) to the reaction mixture of the MCM7 polypeptide and the test substance. If the test substance has the ability to bind the polypeptide, then the formed immune complex will consist of the MCM7 polypeptide, the test substance, and the antibody. On the contrary, if the test substance is devoid of such ability, then the formed immune complex only consists of the MCM7 polypeptide and the antibody. Therefore, the binding ability of a test substance to MCM7 polypeptide can be examined by, for example, measuring the size of the formed immune complex.
  • Any method for detecting the size of a compound can be used, including chromatography, electrophoresis, and such.
  • chromatography electrophoresis
  • Protein A or Protein G sepharose can be used for quantitating the formed immune complex.
  • SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Detection may be achieved using conventional staining method, such as Coomassie staining or silver staining, or, for proteins that is difficult to detect, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, 35 S-methionine or 35 S-cysteine, labeling proteins in the cells, and detecting the proteins.
  • the target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.
  • the MCM7 polypeptide or a fragment thereof used for the screening of substances that bind thereto may be bound to a carrier.
  • carriers that may be used for binding the polypeptides include insoluble polysaccharides, such as agarose, cellulose and dextran; and synthetic resins, such as polyacrylamide, polystyrene and silicon; preferably commercially available beads and plates (e.g., multi-well plates, biosensor chip, etc.) prepared from the above materials may be used. When using beads, they may be filled into a column. Alternatively, the use of magnetic beads is also known in the art, and enables to readily isolate polypeptides and substances bound on the beads via magnetism.
  • binding of a polypeptide to a carrier may be conducted according to routine methods, such as chemical bonding and physical adsorption.
  • a polypeptide may be bound to a carrier via antibodies specifically recognizing the protein.
  • binding of a polypeptide to a carrier can also be conducted by means of interacting molecules, such as the combination of avidin and biotin.
  • Screening using such carrier-bound MCM7 polypeptide or fragments thereof include, for example, contacting a test substance to the carrier-bound polypeptide, incubating the mixture, washing the carrier, and detecting and/or measuring the substance bound to the carrier.
  • the binding may be carried out in buffer, examples of which include, but are not limited to, phosphate buffer and Tris buffer, so long as the buffer does not inhibit the binding.
  • the MCM7 polypeptide or fragments thereof, and a composition are used as the test substance in a screening method
  • a composition e.g., cell extracts, cell lysates, etc.
  • affinity chromatography such method is generally called affinity chromatography.
  • the MCM7 polypeptide may be immobilized on a carrier of an affinity column, and a test substance, containing a substance capable of binding to the polypeptides, is applied to the column. After loading the test substance, the column is washed, and then the substance bound to the polypeptide is eluted with an appropriate buffer.
  • a biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound substance in the present invention.
  • the interaction between the MCM7 polypeptide and a test substance can be observed real-time as a surface plasmon resonance signal, using only a minute amount of the polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide and test substance using a biosensor such as BIAcore.
  • a protein binding to the MCM7 polypeptide can be obtained by preparing first a cDNA library from cells, tissues, organs, or cultured cells (e.g., PC cell lines) expected to express at least one protein binding to the MCM7 polypeptide using a phage vector (e.g., ZAP), expressing the proteins encoded by the vectors of the cDNA library on LB-agarose, fixing the expressed proteins on a filter, reacting the purified and labeled MCM7 polypeptide with the above filter, and detecting the plaques expressing proteins to which the MCM7 polypeptide has bound according to the label of the MCM7 polypeptide.
  • a cDNA library from cells, tissues, organs, or cultured cells (e.g., PC cell lines) expected to express at least one protein binding to the MCM7 polypeptide using a phage vector (e.g., ZAP), expressing the proteins encoded by the vectors of the cDNA library on LB-agarose, fixing the expressed proteins on
  • Labeling substances such as radioisotope (e.g., 3 H, 14 C, 32 P, 33 P, 35 S, 125 I, 131 I), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, beta-glucosidase), fluorescent substances (e.g., fluorescein isothiocyanate (FITC), rhodamine) and biotin/avidin, may be used for the labeling of MCM7 polypeptide in the present method.
  • radioisotope e.g., 3 H, 14 C, 32 P, 33 P, 35 S, 125 I, 131 I
  • enzymes e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, beta-glucosidase
  • fluorescent substances e.g., fluorescein isothiocyanate (FITC), rhodamine
  • the protein when it is labeled with an enzyme, it can be detected or measured by adding a substrate of the enzyme to detect the enzymatic change of the substrate, such as generation of color, with absorptiometer. Further, in case where a fluorescent substance is used as the label, the bound protein may be detected or measured using fluorophotometer.
  • the MCM7 polypeptide bound to the protein can be detected or measured by utilizing an antibody that specifically binds to the MCM7 polypeptide or a peptide or polypeptide (for example, GST) that is fused to the MCM7 polypeptide.
  • the antibody is preferably labeled with one of the labeling substances mentioned above, and detected or measured based on the labeling substance.
  • the antibody against the MCM7 polypeptide may be used as a primary antibody to be detected with a secondary antibody that is labeled with a labeling substance.
  • the antibody bound to the MCM7 polypeptide in the present screening may be detected or measured using protein G or protein A column.
  • Antibodies to be used in the present screening methods can be prepared using techniques well known in the art.
  • Antigens to prepared antibodies may be derived from any animal species, but preferably is derived from a mammal such as a human, mouse, rabbit, or rat, more preferably from a human.
  • the polypeptide used as the antigen can be recombinantly produced or isolated from natural sources.
  • the polypeptides to be used as an immunization antigen may be a complete protein or a partial peptide derived from the complete protein.
  • animals of the order Rodentia, Lagomorpha or Primate are used.
  • Animals of the Rodentia order include, for example, mice, rats and hamsters.
  • Animals of Lagomorpha order include, for example, hares, pikas, and rabbits.
  • Animals of Primate order include, for example, monkeys of Catarrhini (old world monkey) such as Macaca fascicularis, rhesus monkeys, sacred baboons and chimpanzees.
  • antigens may be diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline, etc.
  • PBS phosphate buffered saline
  • the antigen suspension may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, made into emulsion, and then administered to mammalian animals.
  • a standard adjuvant such as Freund's complete adjuvant
  • an appropriately amount of Freund's incomplete adjuvant every 4 to 21 days.
  • An appropriate carrier may also be used for immunization.
  • the serum is examined by a standard method for an increase in the amount of desired antibodies.
  • Polyclonal antibodies may be prepared by collecting blood from the immunized mammal examined for the increase of desired antibodies in the serum, and by separating serum from the blood by any conventional method.
  • Polyclonal antibodies include serum containing the polyclonal antibodies, as well as the fraction containing the polyclonal antibodies isolated from the serum.
  • Immunoglobulin G or M can be prepared from a fraction which recognizes only the objective polypeptide using, for example, an affinity column coupled with the polypeptide, and further purifying this fraction using protein A or protein G column.
  • immune cells are collected from the mammal immunized with the antigen and checked for the increased level of desired antibodies in the serum as described above, and are subjected to cell fusion.
  • the immune cells used for cell fusion are preferably obtained from spleen.
  • Other preferred parental cells to be fused with the above immunocyte include, for example, myeloma cells of mammalians, and more preferably myeloma cells having an acquired property for the selection of fused cells by drugs.
  • the above immunocyte and myeloma cells can be fused according to known methods, for example, the method of Milstein et al., (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)).
  • Resulting hybridomas obtained by the cell fusion may be selected by cultivating them in a standard selection medium, such as HAT medium (hypoxanthine, aminopterin, and thymidine containing medium).
  • HAT medium hyperxanthine, aminopterin, and thymidine containing medium.
  • the cell culture is typically continued in the HAT medium for several days to several weeks, the time being sufficient to allow all the other cells, with the exception of the desired hybridoma, to die. Then, the standard limiting dilution is performed to screen and clone a hybridoma cell producing the desired antibody.
  • human lymphocytes such as those infected by the EB virus, may be immunized with an antigen, cells expressing such antigen, or their lysates in vitro. Then, the immunized lymphocytes are fused with human-derived myeloma cells that are capable of indefinitely dividing, such as U266, to yield a hybridoma producing a desired human antibody that is able to bind to the antigen (Unexamined Published Japanese Patent Application No. (JP-A) Sho 63-17688).
  • JP-A Japanese Patent Application No.
  • the obtained hybridomas may be subsequently transplanted into the abdominal cavity of a mouse and the ascites may be extracted.
  • the obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, DEAE ion exchange chromatography, or an affinity column carrying an objective antigen.
  • Antibodies against the MCM7 polypeptide can be used not only in the present screening method, but also for the detection of the polypeptides as cancer markers in biological samples as described in "II. Diagnosing cancer". They may further serve as candidates for agonists and antagonists of the polypeptides of interest. In addition, such antibodies, serving as candidates for antagonists, can be applied to the antibody treatment for diseases related to the MCM7 polypeptide including lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor as described infra.
  • diseases related to the MCM7 polypeptide including lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor as described infra.
  • Monoclonal antibodies thus obtained can be also recombinantly prepared using genetic engineering techniques (see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD (1990)).
  • a DNA encoding an antibody may be cloned from an immune cell, such as a hybridoma or an immunized lymphocyte producing the antibody, inserted into an appropriate vector, and introduced into host cells to prepare a recombinant antibody.
  • an immune cell such as a hybridoma or an immunized lymphocyte producing the antibody
  • host cells such as a recombinant antibody.
  • Such recombinant antibody can also be used in the context of the present screening.
  • antibodies used in the screening and so on may be fragments of antibodies or modified antibodies, so long as they retain the original binding activity.
  • the antibody fragment may be an Fab, F(ab')2, Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston et al., Proc Natl Acad Sci USA 85: 5879-83 (1988)).
  • an antibody fragment may be generated by treating an antibody with an enzyme, such as papain or pepsin.
  • a gene encoding an antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co et al., J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).
  • An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG). Modified antibodies can be obtained through chemically modification of an antibody. These modification methods are conventional in the field.
  • Antibodies obtained as above may be purified to homogeneity.
  • the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins.
  • the antibody may be separated and isolated by appropriately selected and combined column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and others (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)); however, the present invention is not limited thereto.
  • a protein A column and protein G column can be used as the affinity column.
  • Exemplary protein A columns to be used include, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).
  • Exemplary chromatography includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)).
  • the chromatographic procedures can be carried out by liquid-phase chromatography, such as HPLC and FPLC.
  • two-hybrid system utilizing cells may be used ("MATCHMAKER Two-Hybrid system", “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene); the references “Dalton et al., Cell 1992, 68:597-612” and “Fields et al., Trends Genet 1994, 10:286-92").
  • MCM7 polypeptide or a fragment thereof is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells.
  • a cDNA library is prepared from cells expected to express at least one protein binding to the MCM7 polypeptide such that the library, when expressed, is fused to the VP16 or GAL4 transcriptional activation region.
  • the cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the MCM7 polypeptide is expressed in the yeast cells, the binding of the two activates a reporter gene, making positive clones detectable).
  • a protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E. coli and expressing the protein.
  • a reporter gene for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.
  • the substance isolated by this screening is a candidate for agonists or antagonists of the MCM7 polypeptide.
  • agonist refers to molecules that activate the function of the polypeptide by binding thereto.
  • antagonist refers to molecules that inhibit the function of the polypeptide by binding thereto.
  • an substance isolated by this screening as an antagonist is a candidate that inhibits the in vivo interaction of the MCM7 polypeptide with molecules (including nucleic acids (RNAs and DNAs) and proteins).
  • the MCM7 gene is disclosed to be highly overexpressed in lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor (Fig.1A-C, Fig. 2, 4 Table 5).
  • the suppression of the MCM7 gene by small interfering RNA (siRNA) resulted in growth inhibition and/or cell death of lung and bladder cancer cells (Fig. 3, 5). Accordingly, the MCM7 polypeptide is demonstrated herein to be involved in cancer cell survival, and thus, substances that inhibit a biological activity of the MCM7 polypeptide find utility as candidate agents for cancer therapy.
  • the present invention also provides a method of screening for a candidate substance for treating and/or preventing cancer using the MCM7 polypeptide or fragments thereof including the steps as follows: a) contacting a test substance with an MCM7 polypeptide or a fragment thereof; b) detecting the biological activity of the polypeptide or fragment of the step (a); and c) selecting the test substance that reduces the biological activity of the polypeptide as compared to the biological activity in the absence of the test substance.
  • the therapeutic effect of the test substance on inhibiting the cell growth or a candidate substance for treating and/or preventing MCM7 associated disease may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance capable of inhibiting the cell growth or treating and/or preventing an MCM7-associated disease, using the MCM7 polypeptide or fragments thereof including the steps as follows: a) contacting a test substance with an MCM7 polypeptide or a functional fragment thereof; b) detecting the biological activity of the polypeptide or fragment of step (a); and c) correlating the biological activity of b) with the therapeutic effect of the test substance.
  • the present invention provides a method of evaluating or estimating a therapeutic effect of a test substance in the treatment and/or prevention of cancer and/or in the inhibition of the growth of a cancer associated with the over-expression of MCM7 gene, the method including steps of: (a) contacting a test substance with the MCM7 polypeptide or a functional equivalent thereof; (b) detecting the biological activity of the polypeptide or functional equivalent of step (a); and (c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance suppresses the biological activity of the MCM7 polypeptide or functional equivalent as compared to the biological activity of said polypeptide detected in the absence of the test substance.
  • Such cancer includes lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • the therapeutic effect may be correlated with the biological activity of an MCM7 polypeptide or a functional fragment thereof.
  • the test substance when the test substance suppresses or inhibits the biological activity of an MCM7 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect.
  • the test substance when the test substance does not suppress or inhibit the biological activity of MCM7 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
  • Any polypeptides can be used for the screening method of the present invention so long as they retain a biological activity of the MCM7 polypeptide.
  • biological activity includes, but are not limited to, cell proliferation promoting activity and activity of forming a complex with MCM4 and MCM6 proteins.
  • Naturally occurring human MCM7 polypeptides e.g., polypeptide having an amino acid sequence of SEQ ID NO: 18 or 20
  • polypeptides functionally equivalent to these polypeptide can also be used (see “I. polynucleotide and polypeptides").
  • Such polypeptides may be expressed endogenously or exogenously by cells. Methods for preparing such polypeptides are described above.
  • any substances can be used for the screening method of the present invention so long as they suppress or reduce a biological activity of the MCM7 polypeptide.
  • the phrase "suppress or reduce a biological activity” encompasses at least 10% suppression of the biological activity of MCM7 in comparison with in the absence of the substance, more preferably at least 25%, 50% or 75% suppression and most preferably at 90% suppression. Such suppression can serve an index in the present screening method.
  • the MCM7 polypeptide has been demonstrated to be required for the growth or viability of lung and bladder cancer cells.
  • the biological activities of the MCM7 polypeptide that can be used as an index for the screening include such cell growth promoting activity of the human MCM7 polypeptide.
  • cell growth promoting activity is also referred to as “cell proliferative activity” or “cell proliferation enhancing activity”
  • the biological activity to be detected in the screening method of the present invention is cell growth promoting activity, it can be detected, for example, by preparing cells which express the MCM7 polypeptide or a fragment thereof, culturing the cells in the presence of a test substance, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by detecting wound-healing activity, conducting Matrigel invasion assay and measuring the colony forming activity.
  • the screening method may include the steps of: (a) contacting a test substance with a cell overexpressing MCM7 gene; (b) measuring cell growth promoting activity; and (c) selecting the test substance that reduces the cell growth promoting activity in the comparison with the cell growth promoting activity in the absence of the test substance.
  • the screening method of the present invention may further include the step of: (d) selecting the test substance that has no effect to the cells no or little expressing MCM7 gene.
  • the substance isolated by this screening method is a candidate for antagonists of the polypeptide encoded by MCM7 gene.
  • antagonist refers to molecules that inhibit the function of the polypeptide by binding thereto. This term also refers to molecules that reduce or inhibit expression of the gene encoding MCM7.
  • a substance isolated by this screening is a candidate for substances which inhibit the in vivo interaction of the MCM7 polypeptide with molecules (including DNAs and proteins).
  • control cells that do not express the MCM7 polypeptide may be used.
  • the present invention also provides a method of screening for a candidate substance that inhibits cell growth or a candidate substance for treating and/or preventing an MCM7- associated disease such as cancer, using the MCM7 polypeptide or functional equivalent thereof including the steps as follows: a) culturing cells which express an MCM7 polypeptide or a functional equivalent thereof in the presence or absence of a test substance, and control cells that do not express an MCM7 polypeptide or a functional equivalent thereof in the presence of the test substance; b) detecting a biological activity (e.g., cell growth) of the cells which express the MCM7 polypeptide or the functional equivalent thereof and the control cells; and c) selecting the test substance that inhibits the biological activity of the cells which express the MCM7 polypeptide or the functional equivalent thereof as compared to the biological activity detected in the absence of said test substance and that does not inhibit the biological activity of the control cells.
  • a biological activity e.g., cell
  • suppressing the biological activity of MCM7 polypeptide reduces cell growth.
  • candidate substance that have the potential to treat and/or prevent cancers can be identified.
  • the potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic substance, compounds or agent for cancers. For example, when a substance that inhibits the biological activity of an MCM7 polypeptide also inhibits the activity of a cancer, it may be concluded that such a substance has an MCM7 specific therapeutic effect.
  • heliquinomycin an DNA helicase inhibitor against MCM protein complex composed of MCM4, MCM6 and MCM7 proteins, effectively suppressed the growth of cancer cells such as lung and bladder cancer cells.
  • MCM2-MCM7 six of the MCM proteins, MCM2-MCM7, forms complexes that participate in initiation and elongation steps of DNA replication. They share a conserved 200-amino acid nucleotide-binding region and form different subcomplexes (dimers, trimers and a hexamer) (Koonin EV. et al, Nucleic Acids Res 1993;21:2541-7.).
  • MCM 4, 6 and 7 trimers and MCM2-7 hexamers have ATPase and DNA helicase activities in vitro (lei M. et al, J Cell Sci 2001;114:1447-54.).
  • heliquinomycin an inhibitor of MCM4, 6 and 7 DNA helicase, effectively suppresses the growth of cancer cells in a dose dependent manner (Fig. 6).
  • substances that inhibit DNA helicase activity of MCM protein complex may become candidate agents for cancer therapy.
  • the present invention also provides the method of screening for a candidate substance for treating and/or preventing cancer using DNA helicase activity of MCM protein complex as an index.
  • the present invention further provides the method of screening for a substance that suppresses the proliferation of cancer cells using DNA helicase activity of MCM protein complex as an index.
  • the MCM protein complex is characterized as having the DNA helicase activity.
  • Such MCM protein complexes include, for example, a trimer composed of MCM4, MCM6 and MCM7 proteins and a hexamer composed of MCM2-7 proteins.
  • Substances identified by the screening method of the present invention may be preferably applicable to cancer such as lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  • lung cancer and bladder cancer are suitable cancer for such substances.
  • the present invention provides a method of screening for a candidate substance for treating and/or preventing cancer, including the steps as follows: a) contacting a test substance with an MCM protein complex; b) detecting the biological activity of the complex of the step a); and c) selecting the test substance that reduces the biological activity of the complex as compared to the biological activity in the absence of the test substance.
  • the therapeutic effect of the test substance on cancer may be evaluated in accordance with the present invention. More particularly, the present invention provides a method of screening for a candidate substance capable of inhibiting the cell growth or a candidate substance for treating and/or preventing cancer, using the MCM protein complex including the steps as follows: a) contacting a test substance with an MCM protein complex; b) detecting the biological activity of the complex of step a); and c) correlating the biological activity of b) with the therapeutic effect of the test substance.
  • the present invention further contemplates a method for evaluating or estimating a therapeutic effect of a test substance in connection with the treatment and/or prevention of cancer and/or in the inhibition of the growth of a cancer, the method including steps of: a) contacting a test substance with the MCM protein complex; b) detecting the biological activity of the complex of step a); and c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance suppresses the biological activity of the complex as compared to the biological activity of said complex detected in the absence of the test substance.
  • Such cancer includes lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, particularly lung cancer and bladder cancer.
  • the therapeutic effect may be correlated with the biological activity of an MCM protein complex.
  • the test substance when the test substance suppresses or inhibits the biological activity of an MCM protein complex as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect.
  • the test substance when the test substance does not suppress or inhibit the biological activity of MCM protein complex as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
  • the method of the present invention will be described in more detail below.
  • the findings of the present invention revealed that suppressing the biological activity of MCM protein complex reduces cancer cell growth.
  • candidate substances having therapeutic potential for the treatment and/or prevention of cancer can be identified.
  • the potential of these candidate substances to treat and/or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a substance that suppresses the biological activity of MCM protein complex inhibits an activity of cancer such as cancer cell growth or survival, it may be concluded that such substance has the MCM protein complex specific therapeutic effect.
  • any substances can be used for the screening method of the present invention so long as they suppress or reduce a biological activity of the MCM protein complex.
  • the phrase "suppress or reduce a biological activity” encompasses at least 10% suppression of the biological activity of MCM protein complex in comparison with in the absence of the substance, more preferably at least 25%, 50% or 75% suppression and most preferably at 90% suppression. Such suppression can serve an index in the present screening method.
  • An MCM protein complex can be prepared by methods well known to one skilled in the art (J Biol Chem. 1997 Sep 26;272(39):24508-13.).
  • An MCM protein complex can be purified from cells expressing MCM proteins, such as cancer cells, by ammonium sulfate precipitation, DEAE sepharose column or histone sepharose column.
  • MCM protein complex can be also prepared by immunoprecipitation method etc, using anti-MCM protein antibody.
  • MCM proteins include, for example, lung cancer cell, esophageal cancer cell, colorectal cancer cell, liver cancer cell, pancreatic cancer cell, bladder cancer cell and testicular cancer cell, but are not limited to.
  • an MCM protein complex can be prepared by contacting MCM proteins (e.g., MCM4, MCM6 and MCM7 or MCM2-7) each other under suitable condition for formation of MCM protein complex.
  • DNA helicase activity is the preferred biological activity of the MCM protein complex to be assessed.
  • DNA helicase activity can be detected by methods well known to one skilled in the art (J Biol Chem. 1997 Sep 26;272(39):24508-13.).
  • the DNA helicase activity can be determined by contacting an MCM complex with a substrate (e.g., double-stranded oligonucleotide) and determining quantity of single-stranded oligonucleotide free from the substrate.
  • the screening method of the present invention includes the steps of: (a) contacting an MCM protein complex with a double-stranded oligonucleotide in the presence of a test substance, (b) detecting the quantity of the single-stranded oligonucleotide generated from the double-stranded oligonucleotide; and (c) selecting the test substance that decreases the quantity of the single-stranded oligonucleotide as compared to the quantity of the single-stranded oligonucleotide from the double-stranded oligonucleotide detected in the absence of the test substance.
  • the method may include the steps of: (a) contacting an MCM protein complex with a double-stranded oligonucleotide in the presence of a test substance, (b) detecting the quantity of the double-stranded oligonucleotide; and (c) selecting the test substance that increases the quantity of the double-stranded oligonucleotide as compared to the quantity of the double-stranded oligonucleotide detected in the absence of the test substance.
  • the substrate e.g., double-stranded oligonucleotide
  • DNA helicase can be labeled before using for assay.
  • the double-stranded oligonucleotide can be labeled before using for assay. Labeling substances such as radioisotope (e.g., 3 H, 14 C, 32 P, 33 P, 35 S, 125 I, 131 I), fluorescent substances (e.g., fluorescein isothiocyanate (FITC), rhodamine) and biotin/avidin, may be used for the labeling of a double-stranded oligonucleotide in the present method.
  • radioisotope e.g., 3 H, 14 C, 32 P, 33 P, 35 S, 125 I, 131 I
  • fluorescent substances e.g., fluorescein isothiocyanate (FITC), rhodamine
  • biotin/avidin e.g., biotin/avidin
  • the bound oligonucleotide may be detected or measured using fluorophotometer.
  • substrate e.g. double-stranded oligonucleotide
  • immunoprecipitation may be used as the label.
  • DNA helicase activity of MCM protein complex may be determined using a surface plasmon resonance phenomenon, the reaction between nucleic acid labeled with biotin and agarose beads labeled with avidin or fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • DNA helicase activity can be determined by the quantity of the double-stranded oligonucleotide
  • the higher value indicates the lower DNA helicase activity of MCM protein complex.
  • DNA helicase activity can be determined by the quantity of single-stranded oligonucleotide free from double-stranded oligonucleotide
  • the higher value indicates the higher DNA helicase activity of MCM protein complex.
  • the method of detecting DNA helicase activity can be performed by preparing cells which express the MCM genes, culturing the cells in the presence of a test substance, and measuring cell growth.
  • the substances that reduce DNA helicase activity of the MCM protein complex may be selected as candidate substance for treating and/or preventing cancer. More specifically, the screening method of the present invention may include the steps of: (a) contacting a test substance with cells overexpressing MCM genes; (b) measuring DNA helicase activity in the cells of (a); and (c) selecting the test substance that reduces the DNA helicase activity of (b) in the comparison with the DNA helicase activity in the absence of the test substance. In one preferred embodiment, the screening method of the present invention may further include the steps of: (d) selecting the test substance having no effect to the cells no or little expressing MCM genes.
  • a substance isolated by this screening method is considered to be a candidate antagonist of the MCM protein complex.
  • antagonist refers to molecules that inhibit the function of the complex by binding thereto. This term also refers to molecules that reduce or inhibit activity of the complex.
  • a substance isolated by this screening is a candidate for substances which inhibit the in vivo interaction of the MCM protein complex with molecules (including DNAs and proteins).
  • such a screening method may include, for example, the following steps: a) contacting a test substance with a cell expressing an MCM7 gene; b) detecting the expression level of the MCM7 gene; c) comparing the expression level with the expression level detected in the absence of the test substance; and d) selecting the test substance that reduces the expression level as compared to the expression level in the absence of the test substance as a candidate substance for treating and/or preventing cancer.
  • the therapeutic effect of the test substance for inhibiting the cell growth or a candidate substance for treating or preventing cancer may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance that suppresses the proliferation of cancer cells, as well as a method of screening for a candidate substance for treating and/or preventing cancer.
  • such screening method may include, for example, the following steps: a) contacting a test substance with a cell expressing an MCM7 gene; b) detecting the expression level of the MCM7 gene; and c) correlating the expression level of b) with the therapeutic effect of the test substance.
  • the therapeutic effect may be correlated with the expression level of the MCM7 gene.
  • the test substance when the test substance reduces the expression level of the MCM7 gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect.
  • the test substance when the test substance does not reduce the expression level of the MCM7 gene as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
  • a substance that inhibits the expression of the MCM7 gene can be identified by contacting a cell expressing the MCM7 gene with a test substance and then determining the expression level of the MCM7 gene. Naturally, the identification may also be performed using a population of cells that express the gene in place of a single cell. A decreased expression level detected in the presence of a test substance as compared to the expression level in the absence of the test substance indicates that the test substance is an inhibitor of the MCM7 gene expression, which, in turn, suggests that the test substance may be suitable for inhibiting cancer and therefore have utility in connection with the treatment or prevention of cancer.
  • the expression level of a gene can be estimated by methods well known to one skilled in the art.
  • the expression level of the MCM7 gene can be, for example, determined following the method described above under the item of 'II-1. Method for diagnosing cancer or a predisposition for developing cancer'.
  • the cell or the cell population used for such identification may be any cell or any population of cells, so long as it expresses the MCM7 gene.
  • the cell or cell population may be or contain a lung , esophageal, colorectal , liver, pancreatic, bladder or testicular epithelial cell derived from a cancerous tissue.
  • the cell or cell population may be or contain an immortalized cell derived from a carcinoma cell, including lung cancer cell, esophageal cancer cell, colorectal cancer cell, liver cancer cell, pancreatic cancer cell, bladder cancer cell, testicular cancer cell, acute myeloid leukemia cell, osteosarcoma cell and soft tissue tumor cell.
  • Cells expressing the MCM7 gene may be cell lines established from cancers (e.g., lung, liver, bladder cancer cell lines such as H1780, H1373, LC319, A549, PC-14, SK-MES-1, H2170, H520, H1703, RERF-LCAI, LX1, SBC3, SBC5, DMS273, DMS114, SW780, RT4, SNU475, Huh7 etc.).
  • cancers e.g., lung, liver, bladder cancer cell lines such as H1780, H1373, LC319, A549, PC-14, SK-MES-1, H2170, H520, H1703, RERF-LCAI, LX1, SBC3, SBC5, DMS273, DMS114, SW780, RT4, SNU475, Huh7 etc.
  • the cell or cell population may be or contain a cell which has been transfected with the MCM7 gene.
  • the present method allows screening of various test substances mentioned above and is particularly suited for screening functional nucle
  • the present invention provides a screening method which includes the following steps of: a) contacting a test substance with a cell into which a vector, including a transcriptional regulatory region of an MCM7 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; b) detecting the expression level or activity of said reporter gene; c) comparing the expression level or activity detected in step b) with the expression level or activity detected in the absence of the substance ; and d) selecting the substance that reduces the expression level or activity of said reporter gene as a candidate substance for treating or preventing cancer.
  • the therapeutic effect of the test substance on cancer may be evaluated in accordance with the present invention. More particularly, the present invention provides a method of screening for a candidate substance that suppresses the proliferation of cancer cells, as well as a method of screening for a candidate substance for treating or preventing cancer.
  • the present invention provides a method which includes the following steps of: a) contacting a test substance with a cell into which a vector, composed of a transcriptional regulatory region of an MCM7 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; b) detecting the expression level or activity of said reporter gene; and c) correlating the expression level or activity of b) with the therapeutic effect of the test substance.
  • a therapeutic effect may be correlated with the expression level or activity of said reporter gene.
  • the test substance when the test substance reduces the expression level or activity of said reporter gene as compared to an expression level or activity detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect.
  • the test substance when the test substance does not reduce the expression level or activity of said reporter gene as compared to an expression level or activity detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
  • suppressing the expression of MCM7 gene reduces cell growth.
  • candidate substances that have the potential to treat or prevent cancers can be identified. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers.
  • the reporter construct required for the screening can be prepared using the transcriptional regulatory region of the MCM7 gene, which can be obtained as a nucleotide segment containing the transcriptional regulatory region from a genome library based on the nucleotide sequence information of the gene.
  • the transcriptional regulatory region may be, for example, the promoter sequence of the MCM7 gene.
  • the reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of MCM7 gene.
  • the transcriptional regulatory region of MCM7 gene herein is the region from start codon to at least 500 bp upstream, preferably 1,000 bp, more preferably 5,000 or 10,000 bp upstream.
  • a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).
  • an substance can be identified as inhibiting or enhancing the expression of the MCM7 gene through detecting the expression level of the reporter gene product.
  • Illustrative reporter genes include, but are not limited to, luciferase, green florescence protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed), Chrolamphenicol Acetyltransferase (CAT), lacZ and beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa, Ade2 gene, HIS3 gene, and others well-known in the art. Methods for detection of the expression of these genes are well known in the art.
  • a vector containing a reporter construct may be infected to host cells and the expression or activity of the reporter gene is detected by method well known in the art (e.g., using luminometer, absorption spectrometer, flow cytometer and so on).
  • the phrase "reduces the expression or activity” encompasses at least 10% reduction of the expression or activity of the reporter gene in comparison with in absence of the compound, more preferably at least 25%, 50% or 75% reduction and most preferably at 95% reduction.
  • any substance that is metabolized in a subject to act as an anti-tumor substance can manifest itself by inducing a change in a gene expression pattern in the subject's cells from that characteristic of a cancerous state to a gene expression pattern characteristic of a non cancerous state.
  • the differential expression of the MCM7 gene in cancerous and non-cancerous cells can serve as an index of the putative therapeutic or prophylactic potential of a test substance in a test cell population from a selected subject in order to determine if the substance is a suitable inhibitor of cancer in the subject.
  • test cell populations contain cancer cells expressing the MCM7 gene.
  • the test cell is a lung, esophageal, colorectal, liver, pancreatic, bladder and testicular epithelial cell.
  • a test cell population may be incubated in the presence of a candidate therapeutic substance and the expression of the MCM7 gene in the test cell population may be measured and compared to one or more reference profiles, e.g., a cancerous reference expression profile or a non-cancerous reference expression profile.
  • a decrease in the expression of the MCM7 gene in a test cell population relative to a reference cell population containing cancer indicates that the substance has therapeutic potential.
  • a similarity in the expression of the MCM7 gene in a test cell population relative to a reference cell population not containing cancer indicates that the substance has therapeutic potential.
  • compositions for treating or preventing cancer The substances identified by any of the screening methods of the present invention, as well as antisense nucleic acids and double-stranded molecules (e.g., siRNA) against the MCM7 gene, and antibodies against the MCM7 polypeptide that inhibit or suppress the expression of the MCM7 gene, or the biological activity of the MCM7 polypeptide are expected to inhibit or disrupt cancer cell proliferation and thus find utility in the context of pharmaceutical formulations.
  • the present invention contemplates pharmaceutical compositions formulated for the treatment and/or prevention of cancer, wherein the compositions include substances identified by any of the screening methods of the present invention, antisense nucleic acids or double-stranded molecules against the MCM7 gene, or antibodies against the MCM7 polypeptide.
  • compositions can be used for treating and/or preventing cancer, in particular, cancers such as lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, especially lung cancer and bladder cancer.
  • cancers such as lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, especially lung cancer and bladder cancer.
  • composition is used to refer to a product including that include the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • pharmaceutical when used in relation to the modifier "pharmaceutical” (as in “pharmaceutical composition"), are intended to encompass products including a product that includes the active ingredient(s), and any inert ingredient(s) that make up the carrier, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.
  • pharmaceutical composition refers to any product made by admixing a molecule or compound of the present invention and a pharmaceutically or physiologically acceptable carrier.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier”, as used herein, means a pharmaceutically or physiologically acceptable material, composition, substance or vehicle, including but not limited to, a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
  • active ingredient refers to a substance in composition that is biologically or physiologically active.
  • active ingredient refers to a substance that shows an objective pharmacological effect.
  • active ingredients in the agents or compositions may lead to at least one biological or physiologically action on cancer cells and/or tissues directly or indirectly.
  • such action may include reducing or inhibiting cancer cell growth, damaging or killing cancer cells and/or tissues, and so on.
  • the "active ingredient” may also be referred to as "bulk", “drug substance” or "technical product”.
  • compositions of the present invention are characterized as being at least sterile and pyrogen-free.
  • pharmaceutical formulations include formulations for human and veterinary use.
  • the compositions may be used as pharmaceuticals for humans and other mammals, such as mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees.
  • suitable pharmaceutical formulations for the active ingredients of the present invention include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation.
  • Other formulations include implantable devices and adhesive patches that release a therapeutic agent. When desired, the above-described formulations may be adapted to give sustained release of the active ingredient.
  • compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
  • the preferred route of administration is intravenous delivery.
  • the formulations are optionally packaged in discrete dosage units.
  • compositions suitable for oral administration include capsules, microcapsules, cachets and tablets, each containing a predetermined amount of active ingredient. Suitable formulations also include powders, elixirs, granules, solutions, suspensions and emulsions.
  • the active ingredient is optionally administered as a bolus electuary or paste.
  • the pharmaceutical composition may be administered non-orally, in the form of injections of sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid.
  • the active ingredients of the present invention can be mixed with pharmaceutically acceptable carriers or media, specifically, sterilized water, physiological saline, plant-oils, emulsifiers, suspending substances, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and such, in a unit dose form required for generally accepted drug implementation.
  • pharmaceutically acceptable carriers or media specifically, sterilized water, physiological saline, plant-oils, emulsifiers, suspending substances, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and such, in a unit dose form required for generally accepted drug implementation.
  • the amount of active ingredient contained in such a preparation makes a suitable dosage within the indicated range acquirable.
  • additives that can be admixed into tablets and capsules include, but are not limited to, binders, such as gelatin, corn starch, tragacanth gum and arabic gum; excipients, such as crystalline cellulose; swelling agents, such as corn starch, gelatin and alginic acid; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose or saccharin; and flavoring agents, such as peppermint, Gaultheria adenothrix oil and cherry.
  • a tablet may be made by compression or molding, optionally with one or more formulational ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine in which the active ingredients in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made via molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be coated according to methods well known in the art. The tablets may optionally be formulated so as to provide slow or controlled release of the active ingredient in vivo. A package of tablets may contain one tablet to be taken on each of the month.
  • a liquid carrier such as oil
  • oil can be further included in addition to the above ingredients.
  • Oral fluid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle prior to use.
  • Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) or preservatives.
  • Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use. Alternatively, the formulations may be presented for continuous infusion.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • sterile composites for injection can be formulated following normal drug implementations using vehicles, such as distilled water, suitable for injection.
  • vehicles such as distilled water, suitable for injection.
  • Physiological saline, glucose, and other isotonic liquids, including adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used as aqueous solutions for injection.
  • adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride
  • solubilizers such as alcohol, for example, ethanol
  • polyalcohols such as propylene glycol and polyethylene glycol
  • non-ionic surfactants such as Polysorbate 80 (TM) and HCO-50.
  • Sesame oil or soybean oil can be used as an oleaginous liquid, which may be used in conjunction with benzyl benzoate or benzyl alcohol as a solubilizer, and may be formulated with a buffer, such as phosphate buffer and sodium acetate buffer; a pain-killer, such as procaine hydrochloride; a stabilizer, such as benzyl alcohol and phenol; and/or an anti-oxidant.
  • a prepared injection may be filled into a suitable ampoule.
  • Formulations for rectal administration include suppositories with standard carriers such as cocoa butter or polyethylene glycol.
  • Formulations for topical administration in the mouth include lozenges, which contain the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles including the active ingredient in a base such as gelatin, glycerin, sucrose or acacia.
  • a liquid spray or dispersible powder or in the form of drops may be used. Drops may be formulated with an aqueous or non-aqueous base also including one or more dispersing agents, solubilizing agents or suspending agents.
  • compositions are conveniently delivered from an insufflator, nebulizer, pressurized packs or other convenient means of delivering an aerosol spray.
  • Pressurized packs may include a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichiorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the compositions may take the form of a dry powder composition, for example, a powder mix of an active ingredient and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflators.
  • Other formulations include implantable devices and adhesive patches that release a therapeutic agent.
  • the above-described formulations adapted to give sustained release of the active ingredient, may be employed.
  • the pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants or preservatives.
  • the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question; for example, those suitable for oral administration may include flavoring agents.
  • Preferred unit dosage formulations are those containing an effective dose, as recited under the item of 'VI. Method for treating or preventing cancer' (infra), of each active ingredients of the present invention or an appropriate fraction thereof.
  • compositions containing screened substances The present invention provides compositions for treating or preventing cancers including any of the substances selected by the above-described screening methods of the present invention.
  • An substances screened by the method of the present invention can be directly administered or can be formulated into a dosage form according to any conventional pharmaceutical preparation method detailed above.
  • Double-stranded molecules against the MCM7 gene can be used to reduce the expression level of the MCM7 gene.
  • double-stranded molecule refers to a nucleic acid molecule that inhibits expression of a target gene including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)) as described in "Definitions”.
  • double-stranded molecules include a portion of the sense nucleic acid sequence and the complementary anti-sense nucleic acid sequence of the MCM7 gene.
  • the double-stranded molecule is constructed so that it includes both a portion of the sense and complementary antisense sequences of the target gene (i.e., the MCM7 gene), and may also be a single construct taking a hairpin structure, wherein the sense and antisense strands are linked via a single-strand.
  • the double-stranded molecule serves as a guide for identifying homologous sequences in mRNA for the RISC complex, when the double-stranded molecule is introduced into cells.
  • the identified target RNA is cleaved and degraded by the nuclease activity of Dicer, through which the double-stranded molecule eventually decreases or inhibits production (expression) of the polypeptide encoded by the RNA.
  • a double-stranded molecule of the present invention can be defined by its ability to generate a single-strand that specifically hybridizes to the mRNA of the MCM7 gene under stringent conditions.
  • target sequence or “target nucleic acid” or “target nucleotide”.
  • nucleotide sequence of the “target sequence” can be shown using not only the RNA sequence of the mRNA, but also the DNA sequence of cDNA synthesized from the mRNA.
  • a double-stranded molecule is preferably less than 500, 200, 100, 50, or 25 base pairs in length. More preferably, a double stranded molecule is 19-25 base pairs in length.
  • Exemplary target sequences of double-stranded molecules against the MCM7 gene include the nucleotide sequences of SEQ ID NO: 13 and 15.
  • the pharmaceutical composition of the present invention may include a double-stranded RNA molecule (i.e., siRNA) including the nucleotide sequence 5'- GGCUAAUGGAGAUGUCAA -3' (for SEQ ID NO: 13), and 5'- GAAAGAAGAUGUGAAUGA -3' (for SEQ ID NO: 15) as the sense strand.
  • siRNA double-stranded RNA molecule
  • 3' overhangs can be added to the 3'end of the target sequence in the sense and/or antisense strand.
  • the number of nucleotides to be added is at least 2, generally 2 to 10, preferably 2 to 5.
  • the added nucleotides form a single strand at the 3'end of the sense and/or antisense strand of the double-stranded molecule.
  • the nucleotides to be added is preferably "u" or "t", but are not limited to.
  • a loop sequence composed of an arbitrary nucleotide sequence can be located between the sense and antisense strands in order to form a hairpin loop structure.
  • the double-stranded molecule contained in the pharmaceutical composition of the present invention may take the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence, [B] is an intervening single-strand and [A'] is the antisense strand containing a complementary sequence to the target sequence.
  • the polynucleotide strand which includes a sequence corresponding to a target sequence may be referred to as "sense strand".
  • [A] is the sense strand
  • [B] is a single stranded polynucleotide composed of 3 to 23 nucleotides
  • [A'] is a polynucleotide strand which includes the antisense strand containing a complementary sequence of a target sequence, specifically hybridizing to an mRNA or a cDNA of the MCM7 gene (i.e., a sequence hybridizing to the target sequence of the sense strand [A]).
  • the polynucleotide strand which includes a complementary sequence to a target sequence, specifically hybridizing to an mRNA or a cDNA of the MCM7 gene may be referred to as "antisense strand".
  • the region [A] hybridizes to [A'], and then a loop composed of the region [B] is formed.
  • the loop sequence may be preferably 3 to 23 nucleotides in length.
  • Exemplary loop sequences include, but are not limited to, the following sequences (www.ambion.com/techlib/tb/tb_506.html): CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002, 418: 435-8.
  • UUCG Lee NS et al., Nature Biotechnology 2002, 20:500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003, 100(4):1639-44.
  • UUCAAGAGA Dykxhoorn DM et al., Nature Reviews Molecular Cell Biology 2003, 4:457-67.
  • 'UUCAAGAGA ("ttcaagaga" in DNA)' is a particularly suitable loop sequence.
  • loop sequence composed of 23 nucleotides also provides an active siRNA (Jacque JM et al., Nature 2002, 418:435-8).
  • Exemplary hairpin siRNA suitable for the MCM7 gene include: 5'- GGCUAAUGGAGAUGUCAA -[b]-UUGACAUCUCCAUUAGCC -3' (for target sequence of SEQ ID NO: 13); and 5'- GAAAGAAGAUGUGAAUGA-[b]- UCAUUCACAUCUUCUUUC-3' (for target sequence of SEQ ID NO: 15).
  • Other nucleotide sequences of suitable double-stranded molecules for the present invention can be designed using an siRNA design computer program available from the Ambion website (www.ambion.com/techlib/ misc/siRNA_finder.html). The computer program selects nucleotide sequences for double-stranded molecule synthesis based on the following protocol.
  • Target Sites for double-stranded molecules 1. Beginning with the AUG start codon of the object transcript, scan downstream for AA dinucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential target sites. Tuschl et al. Genes Cev 1999, 13(24):3191-7 don't recommend designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 nucleotides) as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex. 2.
  • the homology search can be performed using BLAST (Altschul SF et al., Nucleic Acids Res 1997, 25:3389-402; J Mol Biol 1990, 215:403-10.), which can be found on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/. 3. Select qualifying target sequences for synthesis. At Ambion, preferably several target sequences can be selected along the length of the gene to evaluate.
  • the method of preparing the double-stranded molecule can use any chemical synthetic method known in the art. According to the chemical synthesis method, sense and antisense single-stranded polynucleotides are separately synthesized and then annealed together via an appropriate method to obtain a double-stranded molecule. Alternatively, a double-stranded molecule or siRNA molecule of the present invention may also be synthesized with in vitro translation. In this embodiment, DNA encoding a nucleotide sequence that comprises the target sequence and antisense thereof is transcribed into the double-stranded molecule in vitro.
  • the synthesized single-stranded polynucleotides are mixed in a molar ratio of at least about 3:7, for example, about 4:6, for example, substantially equimolar amount (i.e., a molar ratio of about 5:5).
  • the mixture is heated to a temperature at which double-stranded molecules dissociate and then is gradually cooled down.
  • the annealed double-stranded polynucleotide can be purified by usually employed methods known in the art.
  • Example of purification methods include methods utilizing agarose gel electrophoresis or wherein remaining single-stranded polynucleotides are optionally removed by, e.g., degradation with appropriate enzyme.
  • the regulatory sequences flanking target sequences can be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner.
  • the double-stranded molecules can be transcribed intracellularly by cloning MCM7 gene template into a vector containing, e.g., an RNA pol III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.
  • snRNA small nuclear RNA
  • Standard techniques are known in the art for introducing a double-stranded molecule into cells.
  • a double-stranded molecule can be directly introduced into the cells in a form that is capable of binding to the mRNA transcripts.
  • the double-stranded molecules are typically modified as described below for antisense molecules.
  • Other modifications are also available, for example, cholesterol-conjugated double-stranded molecule has shown improved pharmacological properties (Song et al., Nature Med 2003, 9:347-51). These conventionally used techniques may also be applied for the double-stranded molecules contained in the present compositions.
  • a DNA encoding the double-stranded molecule may be carried in a vector (hereinafter, also referred to as 'siRNA vector') and the double-stranded molecule may be contained in the present composition in the form of vector which enables expression of the double-stranded molecule in vivo.
  • a vector hereinafter, also referred to as 'siRNA vector'
  • Such vectors may be produced, for example, by cloning a portion of the target sequence sufficient to inhibit the in vivo expression of the target gene into an expression vector having operatively-linked regulatory sequences (e.g., a RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter) flanking the sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nature Biotechnology 2002, 20: 500-5).
  • operatively-linked regulatory sequences e.g., a RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter
  • an RNA molecule that is antisense to mRNA of the target gene is transcribed by a first promoter (e.g., a promoter sequence 3' of the cloned DNA) and an RNA molecule that is the sense strand for the mRNA of the target gene is transcribed by a second promoter (e.g., a promoter sequence 5' of the cloned DNA).
  • the sense and antisense strands hybridize in vivo to generate the double-stranded molecule construct for silencing the expression of the target gene.
  • the sense and antisense strands may be transcribed together with the help of one promoter.
  • the sense and antisense strands may be linked via a polynucleotide sequence to form a single-stranded construct having secondary structure, e.g., hairpin.
  • the present pharmaceutical composition for treating or preventing cancer may include either the double-stranded molecule (e.g., siRNA) or a vector expressing the double-stranded molecule in vivo.
  • the present invention provides pharmaceutical compositions for treating or preventing cancer that include a double-stranded molecule that inhibits the expression of the MCM7 gene, or a vector expressing the double-stranded molecule in vivo.
  • the present invention also provides pharmaceutical compositions for inhibiting cancer cell proliferation, such composition including a double-stranded molecule which inhibits the expression of the MCM7 gene, or a vector expressing the double-stranded molecule in vivo.
  • transfection-enhancing agent For introducing the double-stranded molecule vector into the cell, transfection-enhancing agent can be used. FuGENE6 (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are useful as the transfection-enhancing agent. Therefore, the present pharmaceutical composition may further include such transfection-enhancing agents.
  • the present invention also provides the use of the double-stranded nucleic acid molecules of the present invention or vector encoding thereof in manufacturing a pharmaceutical composition for treating a cancer expressing the MCM7 gene.
  • the present invention relates to a use of double-stranded nucleic acid molecule that inhibits the expression of MCM7 gene in a cell that over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NOs: 13 or 15 for manufacturing a pharmaceutical composition for treating a cancer expressing the MCM7 gene.
  • the double-stranded nucleic acid molecules of the present invention find utility in the treatment of a cancer expressing the MCM7 gene. Accordingly, the present invention provides a method or process for manufacturing a pharmaceutical composition for treating a cancer expressing the MCM7 gene, wherein the method or process includes step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the expression of MCM7 gene in a cell, which over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NOs: 13 or 15 as active ingredients.
  • the present invention provides a method or process for manufacturing a pharmaceutical composition for treating a cancer expressing the MCM7 gene, wherein the method or process includes step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double-stranded nucleic acid molecule inhibiting the expression of MCM7 gene in a cell, which over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NOs: 13 or 15.
  • Antisense nucleic acids targeting the MCM7 gene can be used to reduce the expression level of the gene that is up-regulated in cancerous cells including lung cancer cells, esophageal cancer cells, colorectal cancer cells, liver cancer cells, pancreatic cancer cells, bladder cancer cells, testicular cancer cells, acute myeloid leukemia cells, Osteosarcoma cells and soft tissue tumor cells.
  • cancerous cells including lung cancer cells, esophageal cancer cells, colorectal cancer cells, liver cancer cells, pancreatic cancer cells, bladder cancer cells, testicular cancer cells, acute myeloid leukemia cells, Osteosarcoma cells and soft tissue tumor cells.
  • Such antisense nucleic acids are useful for the treatment of cancer, in particular lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, and thus are also encompassed by the present invention.
  • An antisense nucleic acid acts by binding to the nucleotide sequence of the MCM7 gene, or mRNAs corresponding thereto, thereby inhibiting the transcription or translation of the gene, promoting the degradation of the mRNAs, and/or inhibiting the expression of the protein encoded by the gene.
  • an antisense nucleic acid inhibits the MCM7 protein to function in the cancerous cell.
  • antisense nucleic acids refers to nucleotides that specifically hybridize to a target sequence and includes not only nucleotides that are entirely complementary to the target sequence but also that include mismatches of one or more nucleotides.
  • the antisense nucleic acids of the present invention include polynucleotides that have a homology of at least 70% or higher, preferably of at least 80% or higher, more preferably of at least 90% or higher, even more preferably of at least 95% or higher (up to 99% and 100% homology) over a span of at least 15 continuous nucleotides of the MCM7 gene or the complementary sequence thereof. Algorithms known in the art can be used to determine such homology.
  • Antisense nucleic acids of the present invention act on cells producing proteins encoded by the MCM7 gene by binding to the DNA or mRNA of the gene, inhibiting their transcription or translation, promoting the degradation of the mRNA, and inhibiting the expression of the protein, finally inhibiting the protein to function.
  • Antisense nucleic acids of the present invention can be made into an external preparation, such as a liniment or a poultice, by admixing it with a suitable base material which is inactive against the nucleic acids.
  • the antisense nucleic acids of the present invention can also be formulated into tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops and freeze-drying agents by adding excipients, isotonic agents, solubilizers, stabilizers, preservatives, pain-killers, and such.
  • An antisense-mounting medium can also be used to increase durability and membrane-permeability. Examples include, but are not limited to, liposomes, poly-L-lysine, lipids, cholesterol, lipofectin, or derivatives of these.
  • Such formulations can be prepared in accordance with conventional techniques familiar to those of skill in the art.
  • the antisense nucleic acids of the present invention inhibit the expression of the MCM7 gene and thus find utility in suppressing the biological activity of the protein.
  • expression-inhibitors including antisense nucleic acids of the present invention, are also useful in that they can inhibit the biological activity of the MCM7 protein.
  • the antisense nucleic acids of present invention may encompass modified oligonucleotides. For example, thioated oligonucleotides may be used to confer nuclease resistance to an oligonucleotide.
  • compositions including antibodies
  • An antibody against the MCM7 polypeptide can be mentioned as such a substance and can be used as the active ingredient of a pharmaceutical composition for treating or preventing cancer.
  • the present invention relates to the use of antibodies against a protein encoded by the MCM7 gene, or immunogenic fragments of such antibodies.
  • antibody refers to an immunoglobulin molecule having a specific structure, that interacts (i.e., binds) only with the antigen that was used for synthesizing the antibody (i.e., the gene product of an up-regulated marker) or with an antigen closely related thereto. Molecules including the antigen that was used for synthesizing the antibody and molecules including the epitope of the antigen recognized by the antibody can be mentioned as closely related antigens thereto.
  • an antibody suitable for use as a pharmaceutical composition may be an immunogenic fragment of an antibody or a modified antibody, so long as it binds to the protein encoded by the MCM7 gene (e.g., an immunologically active fragment of anti- MCM7 antibody).
  • the antibody fragment may be Fab, F(ab') 2 , Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston JS et al., Proc Natl Acad Sci USA 1988, 85:5879-83).
  • Such antibody fragments may be generated by treating an antibody with an enzyme, such as papain or pepsin.
  • a gene encoding the antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co MS et al., J Immunol 1994, 152:2968-76; Better M et al., Methods Enzymol 1989, 178:476-96; Pluckthun A et al., Methods Enzymol 1989, 178:497-515; Lamoyi E, Methods Enzymol 1986, 121:652-63; Rousseaux J et al., Methods Enzymol 1986, 121:663-9; Bird RE et al., Trends Biotechnol 1991, 9:132-7).
  • Any antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the present invention includes such modified antibodies.
  • the modified antibody can be obtained by chemically modifying an antibody. Such modification methods are conventional in the field.
  • an antibody of the present invention may be a chimeric antibody having a variable region derived from a non-human antibody against the MCM7 polypeptide and a constant region derived from a human antibody, or a humanized antibody, including a complementarity determining region (CDR) derived from a non-human antibody, a frame work region (FR) and a constant region derived from a human antibody.
  • CDR complementarity determining region
  • FR frame work region
  • Such antibodies can be prepared by using known technologies. Humanization can be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (see e.g., Verhoeyen et al., Science 1988, 239:1534-6). Accordingly, such humanized antibodies are chimeric antibodies, wherein an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • Human antibodies including human variable regions in addition to human framework and constant regions can also be used.
  • Such antibodies can be produced using various techniques known in the art. For example in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage (e.g., Hoogenboom et al., J Mol Biol 1992, 227:381-8).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, e.g., in US Pat. Nos.
  • Antibodies obtained as above may be purified to homogeneity.
  • the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins.
  • the antibody may be separated and isolated by the appropriately selected and combined use of column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and others (Antibodies: A Laboratory Manual. Ed Harlow and D Lane, Cold Spring Harbor Laboratory (1988)), but are not limited thereto.
  • a protein A column and protein G column can be used as the affinity column.
  • Exemplary protein A columns to be used include, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).
  • Exemplary chromatography with the exception of affinity includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)).
  • the chromatographic procedures can be carried out by liquid-phase chromatography, such as HPLC and FPLC.
  • Cancer therapies directed to specific molecular alterations that occur in cancer cells have been validated through clinical development and regulatory approval of anti-tumor pharmaceuticals such as trastuzumab (Herceptin) for the treatment of advanced cancers, imatinib mesylate (Gleevec) for chronic myeloid leukemia, gefitinib (Iressa) for non-small cell lung cancer (NSCLC), and rituximab (anti-CD20 mAb) for B-cell lymphoma and mantle cell lymphoma (Ciardiello F et al., Clin Cancer Res 2001, 7:2958-70, Review; Slamon DJ et al., N Engl J Med 2001, 344:783-92; Rehwald U et al., Blood 2003, 101:420-4; Fang G et al., Blood 2000, 96:2246-53).
  • trastuzumab Herceptin
  • Gleevec imatinib mesylate
  • modulatory methods can be performed ex vivo or in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the methods involve administering a protein or combination of proteins or a nucleic acid molecule or combination of nucleic acid molecules as therapy to counteract aberrant expression of the differentially expressed genes or aberrant activity of their gene products.
  • Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) expression levels or biological activities of genes and gene products, respectively, may be treated with therapeutics that antagonize (i.e., reduce or inhibit) activity of the over-expressed gene.
  • therapeutics that antagonize activity can be administered therapeutically or prophylactically.
  • the dysfunctional antisense molecules are utilized to "knockout" endogenous function of a polypeptide by homologous recombination (see, e.g., Capecchi, Science 1989, 244: 1288 92
  • Increased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a gene whose expression is altered).
  • Methods that are well known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
  • immunoassays e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.
  • hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
  • Prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • Therapeutic methods of the present invention may include the step of administering an agent that modulates one or more of the activities of the MCM7 gene products.
  • agent that modulate protein activity include, but are not limited to, nucleic acids, proteins, naturally occurring cognate ligands of such proteins, peptides, peptidomimetics, and other small molecule.
  • the present invention provides methods for treating or alleviating a symptom of cancer, or preventing cancer in a subject by decreasing the expression of the MCM7 gene or the activity of the gene product.
  • the present method is particularly suited for treating and/or preventing lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, especially lung cancer and bladder cancer.
  • Suitable therapeutics can be administered prophylactically or therapeutically to a subject suffering from or at risk of (or susceptible to) developing cancers. Such subjects can be identified by using standard clinical methods or by detecting an aberrant expression level ("up-regulation” or “over-expression") of the MCM7 gene or aberrant activity of the gene product.
  • substances identified through screening methods of the present invention may be used for treating or preventing cancer.
  • Methods well known to those skilled in the art may be used to administer the substances to patients, for example, as an intra-arterial, intravenous, or percutaneous injection or as an intranasal, transbronchial, intramuscular, or oral administration.
  • the substances are encodable by a DNA
  • the DNA can be inserted into a vector for gene therapy and the vector can be administered to a patient to perform the therapy.
  • the dosage and methods for administration vary according to the body-weight, age, sex, symptom, condition of the patient to be treated and the administration method; however, one skilled in the art can routinely select suitable dosage and administration method.
  • the dose of a substance that binds to an MCM7 polypeptide or regulates the activity of the polypeptide depends on the aforementioned various factors, the dose is generally about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day and more preferably about 1.0 mg to about 20 mg per day, when administered orally to a normal adult human (60 kg weight).
  • the agent When administering the agent parenterally, in the form of an injection to a normal adult human (60 kg weight), although there are some differences according to the patient, target organ, symptoms and methods for administration, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg per day and more preferably about 0.1 to about 10 mg per day. In the case of other animals, the appropriate dosage amount may be routinely calculated by converting to 60 kg of body-weight.
  • compositions of the present invention may be used for treating or preventing cancer.
  • Methods well known to those skilled in the art may be used to administer the compositions to patients, for example, as an intraarterial, intravenous, or percutaneous injection or as an intranasal, transbronchial, intramuscular, or oral administration.
  • the compositions e.g., polypeptides and organic compounds
  • the dose range for adult humans is generally from about 5 mg to about 17.5 g/day, preferably about 5 mg to about 10 g/day, and most preferably about 100 mg to about 3 g/day.
  • Tablets or other unit dosage forms of presentation provided in discrete units may conveniently contain an amount which is effective at such dosage or as a multiple of the same, for instance, units containing about 5 mg to about 500 mg, usually from about 100 mg to about 500 mg.
  • an antisense nucleic acid against the MCM7 gene can be given to the patient by direct application onto the ailing site or by injection into a blood vessel so that it will reach the site of ailment.
  • an antisense nucleic acid derivatives of the present invention can be adjusted suitably according to the patient's condition and used in desired amounts. For example, a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered.
  • the method of the present invention includes the step of administering the double-stranded molecule against the MCM7 gene to a subject.
  • the preferred examples of the double-stranded molecule to be administered are described under the item of " V-2.
  • the double-stranded molecules of the present invention degrade the mRNA of the MCM7 gene in substoichiometric amounts. Without wishing to be bound by any theory, it is believed that the double-stranded molecule of the invention causes degradation of the target mRNA in a catalytic manner. Thus, compared to standard cancer therapies, significantly less a double-stranded molecule needs to be delivered at or near the site of cancer to exert therapeutic effect.
  • an effective amount of the double-stranded molecule of the present invention can readily determine an effective amount of the double-stranded molecule of the present invention to be administered to a given subject, by taking into account factors such as body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is regional or systemic.
  • an effective amount of the double-stranded molecule of the invention is an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM to about 50 nM, more preferably from about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of the double-stranded molecule can be administered.
  • the precise dosage required for a particular circumstance may be readily and routinely determined by one of skill in the art.
  • the present methods can be used to inhibit the growth or metastasis of cancer; for example lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, especially lung cancer and bladder cancer.
  • the double-stranded molecule of the present invention can also be administered to a subject in combination with a pharmaceutical agent different from the double-stranded molecule.
  • the double-stranded molecule of the present invention can be administered to a subject in combination with another therapeutic method designed to treat cancer.
  • the double-stranded molecule of the present invention can be administered in combination with therapeutic methods currently employed for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents).
  • the double-stranded molecule can be administered to the subject either as a naked double-stranded molecule, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector which expresses the double-stranded molecule.
  • Suitable delivery reagents for administration in conjunction with the present a double-stranded molecule include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.
  • a preferred delivery reagent is a liposome.
  • Liposomes can aid in the delivery of the double-stranded molecule to a particular tissue, such as retinal or tumor tissue, and can also increase the blood half-life of the double-stranded molecule.
  • Liposomes suitable for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example as described in Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467; and US Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369, the entire disclosures of which are herein incorporated by reference.
  • the liposomes encapsulating the present double-stranded molecule comprises a ligand molecule that can deliver the liposome to the cancer site.
  • Ligands which bind to receptors prevalent in tumor or vascular endothelial cells such as monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens, are preferred.
  • the liposomes encapsulating the present double-stranded molecule are modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example, by having opsonization-inhibition moieties bound to the surface of the structure.
  • a liposome of the invention can comprise both opsonization-inhibition moieties and a ligand.
  • Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane.
  • an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids.
  • These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); e.g., as described in US Pat. No.
  • Liposomes modified with opsonization-inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes.
  • Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature.
  • target tissue characterized by such microvasculature defects for example, solid tumors, will efficiently accumulate these liposomes; see Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53.
  • the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation in liver and spleen.
  • liposomes of the invention that are modified with opsonization-inhibition moieties can deliver the present double-stranded molecule to tumor cells.
  • Opsonization-inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons.
  • Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM1.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • synthetic polymers such as polyacrylamide or poly N-vinyl
  • Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable.
  • the opsonization-inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide.
  • the opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.
  • the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".
  • the opsonization-inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques.
  • an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane.
  • a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH3 and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratio at 60 degrees C.
  • Vectors expressing a double-stranded molecule of the present invention are discussed in the following item. Such vectors expressing at least one double-stranded molecule of the invention can also be administered directly or in conjunction with a suitable delivery reagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.
  • a suitable delivery reagent including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.
  • a double-stranded molecule of the present invention can be administered to the subject by any means suitable for delivering the double-stranded molecule into cancer sites.
  • the double-stranded molecule can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.
  • Suitable enteral administration routes include oral, rectal, or intranasal delivery.
  • Suitable parenteral administration routes include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation. It is preferred that injections or infusions of the double-stranded molecule or vector be given at or near the site of cancer.
  • intravascular administration e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the
  • a double-stranded molecule of the present invention can be administered in a single dose or in multiple doses.
  • the infusion can be a single sustained dose or can be delivered by multiple infusions.
  • Injection of the agent directly into the tissue is at or near the site of cancer preferred. Multiple injections of the agent into the tissue at or near the site of cancer are particularly preferred.
  • the double-stranded molecule can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site.
  • the double-stranded molecule can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, more preferably from about seven to about ten days.
  • the double-stranded molecule is injected at or near the site of cancer once a day for seven days.
  • a dosage regimen comprises multiple administrations, it is understood that the effective amount of a double-stranded molecule administered to the subject can comprise the total amount of a double-stranded molecule administered over the entire dosage regimen.
  • Double-stranded molecules and vectors encoding them an siRNA including either of the sequences of SEQ ID NOs: 13 or 15 is demonstrated to suppress cell growth or viability of cells expressing the MCM7 gene. Accordingly, double-stranded molecules including any of these sequences and vectors expressing the molecules are considered to serve as preferable pharmaceutics for treating or preventing diseases which involve the proliferation of MCM7 gene expressing cells, for example, cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, more particularly lung cancer and bladder cancer.
  • diseases which involve the proliferation of MCM7 gene expressing cells
  • cancer particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, more particularly lung cancer and bladder cancer.
  • one aspect of the present invention relates to the provision of double-stranded molecules that include a nucleotide sequence corresponding to the target sequence selected from the group consisting of SEQ ID NOs: 13 and 15 and vectors expressing the molecules. More specifically, the present invention provides a double-stranded molecule, when introduced into a cell expressing the MCM7 gene, inhibits expression of the gene, wherein the double-stranded molecule includes a sense strand and an antisense strand, wherein the sense strand includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13 and 15 as a target sequence, and the antisense strand includes a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule.
  • the present invention provides a double-stranded molecule, when introduced into a cell expressing an MCM7 gene, inhibits expression of the gene, wherein the double-stranded molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 13 and 15, and the antisense strand comprises a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule.
  • the target sequence for the MCM7 gene included in the sense strand may be composed of a sequence of a portion of SEQ ID NO: 17 or 19 that is less than about 500, 400, 300, 200, 100, 75, 50 or 25 contiguous nucleotides.
  • the target sequence may be from about 19 to about 25 contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 17 or 19.
  • suitable target sequences include the nucleotide sequences selected from the group consisting of SEQ ID NOs: 13 and 15.
  • the double-stranded molecule of the present invention may be composed of two polynucleotide constructs, i.e., a polynucleotide including the sense strand and a polynucleotide including the antisense strand.
  • the molecule may be composed of one polynucleotide construct; i.e., a polynucleotide including both the sense strand and the antisense strand, wherein the sense and antisense strands are linked via a single-stranded polynucleotide which enables hybridization of the target sequences within the sense and antisense strands by forming a hairpin structure.
  • the single-stranded polynucleotide may also be referred to as "loop sequence” or “single-strand".
  • the single-stranded polynucleotide linking the sense and antisense strands may consist of 3 to 23 nucleotides. See under the item of "V-2.
  • the double-stranded molecules of the present invention may contain one or more modified nucleotides and/or non-phosphodiester linkages.
  • Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule.
  • the skilled person will be aware of other types of chemical modification which may be incorporated into the present molecules (WO03/070744; WO2005/045037).
  • modifications can be used to provide improved resistance to degradation or improved uptake.
  • modifications include, but are not limited to, phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base” nucleotides, 5'-C- methyl nucleotides, and inverted deoxybasic residue incorporation (US20060122137).
  • modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule.
  • Modifications include chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3' or 5' terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2-fluoro modified ribonucleotides and 2'-deoxy ribonucleotides (WO2004/029212).
  • modifications can be used to increased or decreased affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (WO2005/044976).
  • an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine.
  • an unmodified purine can be substituted with a 7-deaza, 7-alkyl, or 7-alkenyl purine.
  • the double-stranded molecule is a double-stranded molecule with a 3' overhang
  • the 3'- terminal nucleotide overhanging nucleotides may be replaced by deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200).
  • published documents such as US20060234970 are available.
  • the present invention is not limited to these examples and any known chemical modifications may be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene.
  • the double-stranded molecules of the invention may include both DNA and RNA, e.g., dsD/R-NA or shD/R-NA.
  • RNA e.g., dsD/R-NA or shD/R-NA.
  • a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability.
  • RNA i.e., a hybrid type double-stranded molecule consisting of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule including both DNA and RNA on any or both of the single strands (polynucleotides), or the like may be formed for enhancing stability of the double-stranded molecule.
  • the hybrid of a DNA strand and an RNA strand may be the hybrid in which either the sense strand is DNA and the antisense strand is RNA, or the opposite so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
  • the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA.
  • the chimera type double-stranded molecule may be either the molecule that both of the sense and antisense strands are composed of DNA and RNA, or the molecule that any one of the sense and antisense strands is composed of DNA and RNA so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
  • the molecule preferably contains as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule is required to be RNA within a range to induce sufficient inhibition of the expression.
  • an upstream partial region i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands
  • the upstream partial region means the 5' side (5'-end) of the sense strand and the 3' side (3'-end) of the antisense strand.
  • a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand consists of RNA.
  • the chimera or hybrid type double-stranded molecule of the present invention include following combinations.
  • sense strand 5'-[---DNA---]-3' 3'-(RNA)-[DNA]-5' :antisense strand
  • sense strand 5'-(RNA)-[DNA]-3' 3'-(RNA)-[DNA]-5' :antisense strand
  • sense strand 5'-(RNA)-[DNA]-3' 3'-(---RNA---)-5' :antisense strand
  • the upstream partial region preferably is a domain composed of 9 to 13 nucleotides counted from the terminus of the target sequence or complementary sequence thereto within the sense or antisense strands of the double-stranded molecules.
  • preferred examples of such chimera type double-stranded molecules include those having a strand length of 19 to 21 nucleotides in which at least the upstream half region (5' side region for the sense strand and 3' side region for the antisense strand) of the polynucleotide is RNA and the other half is DNA. In such a chimera type double-stranded molecule, the effect to inhibit expression of the target gene is much higher when the entire antisense strand is RNA (US20050004064).
  • the double-stranded molecule may form a hairpin, such as a short hairpin RNA (shRNA) and short hairpin composed of DNA and RNA (shD/R-NA).
  • shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • the shRNA or shD/R-NA includes the sense target sequence and the antisense target sequence on a single strand wherein the sequences are separated by a loop sequence.
  • the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the target sequence of the dsRNA or dsD/R-NA.
  • RISC RNA-induced silencing complex
  • the present invention further provides vectors that include a combination of polynucleotide having a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid includes nucleotide sequence of SEQ ID NOs: 13 or 15, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing the MCM7, inhibit expression of said gene.
  • the sense strand of the polynucleotide is an oligonucleotide of between about 19 and 25 nucleotides in length (e.g., contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 17 or 19). More preferably, the combination of polynucleotide includes a single nucleotide transcript having the sense strand and the antisense strand linked via a single-stranded nucleotide sequence.
  • the combination of polynucleotide has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3' wherein [A] is a nucleotide sequence including SEQ ID NO: 13 or 15; [B] is a nucleotide sequence composed of about 3 to about 23 nucleotide; and [A'] is a nucleotide sequence complementary to the target sequence.
  • Vectors of the present invention can be produced by conventional means, for example, by cloning MCM7 sequence into an expression vector so that regulatory sequences are operatively-linked to MCM7 sequence in a manner to allow expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5).
  • RNA molecule that is the antisense to mRNA is transcribed by a first promoter (e.g., a promoter sequence flanking to the 3' end of the cloned DNA) and RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA).
  • a first promoter e.g., a promoter sequence flanking to the 3' end of the cloned DNA
  • RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA).
  • the sense and antisense strands hybridize in vivo to generate a double-stranded molecule constructs for silencing of the gene.
  • two vectors constructs respectively encoding the sense and antisense strands of the double-stranded molecule are utilized to respectively express the sense and anti-sense strands and then forming a double-stranded molecule construct.
  • the cloned sequence may encode a construct having a secondary structure (e.g., hairpin); namely, a single transcript of a vector contains both the sense and complementary antisense sequences of the target gene.
  • the vectors of the present invention may also be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; US Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720.
  • DNA-based delivery technologies include "naked DNA”, facilitated (bupivacaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., US Patent No. 5,922,687).
  • the vectors of the present invention include, for example, viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, such as vaccinia or fowlpox (see, e.g., US Patent No. 4,722,848). This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the double-stranded molecule. Upon introduction into a cell expressing the target gene, the recombinant vaccinia virus expresses the molecule and thereby suppresses the proliferation of the cell.
  • Another example of useable vector includes Bacille Calmette Guerin (BCG). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60.
  • a wide variety of other vectors are useful for therapeutic administration and production of the double-stranded molecules; examples include adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14: 571-85.
  • EXAMPLE1 General Methods Cell lines and lung tissue samples Cancer cell lines used in this study were as follows: lung adenocarcinoma (ADC) NCI-H1781, NCI-H1373, LC319, A549 and PC-14; lung squamous cell carcinoma (SCC) SK-MES-1, NCI-H2170, NCI-H520, NCI-H1703 and RERF-LC-AI; lung large cell carcinoma (LCC) LX1; and small cell lung cancer (SCLC) SBC-3, SBC-5, DMS273 and DMS114; bladder cancer SW780 and RT4; liver cancer SNU475 and Huh7.
  • ADC lung adenocarcinoma
  • SCC lung squamous cell carcinoma
  • LCC lung large cell carcinoma
  • SCLC small cell lung cancer
  • SAEC Human small airway epithelial cells
  • NSCLC Primary non-SCLC
  • tumor samples were selected from patients who fulfilled all of the following criteria: (a) patients suffered primary NSCLC with histologically confirmed stage (only pT1 to pT3, pN0 to pN2, and pM0); (b) patients underwent curative surgery, but did not receive any preoperative treatment; (c) among them, NSCLC patients with positive lymph node metastasis (pN1, pN2) were treated with platinum-based adjuvant chemotherapies after surgical resection, whereas patients with pN0 did not receive adjuvant chemotherapies; and (d) patients whose clinical follow-up data were available.
  • This study and the use of all clinical materials mentioned were approved by individual institutional ethics committees.
  • Bladder tissue samples and RNA preparation Bladder tissue samples and RNA preparation were described previously (hayami S, et al, Int J Cancer 2010, Hayami S, et al, Mol Cancer 2010;9:59, Wallard MJ, et al, Br J Cancer 2006;94:569-77.). Briefly, 136 surgical specimens of primary urothelial carcinoma were collected, either at cystectomy or transurethral resection of bladder tumor (TURBT), and snap frozen in liquid nitrogen. 23 specimens of normal bladder urothelial tissue were collected from areas of macroscopically normal bladder urothelium in patients with no evidence of malignancy. Approximately 10,000 cells were microdissected from both stromal and epithelial/tumor compartments in each tissue.
  • the cDNAs were amplified by RT-PCR using poly (A) + RNAs isolated from various human organs as templates; the lengths of the amplicons ranged from 200 to 1,100 bp, without any repetitive or poly (A) sequences.
  • Many types of tumor and corresponding non-neoplastic tissues were prepared in 8-micrometer, as described previously (Kitahara O, et al, Cancer Res 2001;61:3544-9). A total of 30,000-40,000 cancer or noncancerous cells were collected selectively using the EZ cut system (SL Microtest GmbH, Germany) according to the manufacturer's protocol.
  • RNA twice-amplified RNA
  • siRNA transfection and cell growth assay siRNA oligonucleotide duplexes were purchased from SIGMA Genosys for targeting the human MCM7 transcripts.
  • siEGFP and siNegative control (siNC) which consists of three different oligonucleotide duplexes, were used as control siRNAs.
  • the siRNA sequences are described in Table 2.
  • siRNA duplexes (100 nM final concentration) were transfected into lung and bladder cancer cell lines with Lipofectamine 2000 (Invitrogen) for 72 hours, and cell growth was examined using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan).
  • BrdU labeling and immuocytochemical analysis were performed according to previously reported protocols (Hayami S, et al, Int J Cancer 2010, Hayami S, et al, Mol Cancer 2010;9:59.).
  • A549, SBC5 and SW780 cells were incubated with appropriate media containing 2 microM BrdU (BD Biosciences, Franklin Lakes, NJ) for 20 min, and fixed and permeabilized with 100% methanol for 5 min at room temperature. The cells were washed with PBS, and then blocked by 3% BSA for 1 hour at 37 degrees C. Then, the cells were incubated with an anti-MCM7 antibody in 3% BSA overnight at 4 degrees C.
  • the cells were reacted with Alexa Fluor 594-conjugated goat anti-mouse IgG for 1 hour at 37 degrees C in the blocking solution. They were then re-fixed, treated with 4 M HCl for 30 min at room temperature and incubated with FITC-conjugated anti-BrdU (BD Biosciences), diluted 1:300, for 1 hour at room temperature, followed by observation with confocal microscopy.
  • Alexa Fluor 594-conjugated goat anti-mouse IgG for 1 hour at 37 degrees C in the blocking solution. They were then re-fixed, treated with 4 M HCl for 30 min at room temperature and incubated with FITC-conjugated anti-BrdU (BD Biosciences), diluted 1:300, for 1 hour at room temperature, followed by observation with confocal microscopy.
  • Immunohistochemical staining and tissue microarray Immunohistochemical analysis was performed using a specific mouse-MCM7 antibody as described previously (Sato N, et al, Clin Cancer Res 2010;16:226-39).
  • ENVISION+ kit/horseradish peroxidase (Dako, Glostrup, Denmark) was applied, whereas VECTASTAIN (registered trademark) ABC KIT (VECTOR LABORATORIES, Burlingame, CA) was used for bladder tissue microarray and normal tissue slides.
  • Tumor tissue microarrays were constructed with 331 primary NSCLCs which had been obtained by a single institutional group (please see above) with an identical protocol to collect, fix, and preserve the tissues after resection (Callagy G, et al, Diagn Mol Pathol 2003;12:27-34, Callagy G, et al, J Pathol 2005;205:388-96, Chin SF, et al, Mol Pathol 2003;56:275-9.).
  • a tissue area for sampling was selected based on visual alignment with the corresponding H&E-stained section on a slide.
  • tissue cores Three, four or five tissue cores (diameter, 0.6 mm; depth, 3-4 mm) taken from a donor tumor block were placed into a recipient paraffin block with a tissue microarrayer (Beecher Instruments, Sun Prairie, WI). A core of normal tissue was punched from each case, and 5-micrometer sections of the resulting microarray block were used for immunohistochemical analysis. Three independent investigators semiquantitatively assessed MCM7 positivity without prior knowledge of clinicopathologic data.
  • the intensity of MCM7 staining was semiquantitatively evaluated using the following criteria: negative (no appreciable staining in tumor cells) and positive (brown staining appreciable in the nucleus of tumor cells). Cases were accepted as positive only if all reviewers independently defined them as such.
  • EXAMPLE2 MCM7 expression is significantly high in lung cancer tissues and correlated with poor prognosis in NSCLC It was previously reported that PRMT6, a type I arginine methytransferase, is involved in human carcinogenesis (Yoshimatsu M, et al, Int J Cancer 2010) and identified that MCM7 is a binding partner based on IP-MS analysis (data not shown). Intriguingly, quantitative real-time PCR showed that expression levels of MCM7 in 9 lung cancer tissues (6 NSCLC cases and 3 SCLC cases) were significantly higher than those in 11 normal tissues containing lung, brain, colon, esophagus, eye, liver, rectum, stomach, bladder and kidney (Fig. 1A).
  • MCM7 can also be involved in human carcinogenesis.
  • immunohistochemical analysis was conducted on tissue microarray containing tissue sections from 331 NSCLC patients, who had under gone surgical resection. Immunohistochemistry using an MCM7-specific antibody showed nuclear localization in cancer tissues, but nothing was detected in normal lung tissues (Fig. 1B). Importantly, specific MCM7 signals were not detected in normal brain, heart, lung, liver, pancreas, stomach, testis, kidney and bladder tissues (Fig. 1C), indicating that MCM7 may be specifically overexpressed in cancer tissues. Of 331 cases, MCM7 stained positively in 196 cases (61.1%) and negatively in 135 cases (38.9%; Table 3).
  • EXAMPLE3 MCM7 is overexpressed in bladder and various types of cancers In addition to lung tissues, expression levels of MCM7 in bladder tissues were examined. Quantitative real-time PCR analysis using 23 normal bladder tissues, 124 bladder transitional cell carcinomas (TCCs) and 12 upper tract TCCs showed elevated mRNA levels of MCM7 in bladder and especially upper tract TCCs compared with normal bladder tissues (Fig. 2A). Although there were significant differences between normal bladder and bladder cancers of any pT stages, no significant differences were observed between each pT stages as well as tumor grades (Fig. 2B; data not shown).
  • MCM7 is required for cancer cell proliferation
  • siRNA oligonucleotide duplexes were prepared to specifically suppress the expression of MCM7 (siMCM7#1, #2), and each of them were transfected into cancer cells.
  • the expression profile of MCM7 in normal and cancer cell lines was examined. Quantitative real-time PCR analysis revealed that MCM7 expression levels in cancer cells were significantly higher than those in normal human cells (Fig. 4). Knockdown of MCM7 in A549 cells and SBC5 cells was confirmed by immunoblotting and immunocytochemical analysis as shown in Fig. 3A and 3B.
  • Cancer-related death is on the rise in most countries, and it is a serious public health problem.
  • lung cancer is the leading cause of death from cancer in the United States and Japan, and the median survival of advanced NSCLC patients treated with standard chemotherapy still remains as short as about 8 months (Sawabata N, et al, Nihon Kokyuki Gakkai Zasshi 2010;48:333-44, Jemal A, et al, CA Cancer J Clin 2008;58:71-96, Schiller JH, et al, N Engl J Med 2002;346:92-8.).
  • MCM2-MCM7 Six of the MCM proteins, MCM2-MCM7, form complexes that participate in initiation and elongation steps of DNA replication (Lei M, et al, J Cell Sci 2001;114:1447-54.). They share a conserved 200-amino acid nucleotide-binding region and form different subcomplexes (dimers, trimers and a hexamer) (Koonin EV, et al, Nucleic Acids Res 1993;21:2541-7.). MCM4-MCM6-MCM7 trimers and hexamers (MCM2-MCM7) have ATPase and DNA helicase activities in vitro ( Lei M, et al, J Cell Sci 2001;114:1447-54.).
  • MCM proteins are associated with chromatin in late telophase and at the beginning of the G 1 phase of the cell cycle (Dimitrova DS, et al, J Cell Sci 2002;115:51-9.).
  • S phase MCM proteins are released from origins of replication after initiation of DNA replication and subsequently move with replication folks where they are thought to function as a DNA helicase.
  • Mechanisms that assure the replication of DNA only once per cycle involve the release MCM proteins from chromatin after firing of the origins of replication and prevent the reloading of MCM proteins on chromatin until telophase.
  • MCM proteins are one of the essential regulators in DNA replication, and indeed, it has already been reported that dysregulation of some MCM proteins are apparent in human disease, including cancer (Fujioka S, et al, Lung Cancer 2009;65:223-9, Ramnath N, et al, J Clin 2009;65:223-9, Ramnath N, et al, J Clin Oncol 2001;19:4259-66, Ishimi Y, et al, Eur J Biochem 2003;270:1089-101, Honeycutt KA, et al, Oncogene 2006;25:4027-32, Shohet JM, et al, Cancer Res 2002;62:1123-8, Ren B, et al, Oncogene 2006;25:1090-8.)In this study, it was demonstrated that in various cancer tissues, expression levels of MCM7 were significantly high at both RNA and protein levels whereas MCM7 expression in various normal tissues was hardly detected.
  • MCM7 could therefore be a good indicator enabling us to predict prognosis of NSCLC patients and to conduct a more intensive follow-up according to MCM7 expression status of resected specimens.
  • MCM7 was demonstrated a critical role for MCM7 in the growth regulation of cancer cells.
  • Anti-cancer drugs targeting DNA helicases are now in development (Sharma S, et al, Curr Med Chem Anticancer Agents 2005;5:183-99.). Further validation of present results may affirm the importance of this protein as a promising target for anti-cancer therapy and as a prognostic marker of various cancers.
  • the present invention provides a molecular diagnostic marker for diagnosing or detecting cancers as well as monitoring, determining and/or assessing the prognosis of cancer, in particular, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
  • the data provided herein add to a comprehensive understanding of cancers, facilitate development of novel diagnostic strategies, and provide clues for identification of a molecular target for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of tumorigenesis, and provides indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of cancers.
  • cell growth is suppressed by double-stranded molecules that specifically target the MCM7 gene.
  • these novel double-stranded molecules are useful as anti-cancer pharmaceuticals.
  • the expression of the MCM7 gene is markedly elevated in cancer, specifically lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor, as compared to normal organs. Accordingly, this gene can be conveniently used as a diagnostic marker for cancer, in particular, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor , and the proteins encoded thereby find utility in diagnostic assays for cancer.
  • the methods described herein are also useful in diagnosis of cancer, including lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor Moreover, the present invention provides new therapeutic approaches for treating cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
  • the MCM7 gene is a useful target for the development of anti-cancer pharmaceuticals. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

Abstract

The present invention relates to the discovery that the MCM7 gene can discriminate cancer cells from normal cells. Accordingly, the present invention provides methods for detecting or diagnosing cancer in a subject or a predisposition for developing cancer or assessing or determining the prognosis for a subject so diagnosed using the expression level of the MCM7 genes as an index of disease. The present invention also provides methods of screening for therapeutic agents useful in the treatment of cancer and methods for treating cancer, particularly double-stranded molecules targeting the MCM7 suggested to be useful in the treatment of cancer.

Description

MCM7 AS A TARGET GENE FOR CANCER THERAPY AND DIAGNOSIS
The present invention relates to methods of detecting and diagnosing cancer or a predisposition for developing cancer in a subject, methods of assessing or determining the prognosis of a subject so diagnosed, as well as methods of treating and preventing cancer, particularly cancers associated with the overexpression of MCM7 such as lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor. The present invention also relates to methods of screening for a candidate substance for treating and preventing a MCM7-associated cancer. Moreover, the present invention relates to double-stranded molecules that reduce MCM7 gene expression and uses thereof.
Priority
The present application claims the benefit of U.S. Provisional Applications No. 61/427,563, filed on December 28, 2010, the contents of which are hereby incorporated herein by reference in their entirety for all purposes.
Lung cancer is the leading cause of cancer-related mortality in many countries, accounting for 1.3 million deaths worldwide annually, as of 2004 (World Health Organization, 2006). In Japan, more than 60,000 patients with lung cancer died in 2005. Despite improvements in surgical techniques and disease prognosis and the development of anticancer therapeutics, the survival of patients 5 years after diagnosis with NSCLC, the most common form of lung cancer, remains low, at 46.8%, according to a new report published by the Japanese Joint Committee for Lung Cancer Registration (NPL1). Therefore, it is vitally important for scientists to develop an understanding of the molecular mechanism of lung carcinogenesis so as to develop novel therapeutic methods for lung cancer that maximize efficiency while minimizing negative side-effects.
The concept of specific molecular targeting has been applied to the development of innovative cancer treatment strategies. Two main approaches are presently available in clinical practice: therapeutic monoclonal antibodies and small-molecule agents (NPL2). To date, a number of targeted therapies such as bevacizumab, cetuximab, erlotinib, gefitinib, sorafenib, and sunitinib have been investigated in phase II and phase III trials for the treatment of advanced non-small-cell lung cancer (NSCLC; NPLs 3-6). The addition of therapeutic antibodies against proangiogenic protein vascular endothelial growth factor (bevacizumab) or epidermal growth factor receptor (cetuximab) to conventional chemotherapy has imparted significant survival benefits to patients with NSCLC (NPLs 2-3). Two small-molecule epidermal growth factor receptor tyrosine kinase inhibitors, erlotinib and gefitinib, have been shown to be effective for a subset of advanced NSCLC patients (NPLs 4-5). In addition, Phase II studies done with two oral multi-targeted receptor tyrosine kinase inhibitors, sorafenib (inhibitor for c-RAF, b-RAF, vascular endothelial growth factor receptors 2 and 3, platelet-derived growth factor receptor h, and KIT) and sunitinib (inhibitor for platelet-derived growth factor receptor, KIT, FLT3, and vascular endothelial growth factor receptor) suggested their efficacy in the treatment of advanced NSCLCs (NPL 6).
However, issues of toxicity limit these treatment regimens to selected patients. Furthermore, even if all kinds of available treatments are applied, the proportion of patients exhibiting a positive response is still very limited (NPL 3-6).
DNA replication in eukaryotic cells is a highly regulated process that ensures the accurate duplication of genetic information while preserving genome stability. A large number of molecular players, including mini-chromosome maintenance (MCM) proteins, are involved in DNA replication (NPL 7-9). MCM proteins are essential replication initiation and elongation factors originally found in Saccharomyces cerevisiae that exist in a functional complex composed of MCM2-7. They are evolutionally conserved in all eukaryotes (NPL 10). The MCM protein complex belongs to the AAA+ family (ATPases associated with various cellular activities), and operates to ensure that DNA undergoes a single round of replication per cell cycle through use of a licensing mechanism (NPL 10-14). The MCM4, 6 and 7 subcomplexes possess DNA helicase activity that promotes unwinding of double strand DNA at the replication forks (NPL 15-17). Although it is known that aberrant DNA replication leads to pathological disorders including cancer, exactly how dysregulated MCM proteins contribute to carcinogenesis, and aggressive cancer with poor prognosis is unclear.
[NPL 1] Sawabata N, et al. Nihon Kokyuki Gakkai Zasshi 2010;48: 333-44.
[NPL 2] Thatcher N. Lung Cancer 2007;57 Suppl 2:S18-23
[NPL 3] Sandler A, et al. N Engl J Med 2006;355:2542-50
[NPL 4] Shepherd FA, et al. N Engl J Med 2005;353:123-32
[NPL 5] Thatcher N, et al. Lancet 2005;366:1527-37
[NPL 6] Cesare G, et al. Oncologist 2007;12:191-200
[NPL 7] Branzei D, et al. Nat Rev Mol Cell Biol 2010;11: 208-19.
[NPL 8] Blow JJ, et al.Nat Rev Cancer 2008;8: 799-806.
[NPL 9] Ilves I, et al. Mol Cell 2010;37: 247-58.
[NPL 10] Tye BK. et al. Annu Rev Biochem 1999;68: 649-86.
[NPL 11] Chong JP,et al. Nature 1995;375: 418-21.
[NPL 12] Blow JJ, et al. Nature 1988;332: 546-8.
[NPL 13] Tada S, et al. Biol Chem 1998;379: 941-9.
[NPL 14] Costa A, et al. Biochem Soc Trans 2008;36: 136-40.
[NPL 15] Ishimi Y. J Biol Chem 1997;272: 24508-13.
[NPL 16] You Z,et al.. Mol Cell Biol 1999;19: 8003-15.
[NPL 17] Kelman Z, et al. Proc Natl Acad Sci U S A 1999;96: 14783-8.
The present invention relates to the MCM proteins and the roles they play in carcinogenesis,. Central to the present invention is the discovery of a specific expression pattern of the MCM7 gene in cancerous cells. The present invention further relates to the discovery that an inhibitor of the MCM 4, 6 and 7 complex which has DNA helicase activity effectively suppress the growth of cancer cells.
Through the present invention, the MCM7 gene was revealed to be frequently up-regulated in human tumors, in particular, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor Additionally, the suppression of the MCM7 gene by small interfering RNA (siRNA) was shown to result in growth inhibition and/or cell death of cancer cells, suggesting that the gene may serve as a novel therapeutic target for human tumors, in particular, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, especially lung and bladder cancers.
The results herein suggest that the MCM7 gene, as along with its transcription and translation products, finds diagnostic utility as a marker for lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor and as an oncogene target, the expression and/or activity of which may be altered to treat or alleviate a symptom of cancer. Similarly, by detecting changes in the expression of the MCM7 gene and/or the biological activity of the MCM7 protein that arise from exposure to a test substance, various agents for treating or preventing cancer can be identified.
Accordingly, it is an objective of the present invention to provide a method for diagnosing or determining a predisposition for cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor, in a subject by determining the expression level of the MCM7 gene in a subject-derived biological sample, such as tissue sample. An increase in the level of expression of the test gene as compared to a normal control level indicates that the subject suffers from or is at risk of developing cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
The present invention further relates to the discovery that a high expression level of MCM7 correlates to poor survival rate in a cancer patient, particularly in a lung cancer patient such as an NSCLC patient. Accordingly, it is an objective of the present invention to provide a method for monitoring, assessing or determining the prognosis of a patient diagnosed with cancer, for example, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor cancer, particularly lung cancer such as NSCLC. Such a method includes the steps of detecting the expression level of MCM7 gene, comparing it to a pre-determined reference expression level and determining the prognosis of the patient from the difference between them.
It is another objective of the present invention to provide a kit for diagnosing or detecting cancer or a predisposition therefor, or assessing or determining prognosis of a subject diagnosed with cancer that includes at least one reagent for detecting a transcription or translation product of the MCM7 gene.
It is yet another objective of the present invention to provide a reagent for the diagnosis or detection of cancer or a predisposition therefor, or the assessment or determination of the prognosis of a subject diagnosed with cancer, such a reagent including a nucleic acid that binds to a transcriptional product of the MCM7 gene, or an antibody that binds to a translational product of the MCM7 gene.
It is yet another objective of the present invention to provide utility for a nucleic acid that binds to a transcriptional product of the MCM7 gene, or an antibody that binds to a translational product of the MCM7 gene in the context of the manufacture of a reagent for diagnosis or detection of cancer or a predisposition therefor, or the assessment or determination of the prognosis of a subject diagnosed with cancer.
It is yet another objective of the present invention to provide methods for identifying substances that bind the MCM7 protein. Such methods involve the steps of: contacting the MCM7 protein with a test substance and detecting the binding between the MCM7 protein and the test substance. Test substances that bind the MCM7 protein may prove effective in the treatment and/or the prophylaxis of cancer, e.g., by providing a clinical benefit such as improving prognosis, decreasing mortality levels, reducing tumor marker levels, and/or alleviating detectable symptoms of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
It is yet another objective of the present invention to provide methods for identifying substances that inhibit the biological activity of the MCM7 protein. Such methods involve the steps of: contacting the MCM7 protein with a test substance and detecting the biological activity of the MCM7 protein. The biological activity of the MCM7 protein to be detected is preferably cell proliferative activity (cell proliferation enhancing activity). In the context of the present invention, a decrease in the biological activity of the MCM7 protein as compared to a control level in the absence of the test substance indicates that the test substance may prove effective in the treatment and/or the prophylaxis of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
It is yet another objective of the present invention to provide methods for identifying substances that inhibit the expression of the MCM7 gene or a reporter gene that controlled by the transcription initiation region of the MCM7 gene. Such methods involve the steps of: contacting a test cell expressing the MCM7 gene or a reporter gene that controlled by the transcription initiation region of the MCM7 gene with a test substance and determining the expression level of the MCM7 gene or the reporter gene. The test cell may be an epithelial cell, such as cancerous epithelial cell. In the context of the present invention, a decrease in the expression level of the gene as compared to a control level in the absence of the test substance indicates that the test substance may prove effective in the treatment and/or the prophylaxis of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
It is yet another objective of the present invention to provide methods for identifying substances that inhibit the biological activity of the MCM protein complex. Such methods involve the steps of: contacting the MCM protein complex with a test substance and detecting the biological activity of the MCM protein complex. As an MCM protein complex, a trimer composed of MCM4, MCM6 and MCM7 proteins and a hexamer composed of MCM 2-7 proteins are preferably used. The biological activity of the MCM protein complex to be detected is preferably DNA helicase activity. In the context of the present invention, a decrease in the biological activity of the MCM protein complex as compared to a control level in the absence of the test substance indicates that the test substance may prove effective in the treatment and/or the prophylaxis of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
In the course of the present invention, inhibitory effects of siRNAs for the MCM7 gene were confirmed. In particular, the data presented in the Examples section demonstrates the inhibition of cell proliferation of cancer cells by siRNAs. Thus, the data herein support the utility of the MCM7 gene as a preferred therapeutic target for cancer, particularly lung and bladder cancer and the use of double-stranded molecules (e.g., siRNA) against MCM7 genes in the context of cancer therapy.
Accordingly, it is yet a further objective of the present invention is to provide double-stranded molecules that inhibit the expression of the MCM7 gene as well as siRNAs against the MCM7 gene, and vectors encoding such double-stranded molecules. The double-stranded molecules of the present invention are composed of a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13 and 15 as a target sequence, and the antisense strand comprises a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule. Such double-stranded molecules are shown herein to inhibit expression of the MCM7 gene when introduced into a cell expressing an MCM7 gene and thus may prove effective in the treatment and/or the prophylaxis of cancer.
To that end, the present methods contemplate administration of an siRNA or double-stranded molecule composition to a subject in need thereof with the aim of reducing the expression of the MCM7 gene and thereby prove effective in the treatment and/or the prophylaxis of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, more particularly lung cancer and bladder cancer.
It is yet another objective of the present invention is to provide a pharmaceutical composition formulated for the treatment and/or the prophylaxis of cancer, or the inhibition of cancer cell growth. The pharmaceutical composition of the present invention preferably comprises an antisense nucleotide or double-stranded molecule (e.g., siRNA) against the MCM7 gene, which inhibit the expression of the MCM7 gene.
One advantage of the methods described herein is that the disease may be identified prior to detection of overt clinical symptoms of lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor. Other features and advantages of the invention will be apparent from the following detailed description and the appended claims.
It will be understood by those skilled in the art that one or more aspects of this invention can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all its respects, to every aspect of this invention. As such, the preceding objects can be viewed in the alternative with respect to any one aspect of this invention.
It will also be understood that both the foregoing summary of the present invention and the following detailed description are of exemplified embodiments, and not restrictive of the present invention or other alternate embodiments of the present invention. Other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. In particular, while the invention is described herein with reference to a number of specific embodiments, it will be appreciated that the description is illustrative of the invention and is not constructed as limiting of the invention. Various modifications and applications may occur to those who are skilled in the art, without departing from the spirit and the scope of the invention, as described by the appended claims. Likewise, other objects, features, benefits and advantages of the present invention will be apparent from this summary and certain embodiments described below, and will be readily apparent to those skilled in the art. Such objects, features, benefits and advantages will be apparent from the above in conjunction with the accompanying examples, data, figures and all reasonable inferences to be drawn therefrom, alone or with consideration of the references incorporated herein.
Various aspects and applications of the present invention will become apparent to the skilled artisan upon consideration of the brief description of the figures and the detailed description of the present invention and its preferred embodiments that follows:
Fig. 1 depicts the dysregulation of MCM7 in lung cancer tissues, and the relevance of MCM7 expression to a negative outcome for NSCLC patients after surgical operation.: Part A is a box-whisker plot depicting the overexpression of MCM7 gene in clinical lung cancer tissues [NSCLC (n = 6) and SCLC (n = 3)] as compared with several normal tissues (lung, brain, colon, esophagus, eye, heart, liver, rectum, stomach, bladder and kidney) analyzed by quantitative real-time PCR. Part B includes representative cases for positive MCM7 expression in lung ADC, SCC tissues and normal lung tissues. Original magnification, x100 and x200. Part C depicts the results of immunohistochemical analysis of MCM7 in various normal tissues. No significant staining was observed.
Part D depicts Kaplan-Meier estimates of overall survival time of patients with NSCLC (P = 0.0055, log-rank test).
Fig. 2 depicts the elevated MCM7 expression in bladder cancer : Part A depicts the quantitative real-time PCR results for mRNA expression levels of MCM7 gene in 124 bladder TCCs, 12 upper tract TCCs and 23 normal bladder. Statistical analysis was done using Kruskal-Wallis test:*, P < 0.0001; Mann Whitney U-test: **, P < 0.0001.Part B depicts the correlation between MCM7 gene expression and pathological tumor stages. Statistical analysis was done using Kruskal-Wallis test:*, P < 0.0001; Mann Whitney U-test: **, P < 0.0001.
Part C depicts the results of immunohistochemical analysis of MCM7 in bladder cancer tissues. Original magnification, x200. Part D depicts the results of immunohistochemical analysis of MCM7 in liver cancer tissues. Original magnification, x200.
Fig. 3 depicts the involvement of MCM7 in the proliferation of lung cancer cells.: Part A depicts the results of Western blot analysis of MCM7 in A549 and SBC5 cells after treatment with two MCM7 siRNAs (siMCM7#1 and siMCM7#2) and two control siRNAs (siEGFP and siNC). Part B depicts the results of immunocytochemical analysis of MCM7 in A549 and SBC5 cells after treatment with siRNAs. The nucleus was stained with DAPI, and the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating DNA was used to label proliferating cells. Part C depicts the effects of MCM7 knockdown on the viability of lung cancer cell lines (A549 and SBC5). Statistical analysis was done using Student's t-test. *, P < 0.05.
Fig. 4 depicts mRNA expression levels of MCM7 gene in 15 lung-cancer cell lines, 2 bladder-cancer cell lines, 2 liver-cancer cell lines and 1 normal human cell line examined by quantitative real-time PCR.
Fig. 5 depicts involvement of MCM7 in the growth of bladder cancer. Part A depicts the results of Western blot analysis of MCM7 in SW780 cells after treatment with two MCM7 siRNAs and control siRNAs (siEGFP and siNC). Part B depicts the results of immunocytochemical analysis of MCM7 in SW780 cells after treatment with siRNAs. The nucleus was stained with DAPI, and the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating DNA was used to label proliferating cells. Part C depicts the effects of MCM7 knockdown on the viability of a bladder cancer cell line (SW780). Statistical analysis was done using Student's t-test. *, P < 0.05.
Fig. 6 depicts effects of a DNA helicase inhibitor on the growth of cancer cells. Heliquinomycin reduced growth rate in two lung cancer cell lines (A549 and SBC5) and one bladder cancer cell line (SW780) in a dose-dependent manner. Cell viability was measured by Cell Counting Kit-8 at 72 hours after treatment with indicated concentration of heliquinomycin.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. However, before the present materials and methods are described, it is to be understood that the present invention is not limited to the particular sizes, shapes, dimensions, materials, methodologies, protocols, etc. described herein, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
The disclosure of each publication, patent or patent application mentioned in this specification is specifically incorporated by reference herein in its entirety. However, nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
Further to the summary above, it is an object of the present invention to provide:
[1] A method for detecting or diagnosing cancer or a predisposition for developing cancer in a subject, comprising the step of determining an expression level of an MCM7 gene in a subject-derived biological sample, wherein an increase in said expression level as compared to a normal control level of said gene indicates that said subject suffers from or is at a risk of developing cancer, wherein said expression level is determined by a method selected from a group consisting of:
(a) detecting mRNA of an MCM7 gene;
(b) detecting a protein encoded by an MCM7 gene; and
(c) detecting a biological activity of a protein encoded by an MCM7 gene,
[2] The method of [1], wherein said MCM7 gene expression level is at least 10% greater than the normal control level,
[3] The method of [1] or [2], wherein the subject-derived biological sample is a biopsy, saliva, sputum, blood, serum, plasma, pleural effusion or urine sample,
[4] The method of any one of [1] to [3], wherein the biological activity of the protein encoded by the MCM7 gene is cell proliferative activity,
[5] A method for assessing prognosis of a subject with cancer, wherein the method comprises steps of:
(a) detecting an expression level of MCM7 gene in a subject-derived biological sample;
(b) comparing the detected expression level to a control level; and
(c) determining prognosis of the subject based on the comparison of (b),
[6] The method of [5], wherein the control level is a good prognosis control level and an increase of the expression level compared to the control level indicates poor prognosis,
[7] The method of [6], wherein the MCM7 gene expression level is at least 10% greater than said control level,
[8] A method of screening for a candidate substance for either or both of treating and preventing cancer, wherein said method comprises steps of:
(a) contacting a test substance with an MCM7 polypeptide or a fragment thereof;
(b) detecting binding between the polypeptide or fragment and the test substance; and
(c) selecting a test substance that binds to the polypeptide or fragment as a candidate substance for either or both of treating and preventing cancer,
[9] A method of screening for a candidate substance for either or both of treating and preventing cancer, wherein said method comprises steps of:
(a) contacting a test substance with an MCM7 polypeptide or a fragment thereof;
(b) detecting a biological activity of the polypeptide or fragment;
(c) comparing the biological activity of the polypeptide or fragment with the biological activity detected in the absence of the test substance; and
(d) selecting a test substance that suppresses the biological activity of the polypeptide as a candidate substance for either or both of treating and preventing cancer,
[10] The method of [9], wherein the biological activity of said MCM7 polypeptide or fragment is cell proliferative activity,
[11] A method of screening for a candidate substance for either or both of treating and preventing cancer, wherein said method comprises steps of:
(a) contacting a test substance with a cell expressing an MCM7 gene;
(b) detecting an expression level of the MCM7 gene;
(c) comparing the expression level with the expression level detected in the absence of the test substance; and
(d) selecting a test substance that reduces the expression level as a candidate substance for either or both of treating and preventing cancer,
[12] A method of screening for a candidate substance for either or both of treating and preventing cancer, wherein said method comprises steps of:
(a) contacting a test substance with a cell introduced with a vector that comprises a transcriptional regulatory region of an MCM7 gene and a reporter gene expressed under control of the transcriptional regulatory region;
(b) measuring an expression level or activity of said reporter gene;
(c) comparing the expression level or activity with the expression level or activity detected in the absence of the test substance; and
(d) selecting a test substance that reduces the expression level or activity as a candidate substance for either or both of treating and preventing cancer,
[13] A method of screening for a candidate substance for either or both of treating and preventing cancer, wherein said method comprises steps of:
(a) contacting a test substance with an MCM protein complex ;
(b) detecting a biological activity of the complex;
(c) comparing the biological activity of the complex with the biological activity detected in the absence of the test substance; and
(d) selecting a test substance that suppresses the biological activity of the complex as candidate substance for either or both of treating and preventing cancer,
[14] The method of [13], wherein the biological activity of said MCM protein complex is DNA helicase activity,
[15] An isolated double-stranded molecule that, when introduced into a cell expressing an MCM7 gene, inhibits expression of the gene, wherein said double-stranded molecule comprises a sense strand and an antisense strand, further wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 13 and 15, and the antisense strand comprises a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule,
[16] The double-stranded molecule of [15], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length,
[17] The double-stranded molecule of [15] or [16], wherein said double-stranded molecule is a single polynucleotide construct comprising the sense strand and the antisense strand linked via a single-strand,
[18] The double-stranded molecule of [17], wherein said double-stranded molecule has a general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A], wherein [A] is a sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 13 and 15, [B] is a single-strand and consists of 3 to 23 nucleotides, and [A'] is an antisense strand comprising a nucleotide sequence complementary to the target sequence selected from the group consisting of SEQ ID NO: 13 and 15,
[19] A vector encoding the double-stranded molecule of any one of [15] to [18],
[20] Vectors comprising each of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises a nucleotide sequence corresponding to SEQ ID NO: 13 or 15, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing MCM7 gene, inhibit the cell proliferation,
[21] A method of either or both of the treatment and prevention of cancer in a subject, wherein said method comprises the step of administering to said subject a pharmaceutically effective amount of a double-stranded molecule against an MCM7 gene or a vector encoding said double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell expressing MCM7 gene, inhibits the expression of the MCM7 gene,
[22] The method of [21], wherein the double-stranded molecule is that of any one of [15] to [18],
[23] The method of [21], wherein the vector is that of [19] or [20],
[24] The method of any one of [1] to [14] or [21] or [22], wherein the cancer is selected from the group consisting of lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor,
[25] A composition formulated for either or both of the treatment and prevention of cancer, which comprises a pharmaceutically effective amount of a double-stranded molecule against an MCM7 gene or a vector encoding said double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell expressing MCM7 gene, inhibits the expression of the MCM7 gene, and a pharmaceutically acceptable carrier,
[26] The composition of c[25], wherein the double-stranded molecule is that of any one of [15] to [18],
[27] The composition of [25], wherein the vector is that of [19] or [20],
[28] The composition of any one of [25] to [27], wherein the cancer is selected from the group consisting of lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor,
[29] A kit for diagnosing or detecting cancer or a predisposition therefor, or assessing or determining prognosis of a subject diagnosed with cancer, wherein said kit comprises a reagent for detecting a transcription or translation product of an MCM7 gene,
[30] The kit of [29], wherein the reagent comprises a nucleic acid that binds to a transcription product of MCM7 gene or an antibody that binds to a translation product of an MCM7 gene,
[31] A reagent for diagnosing or detecting cancer or a predisposition therefor, or assessing or determining prognosis of a subject diagnosed with cancer, said reagent comprising a nucleic acid that binds to a transcription product of MCM7 gene or an antibody that binds to a translation product of an MCM7 gene, and
[32] The kit of [29] or [30], or the reagent of [31], wherein the cancer is selected from the group consisting of lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
Definitions
The words "a", "an", and "the" as used herein mean "at least one" unless otherwise specifically indicated.
The terms "isolated" and "purified" used in relation with a substance (e.g., polypeptide, antibody, polynucleotide, etc.) indicates that the substance is substantially free from at least one substance that may else be included in the natural source. Thus, an isolated or purified antibody refers to an antibody that is substantially free of cellular material such as carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When the polypeptide is recombinantly produced, it is also preferably substantially free of culture medium, which includes preparations of polypeptide with culture medium less than about 20%, 10%, or 5% of the volume of the protein preparation. When the polypeptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, which includes preparations of polypeptide with chemical precursors or other chemicals involved in the synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation. That a particular protein preparation contains an isolated or purified polypeptide can be shown, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining or the like of the gel. In a preferred embodiment, antibodies and polypeptides of the present invention are isolated or purified.
As used herein, the phrase "biological sample" refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). "Biological sample" further refers to a homogenate, lysate, extract, cell culture or tissue culture prepared from a whole organism or a subset of its cells, tissues or component parts, or a fraction or portion thereof. Lastly, "biological sample" refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or polynucleotides.
The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly functions to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine). The phrase "amino acid analog" refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium). The phrase "amino acid mimetic" refers to chemical compounds that have different structures but similar functions to general amino acids.
Amino acids may be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
The terms "gene", "polynucleotide", "oligonucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably unless otherwise specifically indicated and are similarly to the amino acids referred to by their commonly accepted single-letter codes. Similar to the amino acids, they encompass both naturally-occurring and non-naturally occurring nucleic acid polymers. The polynucleotide, oligonucleotide, nucleic acid, or nucleic acid molecule may be composed of DNA, RNA or a combination thereof.
As used herein, an "isolated nucleic acid" is a nucleic acid removed from its original environment (e.g., the natural environment if naturally occurring) and thus, synthetically altered from its natural state. Examples of isolated nucleic acid include DNA, RNA, and derivatives thereof, for example, a cDNA molecule, substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
In the context of the present invention, the phrase "MCM7 gene" encompasses the human MCM7 gene as well as equivalents from other animals including, but not limited to, non-human primate, mouse, rat, dog, cat, horse, and cow, and further includes allelic mutants and genes found in other animals as corresponding to the MCM7 gene. The typical nucleotide sequences of the MCM7 gene are shown in SEQ ID NOs 17 and 19. This sequence data is also available via GenBank accession numbers: NM_005916 and NM_182776, respectively. In a similar fashion, typical amino acid sequences encoded the human MCM7 gene are shown in SEQ ID NO: 18 and SEQ ID NO: 20 and available via GenBank accession numbers: MCM7: NP_005907 and NP_877577. However, the present invention should not be construed as limited to those disclosed sequences.
The MCM7 genes and proteins can be obtained from nature as naturally occurring proteins via conventional cloning methods or through chemical synthesis based on the selected nucleotide or amino acid sequence. Methods for cloning genes and proteins using cDNA libraries and such are well known in the art.
Unless otherwise defined, the term "cancer" refers to cancer over-expressing the
MCM7 gene, in particular, lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia (AML), osteosarcoma and soft tissue tumor. "lung cancer" includes non-small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). "non-small cell lung cancer (NSCLC)" includes adenocarcinoma (ADC), squamous-cell carcinoma (SCC) and large-cell carcinoma (LCC).
To the extent that the methods and compositions of the present invention find utility in the context of "prevention" and "prophylaxis", such terms are interchangeably used herein to refer to any activity that reduces the burden of mortality or morbidity from disease. Prevention and prophylaxis can occur "at primary, secondary and tertiary prevention levels". While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g. reducing the proliferation and metastasis of tumors.
To the extent that certain embodiments of the present invention encompass the treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence, such methods may include any of the following steps: the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis. Effective treatment and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer. A treatment may also deem "efficacious" if it leads to clinical benefit such as, reduction in expression of the gene, or a decrease in size, prevalence, or metastatic potential of the cancer in the subject. When the treatment is applied prophylactically, "efficacious" means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer. Efficaciousness is determined in association with any known method for diagnosing or treating the particular tumor type.
As used herein, the term "double-stranded molecule" refers to a nucleic acid molecule that inhibits expression of a target gene, including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)). As used herein, sense strand of a target sequence is a nucleotide sequence within mRNA or cDNA sequence of a gene, which will result in suppress of translation of the whole mRNA if a double-stranded nucleic acid molecule of the invention was introduced within a cell expressing the gene. A nucleotide sequence within mRNA or cDNA sequence of a gene can be determined to be a target sequence when a double-stranded polynucleotide comprising a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene. The double stranded polynucleotide by which suppresses the gene expression may be composed of the target sequence and 3'overhang having 2 to 5 nucleotides in length (e.g., uu).
Herein, "double-stranded molecule" is also referred to as "double-stranded nucleic acid ", " double-stranded nucleic acid molecule", "double-stranded polynucleotide", "double-stranded polynucleotide molecule", "double-stranded oligonucleotide" and "double-stranded oligonucleotide molecule".
As used herein, the term "target sequence" refers to a nucleotide sequence within mRNA or cDNA sequence of a target gene, which will result in suppression of translation of the whole mRNA of the target gene if a double-stranded molecule targeting the sequence is introduced into a cell expressing the target gene. A nucleotide sequence within mRNA or cDNA sequence of a gene can be determined to be a target sequence when a double-stranded molecule including a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene. When a target sequence is shown by cDNA sequence, a sense strand sequence of a double-stranded cDNA, i.e., a sequence that mRNA sequence is converted into DNA sequence, is used for defining a target sequence. A double-stranded molecule is composed of a sense strand that has a sequence corresponding to a target sequence and an antisense strand that has a complementary sequence to the target sequence, and the antisense strand hybridizes with the sense strand at the complementary sequence to form a double-stranded molecule. Herein, the phrase "corresponding to" means converting a target sequence according to the kind of nucleic acid that constitutes a sense strand of a double-stranded molecule. For example, when a target sequence is shown in DNA sequence and a sense strand of a double-stranded molecule has an RNA region, base "t"s within the RNA region is replaced with base "u"s. On the other hand, when a target sequence is shown in an RNA sequence and a sense strand of a double-stranded molecule has a DNA region, base "u"s within the DNA region is replaced with "t"s.
For example, when a target sequence is shown in the RNA sequence of SEQ ID NO: 13 or 15 and the sense strand of the double-stranded molecule has the 3' side half region composed of DNA, "a sequence corresponding to a target sequence" is "5'-GGCUAAUGGAGATGTCAA -3'" (for SEQ ID NO: 13) or "5'-GAAAGAAGATGTGAATGA-3'" (for SEQ ID NO: 15).
Also, a complementary sequence to a target sequence for an antisense strand of a double-stranded molecule can be defined according to the kind of nucleic acid that constitutes the antisense strand.
For example, when a target sequence is shown in the RNA sequence of SEQ ID NO: 13 or 15 and the antisense strand of the double-stranded molecule has the 5' side half region composed of DNA, "a complementary sequence to a target sequence" is 3'- CCGAUUACCTCTACAGTT-5'" (for SEQ ID NO: 13) or "3'-CUUUCUUCTACACTTACT -5'" (for SEQ ID NO: 15).
On the other hand, when a double-stranded molecule is composed of RNA, the sequence corresponding to a target sequence shown in SEQ ID NO: 13 or 15 is the RNA sequence of SEQ ID NO: 13 or 15, and the complementary sequence corresponding to a target sequence shown in SEQ ID NO: 13 or 15 is the RNA sequence of "3'- CCGAUUACCUCUACAGUU-5'" (for SEQ ID NO:13) or "3'- CUUUCUUCUACACUUACU-5'" (for SEQ ID NO:15).
Several nucleotides can be added to 3' end of the sense strand and/or the antisense strand of the target sequence so as to enhance the inhibition activity of the double-stranded molecules. For example, a double-stranded molecule may have one or two 3' overhangs having 2 to 10, preferably 2 to 5 nucleotides in length (and/or a loop sequence that links a sense strand and an antisense strand to form hairpin structure, in addition to a sequence corresponding to a target sequence and complementary sequence thereto. The added nucleotides form a single strand at the 3'-end of the sense strand and/or antisense strand of the double-stranded molecule and are preferably composed of "t" and "u" nucleotides, though are not necessarily limited thereto.
As used herein, the term "siRNA" refers to a double-stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed. The siRNA includes a sense nucleic acid sequence (also referred to as "sense strand"), an antisense nucleic acid sequence (also referred to as "antisense strand") or both. The siRNA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, e.g., a hairpin. The siRNA may either be a dsRNA or shRNA.
As used herein, the term "dsRNA" refers to a construct of two RNA molecules including complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded RNA molecule. The nucleotide sequence of two strands may include not only the "sense" or "antisense" RNAs selected from a protein coding sequence of target gene sequence, but also RNA molecule having a nucleotide sequence selected from non-coding region of the target gene.
The term "shRNA", as used herein, refers to an siRNA having a stem-loop structure, including the first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
As used herein, the term "siD/R-NA" refers to a double-stranded polynucleotide molecule which is composed of both RNA and DNA, and includes hybrids and chimeras of RNA and DNA and prevents translation of a target mRNA. Herein, a hybrid indicates a molecule wherein a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize to each other to form the double-stranded molecule; whereas a chimera indicates that one or both of the strands composing the double stranded molecule may contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used. The siD/R-NA includes a sense nucleic acid sequence (also referred to as "sense strand"), an antisense nucleic acid sequence (also referred to as "antisense strand") or both. The siD/R-NA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences from the target gene, e.g., a hairpin. The siD/R-NA may either be a dsD/R-NA or shD/R-NA.
As used herein, the term "dsD/R-NA" refers to a construct of two molecules including complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded polynucleotide molecule. The nucleotide sequence of two strands may include not only the "sense" or "antisense" polynucleotides sequence selected from a protein coding sequence of target gene sequence, but also polynucleotide having a nucleotide sequence selected from non-coding region of the target gene. One or both of the two molecules constructing the dsD/R-NA are composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules is composed of RNA and the other is composed of DNA (hybrid double-strand).
The term "shD/R-NA", as used herein, refers to an siD/R-NA having a stem-loop structure, including the first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shD/R-NA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".
I. Polynucleotides and Polypeptides
The present invention is based in part on the discovery that the expression of the MCM7 gene is elevated in cancerous cells, particularly cancer cells obtained from patients of lung cancer and various other cancers.
The protein encoded by the MCM7 gene is one of the highly conserved mini-chromosome maintenance proteins (MCM) that are essential for the initiation of eukaryotic genome replication. The hexameric protein complex formed by the MCM proteins is a key component of the pre-replication complex and may be involved in the formation of replication forks and in the recruitment of other DNA replication related proteins. Both the MCM2-MCM7 complex and the complex composed of MCM4, MCM6 and MCM7 have helicase activity and ATPase activity.
As noted above, the typical nucleotide sequences of the MCM7 gene are shown in SEQ ID NOs 17 and 19 and available via GenBank accession numbers: NM_005916 and NM_182776, respectively. Similarly, the typical amino acid sequences encoded the human MCM7 gene are shown in SEQ ID NO: 18 and SEQ ID NO: 20 and available via GenBank accession numbers: MCM7: NP_005907 and NP_877577. However, the present invention is not limited to these disclosed sequences.
In the context of the present invention, the polypeptide encoded by the MCM7 gene is referred to as "MCM7", and sometimes as "MCM7 polypeptide" or "MCM7 protein". One of skill will recognize that MCM7 sequences need not be limited to the sequences mentioned herein and that variants (e.g., functional equivalents and allelic variants) can be used in the present invention as described below.
The present invention contemplates "functional equivalents" and deems such to included in the recitation of "MCM7 proteins". Herein, a "functional equivalent" of a protein is a polypeptide that has a biological activity equivalent to that of the original reference protein. Namely, any polypeptides that retain the biological ability of the MCM7 protein may be used as such functional equivalents of the MCM7 protein in the present invention. Relevant biological activities of the MCM7 protein include, for example, cancer cell proliferation activity (cancer cell proliferation enhancing activity) and activity of forming a complex with MCM4 and MCM6 proteins.
In the context of the present invention, functional equivalents include those polypeptides in which one or more amino acids are substituted, deleted, added, and/or inserted to the natural occurring amino acid sequence of the MCM7 protein. Alternatively, the polypeptide may be one that includes an amino acid sequence having at least about 80% homology (also referred to as sequence identity) to the sequence of the MCM7 protein (e.g., SEQ ID NO: 18 or 20), more preferably at least about 90% to 95% homology, even more preferably 96%, 97%, 98% or 99% homology.
A polypeptide of the present invention may have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has a functional equivalent to that of the human protein of the present invention, it is within the scope of the present invention.
Examples of functional equivalents of MCM7 polypeptide include those wherein one or more amino acids, e.g., 1-5 amino acids, e.g., up to 5% of amino acids, are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the MCM7 protein. In other embodiments, the polypeptide can be encoded by a polynucleotide that hybridizes under stringent conditions to the natural occurring nucleotide sequence of the gene. In some embodiments, the polypeptide is encoded by a polynucleotide that shares at least about 90%, 93%, 95%, 97%, 99% sequence identity to a reference sequence of MCM7, e.g., SEQ ID NO: 17 or 19, as determined using a known sequence comparison algorithm.
The phrase "stringent (hybridization) conditions" refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10 degrees C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times of background, preferably 10 times of background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 degrees C, or, 5x SSC, 1% SDS, incubating at 65 degrees C, with wash in 0.2x SSC, and 0.1% SDS at 50 degrees C.
In the context of the present invention, the optimal condition of hybridization for isolating a DNA encoding a polypeptide functionally equivalent to the human MCM7 protein can be routinely selected by a person skilled in the art. For example, hybridization may be performed by conducting pre-hybridization at 68 degrees C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 degrees C for 1 hour or longer. The following washing step can be conducted, for example, in a low stringent condition. An exemplary low stringent condition may include 42 degrees C, 2x SSC, 0.1% SDS, preferably 50 degrees C, 2x SSC, 0.1% SDS. High stringency conditions are often preferably used. An exemplary high stringency condition may include washing 3 times in 2x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37 degrees C for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50 degrees C for 20 min. However, several factors, such as temperature and salt concentration, can influence the stringency of hybridization and one skilled in the art can routinely adjust these and other factors to arrive at the desired stringency.
It is generally accepted that modification of one, two, or more amino acids in a protein will not influence the function of the protein; in some cases, it may even enhance the desired function of the original protein. In fact, mutated or modified proteins (i.e., peptides composed of an amino acid sequence in which one, two, or several amino acid residues have been modified through substitution, deletion, insertion and/or addition) have been known to retain the original biological activity (Mark et al., Proc Natl Acad Sci USA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10:6487-500 (1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13 (1982)).
Accordingly, one of skill in the art will recognize that individual additions, deletions, insertions, or substitutions to an amino acid sequence that alter a single amino acid or a small percentage of amino acids (i.e., less than 5%, more preferably less than 3%, even more preferably less than 1%) or those considered to be a "conservative modification" wherein the alteration of a protein results in a protein with similar functions, are acceptable in the context of the instant invention. Thus, the peptides of the present invention may have an amino acid sequence wherein one, two or even more amino acids are added, inserted, deleted, and/or substituted in an originally disclosed reference sequence, such as the human MCM7 sequence.
So long as the activity of the protein is maintained, the number of amino acid mutations or modifications is not particularly limited. However, it is generally preferred to alter 5% or less of the amino acid sequence, more preferably less than 3%, even more preferably less than 1%. Accordingly, in a preferred embodiment, the number of amino acids to be mutated in such a mutant is generally 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, more preferably 5 or 6 amino acids or less, and even more preferably 3 or 4 amino acids or less.
An amino acid residue to be mutated is preferably mutated into a different amino acid in which the properties of the amino acid side-chain are conserved (a process known as conservative amino acid substitution). Examples of properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W). Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Aspargine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).
Such conservatively modified polypeptides are included in the present MCM7 protein. However, the present invention is not restricted thereto and the MCM7 protein includes non-conservative modifications so long as the resulting modified peptide retains at least one biological activity of the original MCM7 protein. Furthermore, in the context of the present invention, the modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.
Mutations and/or modifications (i.e., additions, deletions, insertions, or substitutions to an amino acid sequence) may be introduced at the N- and C-terminals as well as intermediate sites. An example of a protein modified by addition of one or more amino acids residues is a fusion protein of the MCM7 protein. Fusion proteins can be made by techniques well known to a person skilled in the art, for example, by linking the DNA encoding the MCM7 gene with a DNA encoding another peptide or protein, so that the frames match, inserting the fusion DNA into an expression vector and expressing it in a host. The "other" component of the fusion protein is typically a small epitope composed of several to a dozen amino acids. There is no restriction as to the peptides or proteins fused to the MCM7 protein so long as the resulting fusion protein retains any one of the objective biological activities of the MCM7 proteins. Exemplary fusion proteins contemplated by the instant invention include fusions of the MCM7 protein and other small peptides or proteins such as FLAG (Hopp TP, et al., Biotechnology 6: 1204-10 (1988)), a polyhistidine (His-tag) such as 6xHis containing six His (histidine) residues or 10xHis containing 10 His residues, Influenza aggregate or agglutinin (HA), human c-myc fragment, Vesicular stomatitis virus glycoprotein (VSV-GP), p18HIV fragment, T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage), SV40T antigen fragment, lck tag, alpha-tubulin fragment, B-tag, Protein C fragment, and the like. Other examples of proteins that can be fused to a protein of the invention include GST (glutathione-S-transferase), Influenza agglutinin (HA), immunoglobulin constant region, beta-galactosidase, MBP (maltose-binding protein), and such. Other examples of modified proteins contemplated by the present invention include polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.
The present invention further contemplates and thus encompasses polynucleotides that encode such functional equivalents of the MCM7 protein and deems such to fall within the scope of "MCM genes". In addition to hybridization, a gene amplification method, for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a polynucleotide encoding a polypeptide functionally equivalent to the protein, using a primer synthesized based on the sequence above information. Polynucleotides and polypeptides that are functionally equivalent to the human gene and protein, respectively, normally have a high homology to the originating nucleotide or amino acid sequence of. "High homology" typically refers to a homology of 40% or higher, preferably 60% or higher, more preferably 80% or higher, even more preferably 90% to 95% or higher. The homology of a particular polynucleotide or polypeptide can be determined by following the algorithm in "Wilbur and Lipman, Proc Natl Acad Sci USA 80: 726-30 (1983)".
Percent sequence identity and sequence similarity can be readily determined using conventional techniques such as the BLAST and BLAST 2.0 algorithms, which are described (Altschul SF, et al., J Mol Biol. 1990 Oct 5; 215 (3):403-10; Nucleic Acids Res. 1997 Sep 1; 25(17):3389-402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the worldwide web at ncbi.nlm.nih.gov/). Such algorithms involve first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them.
The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff S & Henikoff JG. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10915-9).
The present invention also encompasses partial peptides of the MCM7 proteins and contemplates their use in screening methods described in greater detail below. A partial peptide having an amino acid sequence specific to the MCM7 protein is preferably composed of less than about 400 amino acids, usually less than about 200 and often less than about 100 amino acids, and at least about 7 amino acids, for example, about 8 amino acids or more, for example, about 9 amino acids or more.
II. Diagnosing cancer
II-1. Method for diagnosing cancer or a predisposition for developing cancer
As demonstrated herein, the expression of the MCM7 gene is significantly and specifically elevated in cancer tissues, more particularly, in tissue samples derived from lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
Thus, the MCM7 gene, as well as its transcription and translation products, find utility as a diagnostic marker for a cancer over-expressing the MCM7 gene such as lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor. Specifically, by measuring the expression of the MCM7 gene in as subject-derived biological sample such as a cell sample and a tissue sample, cancer can be diagnosed or detected.
Such diagnosis or detection may be performed by comparing the expression level of the MCM7 gene between a subject-derived sample and a normal sample. More particularly, the present invention provides a method for detecting or diagnosing cancer and/or a predisposition for developing cancer in a subject by determining the expression level of the MCM7 gene in the subject-derived biological sample. The MCM7 gene finds further utility in determining the prognosis of a cancer patient as well as in assessing and/or monitoring the efficacy or applicability of a cancer immunotherapy. Preferred cancers to be diagnosed, detected or assessed by the present method include lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor. Lung cancer includes small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC includes adenocarcinoma, squamous cell carcinoma (SCC) and large-cell carcinoma.
The present invention also provides a method for detecting or identifying cancer cells in a subject-derived biological sample such as a tissue sample, the method including the step of determining the expression level of the MCM7 gene in a subject-derived biological sample, wherein an increase in said expression level as compared to a normal control level indicates the presence or suspicion of cancer cells in the tissue.
Such result may be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose that a subject suffers from the disease or is predisposed to developing the disease. Alternatively, the present invention may provide a doctor with useful information to diagnose that the subject suffers from the disease. For example, according to the present invention, when the suspicion or doubt of the presence of cancer cells in the tissue obtained from a subject is indicated, clinical decisions would be made by a doctor with consideration of this observation and another aspect including the pathological finding of the tissue, levels of known tumor marker(s) in blood, or clinical course of the subject, etc. For example, some well-known diagnostic lung cancer markers in blood include ACT, BFP, CA19-9, CA50, CA72-4, CA130, CA602, CEA, IAP, KMO-1, SCC, SLX, SP1, Span-1, STN, TPA, and cytokeratin 19 fragment. Some well-known bladder cancer markers in blood include NMP22, BFP and TPA. Alternatively, diagnostic esophageal tumor markers in blood such as CEA, DUPAN-2, IAP, NSE, SCC, SLX and Span-1 are also well known. Some well-known diagnostic colorectal cancer markers in blood include CA72-4,STN,CA19-9,CEA and NCC-ST-439, liver cancer markers in blood include AFP and PIVKA-2, pancreatic cancer marker in blood include CA19-9, Span-1,SLX and CEA, testicular cancer markers in include AFP and BFP, acute myeloid leukemia markers in blood include ACT and SOD, and osteosarcoma markers in blood include ICTP, NTx, DPD and BAP. Namely, in a particular embodiment, according to the present invention, an intermediate result for examining the condition of a subject may also be provided.
In another embodiment, the present invention provides a method for detecting a diagnostic marker of cancer, the method including the step of detecting the expression level of the MCM7 gene in a subject-derived biological sample as a diagnostic marker of cancer. Preferable cancers to be diagnosed by the present method include lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
In the context of the present invention, the term "diagnosing" is intended to encompass predictions and likelihood analysis. The present method is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria such as disease stages, and disease monitoring and surveillance for cancer. According to the present invention, an intermediate result for examining the condition of a subject may also be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to determine that a subject suffers from the disease. Alternatively, the present invention may be used to detect cancerous cells in a subject-derived tissue, and provide a doctor with useful information to diagnose that the subject suffers from the disease.
A subject to be diagnosed by the present method is preferably a mammal. Exemplary mammals include, but are not limited to, human, non-human primate, mouse, rat, dog, cat, horse, and cow.
It is preferable to collect a biological sample from a subject to be diagnosed. Any biological material can be used as the biological sample for the determination so long as it can include the objective transcription or translation product of the MCM7 gene due to cancer. Examples of suitable biological samples include, but are not limited to, bodily tissues such as biopsy specimen and fluids such as saliva, sputum, blood, serum, plasma, pleural effusion and urine.
Preferably, the biological sample contains a cell population including an epithelial cell, more preferably a epithelial cell derived from tissue suspected to be cancerous. Further, if necessary, the cells may be purified from the obtained bodily tissues and fluids, and then used as the biological sample. Alternatively, biological sample may be a tissue sample collected from an area suspected to be cancerous. Preferably, the tissue sample may be a lung tissue sample for lung cancer, bladder tissue sample for bladder cancer, esophageal tissue sample for esophageal cancer, colorectal tissue sample for colorectal cancer, liver tissue sample for liver cancer, pancreatic tissue sample pancreatic cancer, testicular tissue sample for testicular cancer, myeloid tissue sample for acute myeloid leukemia, osseous tissue sample for osteosarcoma or soft tissue sample for soft tissue tumor.
According to the present invention, the expression level of the MCM7 gene in a subject-derived biological sample is determined and then correlated to a particular healthy or disease state by comparison to a control sample. The expression level can be determined at the transcription product (i.e., mRNA) level, using methods known in the art. For example, the mRNA of the MCM7 gene may be quantified using probes by hybridization methods (e.g., Northern hybridization). The detection may be carried out on a filter, a chip or an array. The use of an array is preferable for detecting the expression level of a plurality of genes (e.g., various cancer specific genes) including the MCM7 gene. Those skilled in the art can prepare such probes utilizing the known sequence information for the MCM7 gene. For example, the cDNA of the MCM7 gene may be used as the probes. If necessary, the probe may be labeled with a suitable label, such as dyes and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.
Alternatively, the transcription product of the MCM7 gene may be quantified using primers by amplification-based detection methods (e.g., RT-PCR). Such primers can also be prepared based on the available sequence information of the gene. For example, the primers used in the Example (SEQ ID NOs: 3 and 4) may be employed for the detection by RT-PCR, but the present invention is not restricted thereto.
A probe or primer suitable for use in the context of the present method will hybridize under stringent, moderately stringent, or low stringent conditions to the mRNA of the MCM7 gene. As used herein, the phrase "stringent (hybridization) conditions" refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degrees C lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees C for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degrees C for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
A probe or primer of the present invention is typically a substantially purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under stringent conditions to at least about 2000, 1000, 500, 400, 350, 300, 250, 200, 150, 100, 50, or 25 bases, consecutive sense strand nucleotide sequence of a nucleic acid including a MCM7 sequence, or an anti sense strand nucleotide sequence of a nucleic acid including a MCM7 sequence, or of a naturally occurring mutant of these sequences. In particular, for example, in a preferred embodiment, an oligonucleotide having 5-50 bases in length can be used as a primer for amplifying the genes, to be detected. More preferably, mRNA or cDNA of a MCM7 gene can be detected with oligonucleotide probe or primer of a specific size, generally 15- 30 bases in length. In preferred embodiments, length of the oligonucleotide probe or primer can be selected from 15-25 bases. Assay procedures, devices, or reagents for the detection of gene by using such oligonucleotide probe or primer are well known (e.g. oligonucleotide microarray or PCR). In these assays, probes or primers can also include tag or linker sequences. Further, probes or primers can be modified with detectable label or affinity ligand to be captured. Alternatively, in hybridization based detection procedures, a polynucleotide having a few hundreds (e.g., about 100-200) bases to a few kilo (e.g., about 1000-2000) bases in length can also be used for a probe (e.g., northern blotting assay or cDNA microarray analysis).
Alternatively, diagnosis may involve detection of a translation product (i.e., protein) of the MCM7 gene. For example, the quantity of the MCM7 protein may be determined and correlated to a disease or normal state. The quantity of the translation products/proteins may be determined using, for example, immunoassay methods that use an antibody specifically recognizing the protein. The antibody may be monoclonal or polyclonal. Furthermore, any antibody fragments or modified antibodies (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) may be used for the detection, so long as they retain the binding ability to the MCM7 protein. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
Alternatively, the intensity of staining may be observed via immunohistochemical analysis using an antibody against the MCM7 protein. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of the MCM7 gene.
The translation product may also be detected based on its biological activity. Specifically, the MCM7 protein was demonstrated herein to be involved in the growth of cancer cells. Thus, the cancer cell growth promoting ability of the MCM7 protein may be used as an index of the MCM7 protein existing in the biological sample. Herein, cell growth promoting ability is also referred to as "cell proliferative activity" or "cell proliferation enhancing activity".
To improve the accuracy of the diagnosis, the expression level of other cancer-associated genes, for example, genes known to be differentially expressed in lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor, may also be determined. Furthermore, in the case where the expression levels of multiple cancer-related genes are compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
In the context of the present invention, methods for detecting or identifying cancer in a subject or cancer cells in a subject-derived sample begin with a determination of MCM7 gene expression level. The expression level may be determined by any of the aforementioned techniques. Once determined, then, this level may be compared to a control level.
In the context of the present invention, the phrase "control level" refers to the expression level of the MCM7 gene detected in a control sample and encompasses both a normal control level and a cancer control level. The phrase "normal control level" refers to a level of the MCM7 gene expression detected in a normal healthy individual or in a population of individuals known not to be suffering from cancer. A normal individual is one with no clinical symptom of cancer. A normal control level can be determined using a normal cell obtained from a non-cancerous tissue. A "normal control level" may also be the expression level of the MCM7 gene detected in a normal healthy tissue or cell of an individual or population known not to be suffering from cancer. On the other hand, the phrase "cancer control level" refers to an expression level of the MCM7 gene detected in the cancerous tissue or cell of an individual or population suffering from cancer.
An increase in the expression level of the MCM7 gene detected in a sample as compared to a normal control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing cancer such as lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor. The subject-derived sample may include epithelial cells, more preferably epithelial cells suspected to be cancerous.
Alternatively, the expression level of the MCM7 gene in a sample can be compared to a cancer control level of the MCM7 gene. A similarity between the expression level of a sample and the cancer control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing cancer. When the expression levels of other cancer-related genes are also measured and compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing cancer.
In the context of the present invention, gene expression levels are deemed to be "altered" or "increased" when the gene expression changes or increases by, for example, 10%, 25%, or 50% from, or at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more compared to a control level. Accordingly, the expression level of cancer marker genes including the MCM7 gene in a biological sample can be considered to be increased if it increases from the normal control level of the corresponding cancer marker gene by, for example, 10% or more, 25% or more, or 50% or more; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
The control level may be determined at the same time with a test biological sample by using a sample(s) previously collected and stored from a subject/subjects whose disease state (cancerous or non-cancerous) is/are known. Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of the MCM7 gene in samples from subjects whose disease state are known. Furthermore, the control level can be a database of expression patterns from previously tested cells. Moreover, according to an aspect of the present invention, the expression level of the MCM7 gene in a biological sample may be compared to multiple control levels, which control levels are determined from multiple reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample. Moreover, it is preferred, to use the standard value of the expression levels of the MCM7 gene in a population with a known disease state. The standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as standard value.
When the expression level of the MCM7 gene is increased compared to the normal control level or is similar to the cancerous control level, the subject may be diagnosed to be suffering from or at a risk of developing cancer. Furthermore, in case where the expression levels of multiple cancer-related genes are compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing cancer.
Difference between the expression levels of a test biological sample and the control level can be normalized to the expression level of control nucleic acids, e.g., housekeeping genes. Genes whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell. Exemplary control genes include, but are not limited to, beta actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.
Furthermore, the present invention provides the use of the MCM7 gene as cancerous markers. The MCM7 gene are particularly useful for lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor as cancerous markers. For example, it can be determined whether a biological sample contains cancerous cells, especially lung cancerous cells, bladder cancerous cells, esophageal cancerous cells, colorectal cancerous cells, liver cancerous cells, pancreatic cancerous cells ,testicular cancerous cells, myeloid tissue tumor cells, osteosarcoma cells or soft tissue tumor cells , by detecting the expression level of the MCM7 gene as cancerous markers. Specifically, increasing the expression level of the MCM7 gene in a biological sample as compared to a normal control level indicates that the biological sample contains cancerous cells. The expression level of the MCM7 gene can be determined by detecting the transcription or translation products of the gene as described above.
The findings of the present invention reveal that MCM7 is not only a useful diagnostic marker, but also suitable target for cancer therapy. Therefore, cancer treatment targeting MCM7 can be achieved by the present invention. In the present invention, the cancer treatment targeting MCM7 refers to suppression or inhibition of MCM7 activity and/or expression in the cancer cells. Any anti-MCM7 agents may be used for the cancer treatment targeting MCM7. In the present invention, a cancer overexpressing MCM7 can be treated with at least one active ingredient selected from the group consisting of:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof.
Accordingly, in a preferred embodiment, the present invention provides a method of (i) diagnosing whether a subject has the cancer to be treated, and/or (ii) selecting a subject for cancer treatment, which method includes the steps of:
a) determining the expression level of MCM7 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated;
b) comparing the expression level of MCM7 with a normal control level;
c) diagnosing the subject as having the cancer to be treated, if the expression level of MCM7 is increased as compared to the normal control level; and
d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
Alternatively, such a method includes the steps of:
a) determining the expression level of MCM7 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated;
b) comparing the expression level of MCM7 with a cancerous control level;
c) diagnosing the subject as having the cancer to be treated, if the expression level of MCM7 is similar or equivalent to the cancerous control level; and
d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
The cancer includes, but is not limited to, lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor. Accordingly, prior to the administration of the double-stranded molecule of the present invention as active ingredient, it is preferable to confirm whether the expression level of MCM7 in the cancer cells or tissues to be treated is enhanced as compared with normal cells of the same organ. Thus, in one embodiment, the present invention provides a method for treating a cancer (over)expressing MCM7, which method may include the steps of:
i) determining the expression level of MCM7 in cancer cells or tissue(s) obtained from a subject with the cancer to be treated;
ii) comparing the expression level of MCM7 with normal control; and
iii) administrating at least one component selected from the group consisting of
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof, to a subject with a cancer overexpressing MCM7 compared with normal control.
Alternatively, the present invention also provides a pharmaceutical composition comprising at least one component selected from the group consisting of:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof,
for use in administrating to a subject having a cancer overexpressing MCM7. In other words, the present invention further provides a method for identifying a subject to be treated with:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, or
(c) a vector encoding thereof,
which method may include the step of determining an expression level of MCM7 in subject-derived cancer cells or tissue(s), wherein an increase of the level compared to a normal control level of the gene indicates that the subject has cancer which may be treated with a double-stranded molecule of the present invention.
The method of treating a cancer of the present invention will be described in more detail below.
A subject to be treated by the present method is preferably a mammal. Exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
According to the present invention, the expression level of MCM7 in cancer cells or tissues obtained from a subject is determined. The expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art. Alternatively, the translation product of the MCM7 gene may be detected for the treatment of the present invention, using methods known in the art. Those methods for determining the expression level of the MCM7 gene were described above.
II-2. Methods for determining or assessing the prognosis of cancer
The present invention relates, in part, to the discovery that MCM7 expression is significantly associated with poorer prognosis of subjects with lung cancer, e.g. NSCLC. Thus, the present invention provides a method for determining, predicting, monitoring or assessing the prognosis of a subject with a cancer caused or promoted in part by the over-expression of MCM7, by detecting the expression level of the MCM7 in a biological sample collected from a subject with cancer; comparing the detected expression level to a control level, wherein an increased expression level of MCM7 in comparison to the normal control level as indicative of poor prognosis (poor survival). Cancers to be assessing or determining the prognosis in the method of the present invention are preferably lung cancer, bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, more preferably lung cancer such as NSCLC. Herein, NSCLC includes adenocarcinoma, squamous cell carcinoma (SCC) and large-cell carcinoma.
In other embodiments, determining a similar or increased expression level of MCM7 gene in comparison to a cancerous control level is indicative of a poor prognosis.
Herein, the term "prognosis" refers to a forecast as to the probable outcome of the disease as well as the prospect of recovery from the disease as indicated by the nature and symptoms of the case. Accordingly, a less favorable, negative, or poor prognosis is defined by a lower post-treatment survival term or survival rate. Conversely, a positive, favorable, or good prognosis is defined by an elevated post-treatment survival term or survival rate.
The terms "assessing the prognosis" refer to the ability of predicting, forecasting or correlating a given detection or measurement with a future outcome of cancer of the subject (e.g., malignancy, likelihood of curing cancer, survival, and the like). For example, a determination of the expression level of MCM7 gene over time enables a predicting of an outcome for the subject (e.g., increase or decrease in malignancy, increase or decrease in grade of a cancer, likelihood of curing cancer, survival, and the like).
In the context of the present invention, the phrase "assessing (or determining or predicting) the prognosis" is intended to encompass predictions and likelihood analysis of cancer progression, particularly cancer recurrence, metastatic spread and disease relapse. The present method for determining or assessing prognosis is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria such as disease staging, and disease monitoring and surveillance for metastasis or recurrence of neoplastic disease.
The subject-derived biological sample used for the method may be any sample derived from the subject to be assessed so long as the expression level of the MCM7 gene can be detected in the sample. The subject-derived biological sample may be any sample derived from a subject, e.g., a subject known to have cancer. Preferably, the biological sample is a cancerous lung tissue sample for lung cancer, a cancerous esophageal tissue sample for esophageal cancer, a cancerous colorectal tissue sample for colorectal cancer, a cancerous liver tissue sample for liver cancer, a cancerous pancreatic tissue sample for pancreatic cancer, a cancerous bladder tissue sample for bladder cancer, a cancerous testicular tissue sample for testicular cancer, a cancerous myeloid tissue sample for acute myelocytic leukemia, a cancerous osterous tissue sample for osteosarcoma or a cancerous soft tissue sample for soft tissue tumor. Furthermore, the biological sample may include bodily fluids such as saliva, sputum, blood, serum, plasma, pleural effusion or urine. Moreover, the sample may be cells purified from a tissue. The biological samples may be obtained from a subject at various time points, including before, during, and/or after a treatment.
According to the present invention, it was shown that the higher the expression level of the MCM7 gene measured in the subject-derived biological sample, the poorer the prognosis for post-treatment remission, recovery, and/or survival and the higher the likelihood of poor clinical outcome. Thus, according to the present methods, the "control level" used for comparison may be, for example, the expression level of the MCM7 gene detected before any kind of treatment in an individual, or a population of individuals who showed good or positive prognosis of cancer after the treatment, which herein is referred to as "good prognosis control level". Alternatively, the "control level" may be the expression level of the MCM7 gene detected before any kind of treatment in an individual, or a population of individuals who showed poor or negative prognosis of cancer after the treatment, which herein will be referred to as "poor prognosis control level". The "control level" may be a single expression pattern derived from a single reference population or from a plurality of expression patterns. Thus, the control level may be determined based on the expression level of the MCM7 gene detected before any kind of treatment of cancer in a subject, or a population of the subjects whose disease state (good or poor prognosis) are known. Preferably, cancer is lung cancer, in particular NSCLC. It is preferred, to use the standard value of the expression levels of the MCM7 gene in a patient group with a known disease state. The standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as standard value.
As noted above, the control level may be determined at the same time with the test biological sample by using a sample(s) previously collected and stored before any kind of treatment from cancer subject(s) (control or control group) whose disease state (good prognosis or poor prognosis) are known.
Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing the expression level of the MCM7 gene in samples previously collected and stored from a control group. Furthermore, the control level can be a database of expression patterns from previously tested cells.
Moreover, according to an aspect of the present invention, the expression level of the MCM7 gene in a biological sample may be compared to multiple control levels, which control levels are determined from multiple reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample.
According to the present invention, a similarity between a measured or calculated the expression level of the MCM7 gene and a level corresponding to a good or positive prognosis control level indicates a more favorable patient prognosis. Likewise, an increase in the expression level as compared to the good or positive prognosis control level indicates a less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome. On the other hand, a decrease in the expression level of the MCM7 as compared to a poor or negative prognosis control level indicates a more favorable prognosis of the patient, with a similarity between the two indicating a less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome. In the context of the present invention, a following cell and/or tissue obtained from a subject who showed good, or poor prognosis of cancer after treatment is a preferable biological sample for good, or poor prognosis control level, respectively:
- a lung cancer cell and/or cancerous lung tissue sample for lung cancer,
- a esophageal cancer cell and/or cancerous esophageal tissue sample for esophageal cancer,
- a colorectal cancer cell and/or cancerous colorectal tissue sample for colorectal cancer,
- a liver cancer cell and/or cancerous liver tissue sample for liver cancer,
- a pancreatic cancer cell and/or cancerous pancreatic tissue sample for pancreatic cancer,
- a bladder cancer cell and/or cancerous bladder tissue sample for bladder cancer,
- a testicular cancer cell and/or cancerous testicular tissue sample for testicular cancer,
- an acute myelocytic leukemia cell and/or cancerous myeloid tissue sample for acute myelocytic leukemia,
- an osteosarcoma cell and/or cancerous osterous tissue sample for osteosarcoma, and
- a soft tissue tumor cell and/or cancerous soft tissue sample for soft tissue tumor.
An expression level of the MCM7 gene in a subject-derived biological sample is considered altered (i.e., increased or decreased) when the expression level differs from the control level by more than 1.0, 1.5, 2.0, 5.0, 10.0, or more fold.
The difference in the expression level between the test biological sample and the control level can be normalized to a control, e.g., housekeeping gene. For example, polynucleotides whose expression levels are known not to differ between the cancerous and non-cancerous cells, including those coding for beta-actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1, may be used to normalize the expression level of the MCM7 gene.
The expression level may be determined by detecting the gene transcript in the patient-derived biological sample using techniques well known in the art, including those described previously, in section II-1. Method for diagnosing cancer or a predisposition for developing cancer, for instance using hybridization, immunoassay, immunostaining, and cell proliferation assays. The gene transcripts of interest detected by the present method (i.e., MCM7 gene transcripts) include both the transcription and translation products, such as mRNA and protein.
For instance, the transcription product of the MCM7 gene can be detected by hybridization, e.g., Northern blot hybridization analyses, that use a MCM7 gene probe to the gene transcript. The detection may be carried out on a chip or an array. The use of an array is preferable for detecting the expression level of a plurality of genes including the MCM7 gene. As another example, amplification-based detection methods, such as reverse-transcription based polymerase chain reaction (RT-PCR) which use primers specific to the MCM7 gene may be employed for the detection (see "EXAMPLES"). The MCM7 gene-specific probe or primers may be designed and prepared using conventional techniques by referring to the whole sequence of the MCM7 gene (e.g., SEQ ID NO: 17 or 19).
For example, the primers (SEQ ID NOs: 3 and 4) used in the "EXAMPLES" may be employed for the detection by RT-PCR, but the present invention is not restricted thereto. Specifically, a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of the MCM7 gene. As used herein, the phrase "stringent (hybridization) conditions" refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degree Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degrees Centigrade for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Alternatively, the translation product may be detected for the assessment of the present invention. For example, the quantity of the MCM7 protein may be determined. A method for determining the quantity of the protein as the translation product includes immunoassay methods that use an antibody specifically recognizing the MCM7 protein. The antibody may be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used for the detection, so long as the fragment retains the binding ability to the MCM7 protein. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
As another method to detect the expression level of the MCM7 gene based on its translation product, the intensity of staining may be observed via immunohistochemical analysis using an antibody against MCM7 protein. Namely, the observation of strong staining indicates increased presence of the MCM7 protein and at the same time high expression level of the MCM7 gene.
Furthermore, herein, the MCM7 protein has been demonstrated to have a cell proliferating activity. Therefore, the expression level of the MCM7 gene can be determined using such cell proliferating activity as an index. For example, cells which express MCM7 are prepared and cultured in the presence of a biological sample, and then by detecting the extent of proliferation in a predetermined time period, or by measuring the cell cycle or the colony forming ability the cell proliferating activity of the biological sample can be determined.
As noted above in the context of prognostic methods, in addition to the expression level of the MCM7 gene, the expression level of other cancer-associated genes, for example, genes known to be differentially expressed in lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and/or soft tissue tumor, especially lung cancer, e.g., NSCLC, may also be determined to improve the accuracy of the assessment. Examples of such other lung cell-associated genes include those described herein and in WO 2004/031413 and WO 2005/090603, the contents of which are incorporated by reference herein.
Alternatively, according to the present invention, an intermediate result may also be provided in addition to other test results for assessing the prognosis of a subject. Such intermediate result may assist a doctor, nurse, or other practitioner to assess, determine, or estimate the prognosis of a subject. Additional information that may be considered, in combination with the intermediate result obtained by the present invention, to assess prognosis includes clinical symptoms and physical conditions of a subject.
The subject to be assessed for the prognosis of cancer according to the method is preferably a mammal and includes human, non-human primate, mouse, rat, dog, cat, horse, and cow.
In other words, the expression level of the MCM7 gene is useful prognostic marker for assessing, predicting or determining the prognosis of a subject suffering from lung cancer (e.g. NSCLC), bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and/or soft tissue tumor. Therefore, the present invention also provides a method for detecting a prognostic marker for assessing, predicting or determining the prognosis of a subject suffering from lung cancer (e.g. NSCLC), bladder cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, which comprises steps of:
a) detecting or determining an expression level of a MCM7 gene in a subject-derived biological sample, wherein the subject-derived biological is collected from a cancerous area, and
b) correlating the expression level detected or determined in step a) with the prognosis of the subject.
In particular, according to the present invention, an increased expression level to the good or positive prognosis control level is indicative of potential or suspicion of poor prognosis (poor survival).
III. Kits and reagents
In addition to diagnosing cancer, assessing the prognosis of cancer, and/or monitoring the efficacy of a cancer therapy, the present invention provides kits for detecting or diagnosing cancer or determining or assessing the prognosis of cancer. Preferably, the cancer to be detected or diagnosing by the present kit is selected from the group consisting of lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor. The kits of the present invention preferably include reagents described bellow.
The present invention also provides reagents for detecting or diagnosing cancer or a predisposition for developing cancer, determining or assessing the prognosis of cancer, or monitoring the efficacy of a cancer therapy i.e., reagents that can detect the transcription or translation product of the MCM7 gene. Examples of such reagents include those capable of:
(a) detecting mRNA of the MCM7 gene;
(b) detecting the MCM7 protein; or
(c) detecting the biological activity of the MCM7 protein,
in a subject-derived biological sample.
Suitable reagents include nucleic acids that specifically bind to or identify a transcription product of the MCM7 gene. For example, a nucleic acid that specifically binds to or identifies a transcription product of the MCM7 gene includes, for example, oligonucleotides (e.g., probes and primers) having a sequence that is complementary to a portion of the MCM7 gene transcription product. Such oligonucleotides are exemplified by primers and probes that are specific to the mRNA of the gene of interest and may be prepared based on methods well known in the art. Alternatively, antibodies can be exemplified as reagents for detecting the translation product of the gene. The probes, primers, and antibodies described above under the item of 'II-1. Method for diagnosing cancer or a predisposition for developing cancer' can be mentioned as suitable examples of such reagents. These reagents may be used for the above-described diagnosis or detection of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor. Also, above-mentioned reagents may be used for the determining or assessing the prognosis of cancer. In this case, the reagents may be preferably used for lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, more preferably lung cancer such as NSCLC. The assay format for using the reagents may be Northern hybridization or sandwich ELISA, both of which are well-known in the art.
The detection reagents may be packaged together in the form of a kit. For example, the detection reagents may be packaged in separate containers. Furthermore, the detection reagents may be packaged with other reagents necessary for the detection. For example, a kit may include a nucleic acid or antibody (either bound to a solid matrix or packaged separately with reagents for binding them to the matrix) as the detection reagent, a control reagent (positive and/or negative), and/or a detectable label. A kit of the present invention may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, and syringes. These reagents and such may be retained in a container with a label. Suitable containers include bottles, vials, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic. Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay may also be included in the kit.
Although the present kit is suited for the detection and diagnosis of cancer, for example, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor, it may also be useful in assessing the prognosis of cancer and/or monitoring the efficacy of a cancer therapy, for example, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor. In cases when the kit is used for assessing the prognosis of cancer, it is preferably applied to lung cancer such as NSCLC.
According to an aspect of the present invention, the kit of the present invention for diagnosing cancer may further include either of positive or negative controls sample, or both. The positive control sample of the present invention may be established a lung cancer cell line(s) (e.g., an NSCLC cell line(s)), esophageal cancer cell line(s), colorectal cancer cell line(s), liver cancer cell line(s), pancreatic cancer cell line(s), bladder cancer cell line(s), testicular cancer cell line(s), acute myeloid leukemia cell line(s), osteosarcoma cell line(s) and soft tissue tumor cell line(s).
Alternatively, the MCM7 positive control samples may also be a tissue(s) selected from the group consisting of:
- a lung cancer tissue(s) obtained from a lung cancer patient(s),
- an esophageal cancer tissue(s) obtained from an esophageal cancer patient(s),
- a colorectal cancer tissue(s) obtained from a colorectal cancer patient(s),
- a liver cancer tissue(s) obtained from a liver cancer patient(s),
- a pancreatic cancer tissue(s) obtained from a pancreatic cancer patient(s),
- a bladder cancer tissue(s) obtained from a bladder cancer patient(s),
- a testicular cancer tissue(s) obtained from a testicular cancer patient(s),
- an acute myeloid leukemia tissue(s) obtained from an acute myeloid leukemia patient(s),
- an osteosarcoma tissue(s) obtained from an osteosarcoma patient(s), and
- a soft tissue tumor tissue(s) obtained from a soft tissue tumor patient(s).
In a preferred embodiment, such lung cancer tissue may be an NSCLC tissue(s) obtained from a NSCLC patient(s). In a more preferred embodiment, such NSCLC tissue(s) may be a clinical lung adenocarcinoma tissue(s) obtained from a lung adenocarcinoma patient(s), a lung squamous cell carcinoma tissue(s) obtained from a lung squamous cell carcinoma patient(s), and/or a lung squamous cell carcinoma tissue(s) obtained from a large cell carcinoma patient(s).
Alternatively, positive control samples may be prepared by determined a cut-off value and preparing a sample containing an amount of an MCM7 mRNA or protein more than the cut-off value. Herein, the phrase "cut-off value" refers to the value dividing between a normal range and a cancerous range. For example, one skilled in the art may be determine a cut-off value using a receiver operating characteristic (ROC) curve. The present kit may include an MCM7 standard sample providing a cut-off value amount of an MCM7 mRNA or polypeptide. On the contrary, negative control samples may be prepared from non-cancerous cell lines or non-cancerous tissues such as normal lung tissues, esophageal tissues, colorectal tissues, liver tissues, pancreatic tissues, bladder tissues, testicular tissues, bone marrow, bone tissues or soft tissue tissues, or may be prepared by preparing a sample containing an MCM7 mRNA or protein less than cut-off value.
In a similar fashion, the kit of the present invention for assessing the prognosis of cancer may further include either of a good or positive prognosis control sample or a poor or negative prognosis control sample, or both. As described in II-2. Methods for determining or assessing the prognosis of cancer, a good or positive control sample may be a cancerous tissue sample obtained from an individual or a population of individuals who showed good or positive prognosis of cancer, after the treatment. Meanwhile, a poor or negative control sample may be a cancerous tissue sample obtained from an individual or a population of individuals who showed poor or negative prognosis of cancer, after the treatment.
In a preferred embodiment, a good or positive prognosis control sample may also be a clinical lung cancer tissue(s) obtained from a lung cancer patient(s) who showed good or positive prognosis of lung cancer, after treatment. Similarly, an esophageal cancer tissue(s), colorectal cancer tissue(s), liver cancer tissue(s), pancreatic cancer tissue(s), bladder cancer tissue(s), testicular cancer tissue(s), acute myeloid leukemia tissue(s), osteosarcoma tissue(s), or soft tissue tumor tissue(s) obtained from a respective cancer patient(s) who showed good or positive prognosis of cancer, after the treatment may also be a preferable good or positive prognosis control sample for respective cancer.
In a preferred embodiment, such lung cancer tissue may be an NSCLC tissue(s) obtained from an NSCLC cancer patient(s). In a more preferred embodiment, such NSCLC tissue may be a lung adenocarcinoma (ADC) tissue(s), a lung squamous cell carcinoma (SCC) tissue(s), and/or a large cell carcinoma tissue(s).
Alternatively, a good or positive prognosis control sample may be prepared by determined a cut-off value and preparing a sample containing an amount of an MCM7 mRNA or protein less than the cut-off value. Herein, the phrase "cut-off value" refers to the value dividing between a good prognosis range and a poor prognosis range. For example, one skilled in the art may be determine a cut-off value using a receiver operating characteristic (ROC) curve. The present kit may include an MCM7 standard sample providing a cut-off value amount of an MCM7 mRNA or polypeptide.
On the contrary, a poor or negative prognosis control sample may be a clinical lung cancer tissue(s) obtained from a lung cancer patient(s) who showed poor or negative prognosis of lung cancer, after the treatment. Similarly, an esophageal cancer tissue(s), colorectal cancer tissue(s), liver cancer tissue(s), pancreatic cancer tissue(s), bladder cancer tissue(s), testicular cancer tissue(s), acute myeloid leukemia tissue(s), osteosarcoma tissue(s), or soft tissue tumor tissue(s) obtained from a respective cancer patient(s) who showed poor or negative prognosis of cancer, after the treatment may also be a preferable poor or negative prognosis control sample for respective cancer.
In a preferred embodiment, such lung cancer tissue may be an NSCLC tissue(s) obtained from an NSCLC cancer patient(s). In a more preferred embodiment, such NSCLC tissue may be a lung adenocarcinoma (ADC) tissue(s), a lung squamous cell carcinoma (SCC) tissue(s), and/or a large cell carcinoma tissue(s).
Alternatively, a poor or negative prognosis control sample may be prepared by determined a cut-off value and preparing a sample containing an amount of an MCM7 mRNA or protein more than the cut-off value.
As an aspect of the present invention, the reagents for diagnosing or detecting cancer, or determining or assessing the prognosis of cancer may be immobilized on a solid matrix, such as a porous strip, to form at least one site for detecting cancer. The measurement or detection region of the porous strip may include a plurality of sites, each containing a detection reagent (e.g., nucleic acid). A test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a separate strip from the test strip. Optionally, the different detection sites may contain different amounts of immobilized detection reagents (e.g., nucleic acid), i.e., a higher amount in the first detection site and lesser amounts in subsequent sites. Upon the addition of test biological sample, the number of sites displaying a detectable signal provides a quantitative indication of the expression level of the MCM7 gene in the sample. The detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
IV. Screening methods
Through the present invention, it has been demonstrated that MCM7 is involved in cancer cell growth. Accordingly, substances that suppress an expression level of the MCM7 gene and/or a biological activity of the MCM7 polypeptide are expected to be useful for the treatment or prevention of cancer. Using the MCM7 gene, a polypeptide encoded by the gene or fragment thereof, or a transcriptional regulatory region of the gene, it is possible to screen substances that alter the expression of the gene or the biological activity of a polypeptide encoded by the gene. Such substances may be used as pharmaceuticals for treating or preventing cancer, in particular, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor,. Thus, the present invention provides methods of screening for candidate substances for treating or preventing cancer using the MCM7 gene, a polypeptide encoded by the gene or fragments thereof, or a transcriptional regulatory region of the gene.
A substance isolated and identified by the screening method of the present invention is expected to inhibit the expression of the MCM7 gene, or the activity of the translation product of the gene, and thus, is a candidate for the treatment or prevention of diseases attributed to MCM7, for example, cell proliferative diseases, such as cancer (in particular, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor). Namely, the substances identified through the screening methods of the present invention are deemed to have a clinical benefit and can be further tested for the ability to prevent cancer cell growth in animal models or test subjects.
In the context of the present invention, substances identified through the present screening methods may be any substance or composition and may in fact be a combination of several substances. When a combination of substances is used in the methods, the substances may be contacted sequentially or simultaneously.
Any test substances, for example, cell extracts, cell culture supernatants, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds (including nucleic acid constructs, such as antisense RNA, siRNA, Ribozymes, etc.) and natural compounds can be used in the screening methods of the present invention.
Test substances useful in the screenings described herein can also be antibodies that specifically bind to a protein of interest or a partial peptide thereof that lacks the biological activity of the original proteins in vivo.
Test substances of the present invention can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including:
(1) biological libraries,
(2) spatially addressable parallel solid phase or solution phase libraries,
(3) synthetic library methods requiring deconvolution,
(4) the "one-bead one-compound" library method and
(5) synthetic library methods using affinity chromatography selection.
The biological library methods using affinity chromatography selection is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des 1997, 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al., Proc Natl Acad Sci USA 1993, 90: 6909-13; Erb et al., Proc Natl Acad Sci USA 1994, 91: 11422-6; Zuckermann et al., J Med Chem 37: 2678-85, 1994; Cho et al., Science 1993, 261: 1303-5; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2059; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2061; Gallop et al., J Med Chem 1994, 37: 1233-51). Libraries of compounds may be presented in solution (see Houghten, Bio/Techniques 1992, 13: 412-21) or on beads (Lam, Nature 1991, 354: 82-4), chips (Fodor, Nature 1993, 364: 555-6), bacteria (US Pat. No. 5,223,409), spores (US Pat. No. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 1992, 89: 1865-9) or phage (Scott and Smith, Science 1990, 249: 386-90; Devlin, Science 1990, 249: 404-6; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Felici, J Mol Biol 1991, 222: 301-10; US Pat. Application 2002103360).
Although the construction of test substance libraries is well known in the art, herein below, additional guidance in identifying test substances and construction libraries of such substances for the present screening methods are provided.
A. Molecular Modeling:
Construction of test substance libraries is facilitated by knowledge of the molecular structure of compounds known to have the properties sought, and/or the molecular structure of MCM7 protein. One approach to preliminary screening of test substances suitable for further evaluation utilizes computer modeling of the interaction between the test substance and its target.
Computer modeling technology allows for the visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule. The three-dimensional construct typically depends on data from x-ray crystallographic analysis or NMR imaging of the selected molecule. The molecular dynamics require force field data. The computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.
An example of the molecular modeling system described generally above includes the CHARMM and QUANTA programs, Polygen Corporation, Waltham, Mass. CHARMM performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
A number of articles have been published on the subject of computer modeling of drugs interactive with specific proteins, examples of which include Rotivinen et al. Acta Pharmaceutica Fennica 1988, 97: 159-66; Ripka, New Scientist 1988, 54-8; McKinlay & Rossmann, Annu Rev Pharmacol Toxiciol 1989, 29: 111-22; Perry & Davies, Prog Clin Biol Res 1989, 291: 189-93; Lewis & Dean, Proc R Soc Lond 1989, 236: 125-40, 141-62; and, with respect to a model receptor for nucleic acid components, Askew et al., J Am Chem Soc 1989, 111: 1082-90.
Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. See, e.g., DesJarlais et al., J Med Chem 1988, 31: 722-9; Meng et al., J Computer Chem 1992, 13: 505-24; Meng et al., Proteins 1993, 17: 266-78; Shoichet et al., Science 1993, 259: 1445-50.
Once a putative inhibitor has been identified, combinatorial chemistry techniques can be employed to construct any number of variants based on the chemical structure of the identified putative inhibitor, as detailed below. The resulting library of putative inhibitors may be screened using the methods of the present invention to identify test substances suited to the treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence of cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
B. Combinatorial Chemical Synthesis:
Combinatorial libraries of test substances may be produced as part of a rational drug design program involving knowledge of core structures existing in known inhibitors. This approach allows the library to be maintained at a reasonable size, facilitating high throughput screening. Alternatively, simple, particularly short, polymeric molecular libraries may be constructed by simply synthesizing all permutations of the molecular family making up the library. An example of this latter approach would be a library of all peptides six amino acids in length. Such a peptide library could include every 6 amino acid sequence permutation. This type of library is termed a linear combinatorial chemical library.
Preparation of combinatorial chemical libraries is well known to those of skill in the art, and may be generated by either chemical or biological synthesis. Combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptides (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., WO 93/20242), random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g., US Patent 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90:6909-13), vinylogous polypeptides (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J Amer Chem Soc 1992, 114: 9217-8), analogous organic syntheses of small compound libraries (Chen et al., J. Amer Chem Soc 1994, 116: 2661), oligocarbamates (Cho et al., Science 1993, 261: 1303), and/or peptidylphosphonates (Campbell et al., J Org Chem 1994, 59: 658), nucleic acid libraries (see Ausubel, Current Protocols in Molecular Biology 1995 supplement; Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory, New York, USA), peptide nucleic acid libraries (see, e.g., US Patent 5,539,083), antibody libraries (see, e.g., Vaughan et al., Nature Biotechnology 1996, 14(3):309-14 and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 1996, 274: 1520-22; US Patent 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Gordon EM. Curr Opin Biotechnol. 1995 Dec 1;6(6):624-31.; isoprenoids, US Patent 5,569,588; thiazolidinones and metathiazanones, US Patent 5,549,974; pyrrolidines, US Patents 5,525,735 and 5,519,134; morpholino compounds, US Patent 5,506,337; benzodiazepines, 5,288,514, and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).
C. Other Candidates:
Another approach uses recombinant bacteriophage to produce libraries. Using the "phage method" (Scott & Smith, Science 1990, 249: 386-90; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Devlin et al., Science 1990, 249: 404-6), very large libraries can be constructed (e.g., 106 -108 chemical entities). A second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 1986, 23: 709-15; Geysen et al., J Immunologic Method 1987, 102: 259-74); and the method of Fodor et al. (Science 1991, 251: 767-73) are examples. Furka et al. (14th International Congress of Biochemistry 1988, Volume #5, Abstract FR:013; Furka, Int J Peptide Protein Res 1991, 37: 487-93), Houghten (US Patent 4,631,211) and Rutter et al. (US Patent 5,010,175) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.
Aptamers are macromolecules composed of nucleic acid that bind tightly to a specific molecular target. Tuerk and Gold (Science. 249:505-510 (1990)) disclose SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method for selection of aptamers. In the SELEX method, a large library of nucleic acid molecules (e.g., 1015 different molecules) can be used for screening.
A compound in which a part of the structure of the compound screened by any of the present screening methods is converted by addition, deletion and/or replacement, is included in the substances obtained by the screening methods of the present invention.
Furthermore, when the screened test substance is a protein, for obtaining a DNA encoding the protein, either the whole amino acid sequence of the protein may be determined to deduce the nucleic acid sequence coding for the protein, or partial amino acid sequence of the obtained protein may be analyzed to prepare an oligo DNA as a probe based on the sequence, and screen cDNA libraries with the probe to obtain a DNA encoding the protein. The obtained DNA finds use in preparing the test substance which is a candidate for treating or preventing cancer.
IV-1. Protein based screening methods
According to the present invention, the expression of the MCM7 gene is crucial for the growth and/or survival of cancer cells, in particular lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor cells.
Accordingly, substances that suppress the function of the MCM7 polypeptide are presumed to inhibit the growth and/or survival of cancer cells, and therefore find utility in either or both of the treatment and prevention of cancer. Thus, the present invention provides methods of screening a candidate substance for treating or preventing cancer, using the MCM7 polypeptide. Further, the present invention also provides methods of screening a candidate substance capable of inhibiting the growth and/or survival of cancer cells, using the MCM7 polypeptide.
In addition to the MCM7 polypeptide, fragments of the polypeptides may be used for the present screening, so long as it retains at least one biological activity of the naturally occurring MCM7 polypeptide. The polypeptides or fragments thereof may be further linked to other substances, so long as the polypeptides and fragments retain at least one of their biological activities. Usable substances include: peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc. These kinds of modifications may be performed to confer additional functions or to stabilize the polypeptide and fragments.
The polypeptides or fragments used for the present method may be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on the selected amino acid sequence. For example, conventional peptide synthesis methods that can be adopted for the synthesis include:
1) Peptide Synthesis, Interscience, New York, 1966;
2) The Proteins, Vol. 2, Academic Press, New York, 1976;
3) Peptide Synthesis (in Japanese), Maruzen Co., 1975;
4) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985;
5) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;
6) WO99/67288; and
7) Barany G. & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.
Alternatively, the proteins may be obtained through any known genetic engineering methods for producing polypeptides (e.g., Morrison J., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62). For example, first, a suitable vector including a polynucleotide encoding the objective protein in an expressible form (e.g., downstream of a regulatory sequence including a promoter) is prepared, transformed into a suitable host cell, and then the host cell is cultured to produce the protein. More specifically, a gene encoding the MCM7 polypeptide is expressed in host (e.g., animal) cells and such by inserting the gene into a vector for expressing foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8. A promoter may be used for the expression. Any commonly used promoters may be employed including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 1982, 83-141), the EF-alpha promoter (Kim et al., Gene 1990, 91:217-23), the CAG promoter (Niwa et al., Gene 1991, 108:193), the RSV LTR promoter (Cullen, Methods in Enzymology 1987, 152:684-704), the SR alpha promoter (Takebe et al., Mol Cell Biol 1988, 8:466), the CMV immediate early promoter (Seed et al., Proc Natl Acad Sci USA 1987, 84:3365-9), the SV40 late promoter (Gheysen et al., J Mol Appl Genet 1982, 1:385-94), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 1989, 9:946), the HSV TK promoter, and such. The introduction of the vector into host cells to express the MCM7 gene can be performed according to any methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 1987, 15:1311-26), the calcium phosphate method (Chen et al., Mol Cell Biol 1987, 7:2745-52), the DEAE dextran method (Lopata et al., Nucleic Acids Res 1984, 12:5707-17; Sussman et al., Mol Cell Biol 1985, 4:1641-3), the Lipofectin method (Derijard B, Cell 1994, 7:1025-37; Lamb et al., Nature Genetics 1993, 5:22-30; Rabindran et al., Science 1993, 259:230-4), and such.
The polypeptides may be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C- terminus of the polypeptide. Alternatively, a commercially available epitope-antibody system may be used (Experimental Medicine 13: 85-90 (1995)). Vectors which are capable of expressing a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP), and so on, by the use of its multiple cloning sites are commercially available.
A fusion protein, prepared by introducing only small epitopes composed of several to a dozen amino acids so as not to change the property of the original polypeptide by the fusion, is also provided herein. Epitopes, such as polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such, and antibodies recognizing them may be used as the epitope-antibody system for detecting the binding activity between the polypeptides (Experimental Medicine 13: 85-90 (1995)).
The MCM7 protein may also be produced in vitro adopting an in vitro translation system.
IV-1-1. Identifying substances that bind to the polypeptides
In context of the present invention, over-expression of MCM7 was detected in various cancers, in spite of no expression in normal organs (Fig. 1, 2, 4, Table 5). In addition, knock-down of the MCM7 expression by siRNAs led to suppression of the cell growth in cancer cell lines (Fig. 3, 5). Accordingly, substances that suppress the expression of the MCM7 gene or the biological activity of the MCM7 protein can be good candidates for cancer therapeutic drugs. As a substance that binds to a protein is likely to alter the expression of the gene coding for the protein or the biological activity of the protein, substances that bind to the MCM7 polypeptide can be also good candidates for cancer therapeutic drugs. Thus, as an aspect, the present invention provides a method of screening for a candidate substance for either or both of treating and preventing cancer, which includes steps of:
a) contacting a test substance with an MCM7 polypeptide or a fragment thereof;
b) detecting binding (or binding activity) between the polypeptide or fragment and the test substance; and
c) selecting the test substance that binds to the polypeptide as a candidate substance for either or both of treating and preventing cancer.
Identified substances by the screening method of the present invention can be applied to any cancer that over-expresses the MCM7 gene, such as lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor cells.
The potential therapeutic effect of the test substance in inhibiting the cell growth or treating or preventing MCM7 associated disease (e.g., cancer) may be evaluated or estimated using methods described herein. In particular, the present invention also provides a method of screening for a candidate substance capable of inhibiting the cell growth or a candidate substance for treating or preventing MCM7 associating disease (e.g., cancer), using the MCM7 polypeptide or fragments thereof including the steps as follows:
a) contacting a test substance with an MCM7 polypeptide or a fragment thereof;
b) detecting the binding (or binding activity) between the polypeptide or fragment and the test substance; and
c) correlating the binding of b) with the therapeutic effect of the test substance.
In the context of the present invention, the therapeutic effect may be correlated with the binding level to MCM7 polypeptide or a functional fragment thereof. For example, when the test substance binds to MCM7 polypeptide or a functional fragment thereof, the test substance may identified or selected as the candidate substance having the requisite therapeutic effect. Alternatively, when the test substance does not bind to an MCM7 polypeptide or a functional fragment thereof, the test substance may identified as the substance having no significant therapeutic effect.
In the course of the present invention, it was discovered that suppressing the expression of MCM7 reduces cancer cell growth. Thus, by screening for candidate substances that bind to MCM7 polypeptide, candidate substances that have the potential to treat or prevent cancers can be identified. The potential of these candidate substances to treat and/or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers.
The binding of a test substance to the MCM7 polypeptide may be, for example, detected by immunoprecipitation using an antibody against the polypeptide. Therefore, for the purpose for such detection, it is preferred that the MCM7 polypeptide or fragments thereof used for the screening contains an antibody recognition site. The antibody used for the screening may be one that recognizes an antigenic region (e.g., epitope) of the MCM7 polypeptide. Preparation methods for such antibodies are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
Alternatively, the MCM7 polypeptide or a fragment thereof may be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody at its N- or C-terminus. The specificity of the antibody has been revealed, to the N- or C- terminus of the polypeptide. A commercially available epitope-antibody system can be used (Experimental Medicine 1995, 13:85-90). Vectors which can express a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP), and such by the use of its multiple cloning sites are commercially available and can be used for the present invention. Furthermore, fusion proteins containing much smaller epitopes to be detected by immunoprecipitation with an antibody against the epitopes are also known in the art (Experimental Medicine 1995, 13:85-90). Such epitopes, composed of several to a dozen amino acids so as not to change the property of the MCM7 polypeptide or fragments thereof, can also be used in the present invention. Examples include polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage), and such and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the MCM7 polypeptide (Experimental Medicine 13: 85-90 (1995)).
Glutathione S-transferase (GST) is also well-known as the counterpart of the fusion protein to be detected by immunoprecipitation. When GST is used as the protein to be fused with the MCM7 polypeptide or fragment thereof to form a fusion protein, the fusion protein can be detected either with an antibody against GST or a substance specifically binding to GST, i.e., such as glutathione (e.g., glutathione-Sepharose 4B).
In immunoprecipitation, an immune complex is formed by adding an antibody (recognizing the MCM7 polypeptide or a fragment thereof itself, or an epitope tagged to the polypeptide or fragment) to the reaction mixture of the MCM7 polypeptide and the test substance. If the test substance has the ability to bind the polypeptide, then the formed immune complex will consist of the MCM7 polypeptide, the test substance, and the antibody. On the contrary, if the test substance is devoid of such ability, then the formed immune complex only consists of the MCM7 polypeptide and the antibody. Therefore, the binding ability of a test substance to MCM7 polypeptide can be examined by, for example, measuring the size of the formed immune complex. Any method for detecting the size of a compound can be used, including chromatography, electrophoresis, and such. For example, when mouse IgG antibody is used for the detection, Protein A or Protein G sepharose can be used for quantitating the formed immune complex.
For more details on immunoprecipitation see, for example, Harlow et al., Antibodies, Cold Spring Harbor Laboratory publications, New York, 1988, 511-52. SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Detection may be achieved using conventional staining method, such as Coomassie staining or silver staining, or, for proteins that is difficult to detect, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, 35S-methionine or 35S-cysteine, labeling proteins in the cells, and detecting the proteins. The target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.
Furthermore, the MCM7 polypeptide or a fragment thereof used for the screening of substances that bind thereto may be bound to a carrier. Example of carriers that may be used for binding the polypeptides include insoluble polysaccharides, such as agarose, cellulose and dextran; and synthetic resins, such as polyacrylamide, polystyrene and silicon; preferably commercially available beads and plates (e.g., multi-well plates, biosensor chip, etc.) prepared from the above materials may be used. When using beads, they may be filled into a column. Alternatively, the use of magnetic beads is also known in the art, and enables to readily isolate polypeptides and substances bound on the beads via magnetism.
The binding of a polypeptide to a carrier may be conducted according to routine methods, such as chemical bonding and physical adsorption. Alternatively, a polypeptide may be bound to a carrier via antibodies specifically recognizing the protein. Moreover, binding of a polypeptide to a carrier can also be conducted by means of interacting molecules, such as the combination of avidin and biotin.
Screening using such carrier-bound MCM7 polypeptide or fragments thereof include, for example, contacting a test substance to the carrier-bound polypeptide, incubating the mixture, washing the carrier, and detecting and/or measuring the substance bound to the carrier. The binding may be carried out in buffer, examples of which include, but are not limited to, phosphate buffer and Tris buffer, so long as the buffer does not inhibit the binding.
When such carrier-bound MCM7 polypeptide or fragments thereof, and a composition (e.g., cell extracts, cell lysates, etc.) are used as the test substance in a screening method, such method is generally called affinity chromatography. For example, the MCM7 polypeptide may be immobilized on a carrier of an affinity column, and a test substance, containing a substance capable of binding to the polypeptides, is applied to the column. After loading the test substance, the column is washed, and then the substance bound to the polypeptide is eluted with an appropriate buffer.
A biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound substance in the present invention. When such a biosensor is used, the interaction between the MCM7 polypeptide and a test substance can be observed real-time as a surface plasmon resonance signal, using only a minute amount of the polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide and test substance using a biosensor such as BIAcore.
Methods of screening for molecules that bind to a specific protein among synthetic chemical compounds, or molecules in natural substance banks or a random phage peptide display library by exposing the specific protein immobilized on a carrier to the molecules, and methods of high-throughput screening based on combinatorial chemistry techniques (Wrighton et al., Science 1996, 273:458-64; Verdine, Nature 1996, 384:11-3) to isolate not only proteins but chemical compounds are also well-known to those skilled in the art. These methods can also be used for screening substances (including agonist and antagonist) that bind to the MCM7 protein or fragments thereof.
When the test substance is a protein, for example, West-Western blotting analysis (Skolnik et al., Cell 1991, 65:83-90) can be used for the present method. Specifically, a protein binding to the MCM7 polypeptide can be obtained by preparing first a cDNA library from cells, tissues, organs, or cultured cells (e.g., PC cell lines) expected to express at least one protein binding to the MCM7 polypeptide using a phage vector (e.g., ZAP), expressing the proteins encoded by the vectors of the cDNA library on LB-agarose, fixing the expressed proteins on a filter, reacting the purified and labeled MCM7 polypeptide with the above filter, and detecting the plaques expressing proteins to which the MCM7 polypeptide has bound according to the label of the MCM7 polypeptide.
Labeling substances such as radioisotope (e.g., 3H, 14C, 32P, 33P, 35S, 125I, 131I), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, beta-glucosidase), fluorescent substances (e.g., fluorescein isothiocyanate (FITC), rhodamine) and biotin/avidin, may be used for the labeling of MCM7 polypeptide in the present method. When the protein is labeled with radioisotope, the detection or measurement can be carried out by liquid scintillation. Alternatively, when the protein is labeled with an enzyme, it can be detected or measured by adding a substrate of the enzyme to detect the enzymatic change of the substrate, such as generation of color, with absorptiometer. Further, in case where a fluorescent substance is used as the label, the bound protein may be detected or measured using fluorophotometer.
Moreover, the MCM7 polypeptide bound to the protein can be detected or measured by utilizing an antibody that specifically binds to the MCM7 polypeptide or a peptide or polypeptide (for example, GST) that is fused to the MCM7 polypeptide. In case of using an antibody in the present screening, the antibody is preferably labeled with one of the labeling substances mentioned above, and detected or measured based on the labeling substance. Alternatively, the antibody against the MCM7 polypeptide may be used as a primary antibody to be detected with a secondary antibody that is labeled with a labeling substance. Furthermore, the antibody bound to the MCM7 polypeptide in the present screening may be detected or measured using protein G or protein A column.
Antibodies to be used in the present screening methods can be prepared using techniques well known in the art. Antigens to prepared antibodies may be derived from any animal species, but preferably is derived from a mammal such as a human, mouse, rabbit, or rat, more preferably from a human. The polypeptide used as the antigen can be recombinantly produced or isolated from natural sources. The polypeptides to be used as an immunization antigen may be a complete protein or a partial peptide derived from the complete protein.
Any mammalian animal may be immunized with the antigen; however, the compatibility with parental cells used for cell fusion is preferably taken into account. In general, animals of the order Rodentia, Lagomorpha or Primate are used. Animals of the Rodentia order include, for example, mice, rats and hamsters. Animals of Lagomorpha order include, for example, hares, pikas, and rabbits. Animals of Primate order include, for example, monkeys of Catarrhini (old world monkey) such as Macaca fascicularis, rhesus monkeys, sacred baboons and chimpanzees.
Methods for immunizing animals with antigens are well known in the art. Intraperitoneal injection or subcutaneous injection of antigens is a standard method for immunizing mammals. More specifically, antigens may be diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline, etc. If desired, the antigen suspension may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, made into emulsion, and then administered to mammalian animals. Preferably, it is followed by several administrations of the antigen mixed with an appropriately amount of Freund's incomplete adjuvant every 4 to 21 days. An appropriate carrier may also be used for immunization. After immunization as above, the serum is examined by a standard method for an increase in the amount of desired antibodies.
Polyclonal antibodies may be prepared by collecting blood from the immunized mammal examined for the increase of desired antibodies in the serum, and by separating serum from the blood by any conventional method. Polyclonal antibodies include serum containing the polyclonal antibodies, as well as the fraction containing the polyclonal antibodies isolated from the serum. Immunoglobulin G or M can be prepared from a fraction which recognizes only the objective polypeptide using, for example, an affinity column coupled with the polypeptide, and further purifying this fraction using protein A or protein G column.
To prepare monoclonal antibodies, immune cells are collected from the mammal immunized with the antigen and checked for the increased level of desired antibodies in the serum as described above, and are subjected to cell fusion. The immune cells used for cell fusion are preferably obtained from spleen. Other preferred parental cells to be fused with the above immunocyte include, for example, myeloma cells of mammalians, and more preferably myeloma cells having an acquired property for the selection of fused cells by drugs.
The above immunocyte and myeloma cells can be fused according to known methods, for example, the method of Milstein et al., (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)).
Resulting hybridomas obtained by the cell fusion may be selected by cultivating them in a standard selection medium, such as HAT medium (hypoxanthine, aminopterin, and thymidine containing medium). The cell culture is typically continued in the HAT medium for several days to several weeks, the time being sufficient to allow all the other cells, with the exception of the desired hybridoma, to die. Then, the standard limiting dilution is performed to screen and clone a hybridoma cell producing the desired antibody.
In addition to the above method, in which a non-human animal is immunized with an antigen for preparing hybridoma, human lymphocytes, such as those infected by the EB virus, may be immunized with an antigen, cells expressing such antigen, or their lysates in vitro. Then, the immunized lymphocytes are fused with human-derived myeloma cells that are capable of indefinitely dividing, such as U266, to yield a hybridoma producing a desired human antibody that is able to bind to the antigen (Unexamined Published Japanese Patent Application No. (JP-A) Sho 63-17688).
The obtained hybridomas may be subsequently transplanted into the abdominal cavity of a mouse and the ascites may be extracted. The obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, DEAE ion exchange chromatography, or an affinity column carrying an objective antigen.
Antibodies against the MCM7 polypeptide can be used not only in the present screening method, but also for the detection of the polypeptides as cancer markers in biological samples as described in "II. Diagnosing cancer". They may further serve as candidates for agonists and antagonists of the polypeptides of interest. In addition, such antibodies, serving as candidates for antagonists, can be applied to the antibody treatment for diseases related to the MCM7 polypeptide including lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor as described infra.
Monoclonal antibodies thus obtained can be also recombinantly prepared using genetic engineering techniques (see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD (1990)). For example, a DNA encoding an antibody may be cloned from an immune cell, such as a hybridoma or an immunized lymphocyte producing the antibody, inserted into an appropriate vector, and introduced into host cells to prepare a recombinant antibody. Such recombinant antibody can also be used in the context of the present screening.
Furthermore, antibodies used in the screening and so on may be fragments of antibodies or modified antibodies, so long as they retain the original binding activity. For instance, the antibody fragment may be an Fab, F(ab')2, Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston et al., Proc Natl Acad Sci USA 85: 5879-83 (1988)). More specifically, an antibody fragment may be generated by treating an antibody with an enzyme, such as papain or pepsin. Alternatively, a gene encoding an antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co et al., J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).
An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG). Modified antibodies can be obtained through chemically modification of an antibody. These modification methods are conventional in the field.
Antibodies obtained as above may be purified to homogeneity. For example, the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins. For example, the antibody may be separated and isolated by appropriately selected and combined column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and others (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)); however, the present invention is not limited thereto. A protein A column and protein G column can be used as the affinity column. Exemplary protein A columns to be used include, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).
Exemplary chromatography, with the exception of affinity, includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). The chromatographic procedures can be carried out by liquid-phase chromatography, such as HPLC and FPLC.
Alternatively, in another embodiment of the screening method of the present invention, two-hybrid system utilizing cells may be used ("MATCHMAKER Two-Hybrid system", "Mammalian MATCHMAKER Two-Hybrid Assay Kit", "MATCHMAKER one-Hybrid system" (Clontech); "HybriZAP Two-Hybrid Vector System" (Stratagene); the references "Dalton et al., Cell 1992, 68:597-612" and "Fields et al., Trends Genet 1994, 10:286-92"). In two-hybrid system, MCM7 polypeptide or a fragment thereof is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells. A cDNA library is prepared from cells expected to express at least one protein binding to the MCM7 polypeptide such that the library, when expressed, is fused to the VP16 or GAL4 transcriptional activation region. The cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the MCM7 polypeptide is expressed in the yeast cells, the binding of the two activates a reporter gene, making positive clones detectable). A protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E. coli and expressing the protein.
As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.
The substance isolated by this screening is a candidate for agonists or antagonists of the MCM7 polypeptide. The term "agonist" refers to molecules that activate the function of the polypeptide by binding thereto. On the other hand, the term "antagonist" refers to molecules that inhibit the function of the polypeptide by binding thereto. Moreover, an substance isolated by this screening as an antagonist is a candidate that inhibits the in vivo interaction of the MCM7 polypeptide with molecules (including nucleic acids (RNAs and DNAs) and proteins).
IV-1-2. Identifying substances by detecting biological activity of the polypeptides
In the present invention, the MCM7 gene is disclosed to be highly overexpressed in lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor (Fig.1A-C, Fig. 2, 4 Table 5). In addition, the suppression of the MCM7 gene by small interfering RNA (siRNA) resulted in growth inhibition and/or cell death of lung and bladder cancer cells (Fig. 3, 5). Accordingly, the MCM7 polypeptide is demonstrated herein to be involved in cancer cell survival, and thus, substances that inhibit a biological activity of the MCM7 polypeptide find utility as candidate agents for cancer therapy.
Thus, the present invention also provides a method of screening for a candidate substance for treating and/or preventing cancer using the MCM7 polypeptide or fragments thereof including the steps as follows:
a) contacting a test substance with an MCM7 polypeptide or a fragment thereof;
b) detecting the biological activity of the polypeptide or fragment of the step (a); and
c) selecting the test substance that reduces the biological activity of the polypeptide as compared to the biological activity in the absence of the test substance.
According to the present invention, the therapeutic effect of the test substance on inhibiting the cell growth or a candidate substance for treating and/or preventing MCM7 associated disease may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance capable of inhibiting the cell growth or treating and/or preventing an MCM7-associated disease, using the MCM7 polypeptide or fragments thereof including the steps as follows:
a) contacting a test substance with an MCM7 polypeptide or a functional fragment thereof;
b) detecting the biological activity of the polypeptide or fragment of step (a); and
c) correlating the biological activity of b) with the therapeutic effect of the test substance.
Alternatively, in some embodiments, the present invention provides a method of evaluating or estimating a therapeutic effect of a test substance in the treatment and/or prevention of cancer and/or in the inhibition of the growth of a cancer associated with the over-expression of MCM7 gene, the method including steps of:
(a) contacting a test substance with the MCM7 polypeptide or a functional equivalent thereof;
(b) detecting the biological activity of the polypeptide or functional equivalent of step (a); and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance suppresses the biological activity of the MCM7 polypeptide or functional equivalent as compared to the biological activity of said polypeptide detected in the absence of the test substance. Such cancer includes lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
In the present invention, the therapeutic effect may be correlated with the biological activity of an MCM7 polypeptide or a functional fragment thereof. For example, when the test substance suppresses or inhibits the biological activity of an MCM7 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not suppress or inhibit the biological activity of MCM7 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
The method of the present invention will be described in more detail below.
Any polypeptides can be used for the screening method of the present invention so long as they retain a biological activity of the MCM7 polypeptide. Such biological activity includes, but are not limited to, cell proliferation promoting activity and activity of forming a complex with MCM4 and MCM6 proteins.
For example, naturally occurring human MCM7 polypeptides (e.g., polypeptide having an amino acid sequence of SEQ ID NO: 18 or 20) can be used and polypeptides functionally equivalent to these polypeptide can also be used (see "I. polynucleotide and polypeptides"). Such polypeptides may be expressed endogenously or exogenously by cells. Methods for preparing such polypeptides are described above.
Any substances can be used for the screening method of the present invention so long as they suppress or reduce a biological activity of the MCM7 polypeptide. In the context of the instant invention, the phrase "suppress or reduce a biological activity" encompasses at least 10% suppression of the biological activity of MCM7 in comparison with in the absence of the substance, more preferably at least 25%, 50% or 75% suppression and most preferably at 90% suppression. Such suppression can serve an index in the present screening method.
According to the present invention, the MCM7 polypeptide has been demonstrated to be required for the growth or viability of lung and bladder cancer cells. The biological activities of the MCM7 polypeptide that can be used as an index for the screening include such cell growth promoting activity of the human MCM7 polypeptide. Herein, cell growth promoting activity is also referred to as "cell proliferative activity" or "cell proliferation enhancing activity"
When the biological activity to be detected in the screening method of the present invention is cell growth promoting activity, it can be detected, for example, by preparing cells which express the MCM7 polypeptide or a fragment thereof, culturing the cells in the presence of a test substance, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by detecting wound-healing activity, conducting Matrigel invasion assay and measuring the colony forming activity.
More specifically, the screening method may include the steps of:
(a) contacting a test substance with a cell overexpressing MCM7 gene;
(b) measuring cell growth promoting activity; and
(c) selecting the test substance that reduces the cell growth promoting activity in the comparison with the cell growth promoting activity in the absence of the test substance.
In preferable embodiments, the screening method of the present invention may further include the step of:
(d) selecting the test substance that has no effect to the cells no or little expressing MCM7 gene.
The substance isolated by this screening method is a candidate for antagonists of the polypeptide encoded by MCM7 gene. The term "antagonist" refers to molecules that inhibit the function of the polypeptide by binding thereto. This term also refers to molecules that reduce or inhibit expression of the gene encoding MCM7. Moreover, a substance isolated by this screening is a candidate for substances which inhibit the in vivo interaction of the MCM7 polypeptide with molecules (including DNAs and proteins).
When the cell growth promoting activity is evaluated, control cells that do not express the MCM7 polypeptide may be used. Accordingly, the present invention also provides a method of screening for a candidate substance that inhibits cell growth or a candidate substance for treating and/or preventing an MCM7- associated disease such as cancer, using the MCM7 polypeptide or functional equivalent thereof including the steps as follows:
a) culturing cells which express an MCM7 polypeptide or a functional equivalent thereof in the presence or absence of a test substance, and control cells that do not express an MCM7 polypeptide or a functional equivalent thereof in the presence of the test substance;
b) detecting a biological activity (e.g., cell growth) of the cells which express the MCM7 polypeptide or the functional equivalent thereof and the control cells; and
c) selecting the test substance that inhibits the biological activity of the cells which express the MCM7 polypeptide or the functional equivalent thereof as compared to the biological activity detected in the absence of said test substance and that does not inhibit the biological activity of the control cells.
As revealed herein, suppressing the biological activity of MCM7 polypeptide reduces cell growth. Thus, by screening for a substance that inhibits the biological activity of MCM7 polypeptide, candidate substance that have the potential to treat and/or prevent cancers can be identified. The potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic substance, compounds or agent for cancers. For example, when a substance that inhibits the biological activity of an MCM7 polypeptide also inhibits the activity of a cancer, it may be concluded that such a substance has an MCM7 specific therapeutic effect.
IV-1-3 Identifying substances by detecting biological activity of MCM complex
In the present invention, heliquinomycin, an DNA helicase inhibitor against MCM protein complex composed of MCM4, MCM6 and MCM7 proteins, effectively suppressed the growth of cancer cells such as lung and bladder cancer cells.
It is known that six of the MCM proteins, MCM2-MCM7, forms complexes that participate in initiation and elongation steps of DNA replication. They share a conserved 200-amino acid nucleotide-binding region and form different subcomplexes (dimers, trimers and a hexamer) (Koonin EV. et al, Nucleic Acids Res 1993;21:2541-7.). Additionally, it is known that MCM 4, 6 and 7 trimers and MCM2-7 hexamers have ATPase and DNA helicase activities in vitro (lei M. et al, J Cell Sci 2001;114:1447-54.). In the present invention, it has been revealed that heliquinomycin, an inhibitor of MCM4, 6 and 7 DNA helicase, effectively suppresses the growth of cancer cells in a dose dependent manner (Fig. 6). Thus, substances that inhibit DNA helicase activity of MCM protein complex may become candidate agents for cancer therapy.
Accordingly, the present invention also provides the method of screening for a candidate substance for treating and/or preventing cancer using DNA helicase activity of MCM protein complex as an index. The present invention further provides the method of screening for a substance that suppresses the proliferation of cancer cells using DNA helicase activity of MCM protein complex as an index.
In the context of the present invention, the MCM protein complex is characterized as having the DNA helicase activity. Such MCM protein complexes include, for example, a trimer composed of MCM4, MCM6 and MCM7 proteins and a hexamer composed of MCM2-7 proteins.
Substances identified by the screening method of the present invention may be preferably applicable to cancer such as lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor. In particular, lung cancer and bladder cancer are suitable cancer for such substances.
Specifically, the present invention provides a method of screening for a candidate substance for treating and/or preventing cancer, including the steps as follows:
a) contacting a test substance with an MCM protein complex;
b) detecting the biological activity of the complex of the step a); and
c) selecting the test substance that reduces the biological activity of the complex as compared to the biological activity in the absence of the test substance.
The therapeutic effect of the test substance on cancer may be evaluated in accordance with the present invention. More particularly, the present invention provides a method of screening for a candidate substance capable of inhibiting the cell growth or a candidate substance for treating and/or preventing cancer, using the MCM protein complex including the steps as follows:
a) contacting a test substance with an MCM protein complex;
b) detecting the biological activity of the complex of step a); and
c) correlating the biological activity of b) with the therapeutic effect of the test substance.
The present invention further contemplates a method for evaluating or estimating a therapeutic effect of a test substance in connection with the treatment and/or prevention of cancer and/or in the inhibition of the growth of a cancer, the method including steps of:
a) contacting a test substance with the MCM protein complex;
b) detecting the biological activity of the complex of step a); and
c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance suppresses the biological activity of the complex as compared to the biological activity of said complex detected in the absence of the test substance. Such cancer includes lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, particularly lung cancer and bladder cancer.
In the context of present invention, the therapeutic effect may be correlated with the biological activity of an MCM protein complex. For example, when the test substance suppresses or inhibits the biological activity of an MCM protein complex as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not suppress or inhibit the biological activity of MCM protein complex as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
The method of the present invention will be described in more detail below.
The findings of the present invention revealed that suppressing the biological activity of MCM protein complex reduces cancer cell growth. Thus, by screening for substances that suppress the biological activity of the complex, candidate substances having therapeutic potential for the treatment and/or prevention of cancer can be identified. The potential of these candidate substances to treat and/or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a substance that suppresses the biological activity of MCM protein complex inhibits an activity of cancer such as cancer cell growth or survival, it may be concluded that such substance has the MCM protein complex specific therapeutic effect.
Any substances can be used for the screening method of the present invention so long as they suppress or reduce a biological activity of the MCM protein complex. In the context of the instant invention, the phrase "suppress or reduce a biological activity" encompasses at least 10% suppression of the biological activity of MCM protein complex in comparison with in the absence of the substance, more preferably at least 25%, 50% or 75% suppression and most preferably at 90% suppression. Such suppression can serve an index in the present screening method.
An MCM protein complex can be prepared by methods well known to one skilled in the art (J Biol Chem. 1997 Sep 26;272(39):24508-13.). An MCM protein complex can be purified from cells expressing MCM proteins, such as cancer cells, by ammonium sulfate precipitation, DEAE sepharose column or histone sepharose column. Alternatively, MCM protein complex can be also prepared by immunoprecipitation method etc, using anti-MCM protein antibody.
Cells expresses MCM proteins include, for example, lung cancer cell, esophageal cancer cell, colorectal cancer cell, liver cancer cell, pancreatic cancer cell, bladder cancer cell and testicular cancer cell, but are not limited to. Alternatively, an MCM protein complex can be prepared by contacting MCM proteins (e.g., MCM4, MCM6 and MCM7 or MCM2-7) each other under suitable condition for formation of MCM protein complex.
In the context of the present invention, DNA helicase activity is the preferred biological activity of the MCM protein complex to be assessed. DNA helicase activity can be detected by methods well known to one skilled in the art (J Biol Chem. 1997 Sep 26;272(39):24508-13.). For example, the DNA helicase activity can be determined by contacting an MCM complex with a substrate (e.g., double-stranded oligonucleotide) and determining quantity of single-stranded oligonucleotide free from the substrate.
More specifically, the screening method of the present invention includes the steps of:
(a) contacting an MCM protein complex with a double-stranded oligonucleotide in the presence of a test substance,
(b) detecting the quantity of the single-stranded oligonucleotide generated from the double-stranded oligonucleotide; and
(c) selecting the test substance that decreases the quantity of the single-stranded oligonucleotide as compared to the quantity of the single-stranded oligonucleotide from the double-stranded oligonucleotide detected in the absence of the test substance.
Alternatively, the method may include the steps of:
(a) contacting an MCM protein complex with a double-stranded oligonucleotide in the presence of a test substance,
(b) detecting the quantity of the double-stranded oligonucleotide; and
(c) selecting the test substance that increases the quantity of the double-stranded oligonucleotide as compared to the quantity of the double-stranded oligonucleotide detected in the absence of the test substance. The substrate (e.g., double-stranded oligonucleotide) to be catalyzed by DNA helicase can be labeled before using for assay.
The double-stranded oligonucleotide can be labeled before using for assay. Labeling substances such as radioisotope (e.g., 3H, 14C, 32P, 33P, 35S, 125I, 131I), fluorescent substances (e.g., fluorescein isothiocyanate (FITC), rhodamine) and biotin/avidin, may be used for the labeling of a double-stranded oligonucleotide in the present method. When the double-stranded oligonucleotide is labeled with radioisotope (preferably 32P,e.g. [gamma32P]-ATP), the detection or measurement can be carried out, for example, by SDS-polyacrylamide gel electrophoresis, autoradiography and liquid scintillation.
Alternatively, in case where a fluorescent substance is used as the label, the bound oligonucleotide may be detected or measured using fluorophotometer.
Moreover, such substrate (e.g. double-stranded oligonucleotide) may be separated from MCM protein complex by conventional methods such as gel filtration and immunoprecipitation.
Alternatively, DNA helicase activity of MCM protein complex may be determined using a surface plasmon resonance phenomenon, the reaction between nucleic acid labeled with biotin and agarose beads labeled with avidin or fluorescence resonance energy transfer (FRET).
When DNA helicase activity can be determined by the quantity of the double-stranded oligonucleotide, the higher value indicates the lower DNA helicase activity of MCM protein complex. When DNA helicase activity can be determined by the quantity of single-stranded oligonucleotide free from double-stranded oligonucleotide, the higher value indicates the higher DNA helicase activity of MCM protein complex.
Furthermore, the method of detecting DNA helicase activity can be performed by preparing cells which express the MCM genes, culturing the cells in the presence of a test substance, and measuring cell growth.
The substances that reduce DNA helicase activity of the MCM protein complex may be selected as candidate substance for treating and/or preventing cancer.
More specifically, the screening method of the present invention may include the steps of:
(a) contacting a test substance with cells overexpressing MCM genes;
(b) measuring DNA helicase activity in the cells of (a); and
(c) selecting the test substance that reduces the DNA helicase activity of (b) in the comparison with the DNA helicase activity in the absence of the test substance.
In one preferred embodiment, the screening method of the present invention may further include the steps of:
(d) selecting the test substance having no effect to the cells no or little expressing MCM genes.
A substance isolated by this screening method is considered to be a candidate antagonist of the MCM protein complex. The term "antagonist" refers to molecules that inhibit the function of the complex by binding thereto. This term also refers to molecules that reduce or inhibit activity of the complex. Moreover, a substance isolated by this screening is a candidate for substances which inhibit the in vivo interaction of the MCM protein complex with molecules (including DNAs and proteins).
IV-2. Nucleotide based screening methods
IV-2-1. Screening method using MCM7 gene
As discussed in detail above, by controlling the expression level of the MCM7 gene, one can control the onset and progression of cancer. Accordingly, screening methods that use the expression level of the MCM7 gene as an index can result in the identification of candidate substance for the treatment and/or prevention of cancer. In the context of the present invention, such a screening method may include, for example, the following steps:
a) contacting a test substance with a cell expressing an MCM7 gene;
b) detecting the expression level of the MCM7 gene;
c) comparing the expression level with the expression level detected in the absence of the test substance; and
d) selecting the test substance that reduces the expression level as compared to the expression level in the absence of the test substance as a candidate substance for treating and/or preventing cancer.
According to the present invention, the therapeutic effect of the test substance for inhibiting the cell growth or a candidate substance for treating or preventing cancer may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance that suppresses the proliferation of cancer cells, as well as a method of screening for a candidate substance for treating and/or preventing cancer.
In the context of the present invention, such screening method may include, for example, the following steps:
a) contacting a test substance with a cell expressing an MCM7 gene;
b) detecting the expression level of the MCM7 gene; and
c) correlating the expression level of b) with the therapeutic effect of the test substance.
In the context of the present invention, the therapeutic effect may be correlated with the expression level of the MCM7 gene. For example, when the test substance reduces the expression level of the MCM7 gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the expression level of the MCM7 gene as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
Herein, it was revealed that suppressing the expression of MCM7 gene reduces cancer cell growth. Thus, by screening for substances that reduce the expression level of MCM7gene, candidate substances that have the potential to treat or prevent cancers can be identified. Potential of these candidate substance to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a substance that reduces the expression level of the MCM7 gene inhibits an activity of the cancer such as cancer cell growth or survival, it may be concluded that such substance has the MCM7 specific therapeutic effect.
A substance that inhibits the expression of the MCM7 gene can be identified by contacting a cell expressing the MCM7 gene with a test substance and then determining the expression level of the MCM7 gene. Naturally, the identification may also be performed using a population of cells that express the gene in place of a single cell. A decreased expression level detected in the presence of a test substance as compared to the expression level in the absence of the test substance indicates that the test substance is an inhibitor of the MCM7 gene expression, which, in turn, suggests that the test substance may be suitable for inhibiting cancer and therefore have utility in connection with the treatment or prevention of cancer.
The expression level of a gene can be estimated by methods well known to one skilled in the art. The expression level of the MCM7 gene can be, for example, determined following the method described above under the item of 'II-1. Method for diagnosing cancer or a predisposition for developing cancer'.
The cell or the cell population used for such identification may be any cell or any population of cells, so long as it expresses the MCM7 gene. For example, the cell or cell population may be or contain a lung , esophageal, colorectal , liver, pancreatic, bladder or testicular epithelial cell derived from a cancerous tissue. Alternatively, the cell or cell population may be or contain an immortalized cell derived from a carcinoma cell, including lung cancer cell, esophageal cancer cell, colorectal cancer cell, liver cancer cell, pancreatic cancer cell, bladder cancer cell, testicular cancer cell, acute myeloid leukemia cell, osteosarcoma cell and soft tissue tumor cell. Cells expressing the MCM7 gene may be cell lines established from cancers (e.g., lung, liver, bladder cancer cell lines such as H1780, H1373, LC319, A549, PC-14, SK-MES-1, H2170, H520, H1703, RERF-LCAI, LX1, SBC3, SBC5, DMS273, DMS114, SW780, RT4, SNU475, Huh7 etc.). Furthermore, the cell or cell population may be or contain a cell which has been transfected with the MCM7 gene.
The present method allows screening of various test substances mentioned above and is particularly suited for screening functional nucleic acid molecules including antisense RNA, siRNA, and such.
IV-2-2. Screening method using transcriptional regulatory region of MCM7 gene
According to another aspect, the present invention provides a screening method which includes the following steps of:
a) contacting a test substance with a cell into which a vector, including a transcriptional regulatory region of an MCM7 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
b) detecting the expression level or activity of said reporter gene;
c) comparing the expression level or activity detected in step b) with the expression level or activity detected in the absence of the substance ; and
d) selecting the substance that reduces the expression level or activity of said reporter gene as a candidate substance for treating or preventing cancer.
The therapeutic effect of the test substance on cancer may be evaluated in accordance with the present invention. More particularly, the present invention provides a method of screening for a candidate substance that suppresses the proliferation of cancer cells, as well as a method of screening for a candidate substance for treating or preventing cancer.
According to another aspect, the present invention provides a method which includes the following steps of:
a) contacting a test substance with a cell into which a vector, composed of a transcriptional regulatory region of an MCM7 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
b) detecting the expression level or activity of said reporter gene; and
c) correlating the expression level or activity of b) with the therapeutic effect of the test substance.
In the context of the present invention, a therapeutic effect may be correlated with the expression level or activity of said reporter gene. For example, when the test substance reduces the expression level or activity of said reporter gene as compared to an expression level or activity detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the expression level or activity of said reporter gene as compared to an expression level or activity detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.
As noted previously, suppressing the expression of MCM7 gene reduces cell growth. Thus, by screening for substances that reduce the expression level or activity of the reporter gene, candidate substances that have the potential to treat or prevent cancers can be identified. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers.
Suitable reporter genes and host cells are well known in the art. The reporter construct required for the screening can be prepared using the transcriptional regulatory region of the MCM7 gene, which can be obtained as a nucleotide segment containing the transcriptional regulatory region from a genome library based on the nucleotide sequence information of the gene.
The transcriptional regulatory region may be, for example, the promoter sequence of the MCM7 gene. The reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of MCM7 gene. The transcriptional regulatory region of MCM7 gene herein is the region from start codon to at least 500 bp upstream, preferably 1,000 bp, more preferably 5,000 or 10,000 bp upstream. A nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).
When a cell(s) transfected with a reporter gene that is operably linked to the regulatory sequence (e.g., promoter sequence) of the MCM7 gene is used, an substance can be identified as inhibiting or enhancing the expression of the MCM7 gene through detecting the expression level of the reporter gene product.
Illustrative reporter genes include, but are not limited to, luciferase, green florescence protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed), Chrolamphenicol Acetyltransferase (CAT), lacZ and beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa, Ade2 gene, HIS3 gene, and others well-known in the art. Methods for detection of the expression of these genes are well known in the art.
A vector containing a reporter construct may be infected to host cells and the expression or activity of the reporter gene is detected by method well known in the art (e.g., using luminometer, absorption spectrometer, flow cytometer and so on). In the context of the instant invention, the phrase "reduces the expression or activity" encompasses at least 10% reduction of the expression or activity of the reporter gene in comparison with in absence of the compound, more preferably at least 25%, 50% or 75% reduction and most preferably at 95% reduction.
IV-3. Selecting therapeutic substances that are appropriate for a particular individual
Differences in the genetic makeup of individuals can result in differences in their relative abilities to metabolize various drugs. Any substance that is metabolized in a subject to act as an anti-tumor substance can manifest itself by inducing a change in a gene expression pattern in the subject's cells from that characteristic of a cancerous state to a gene expression pattern characteristic of a non cancerous state. Accordingly, the differential expression of the MCM7 gene in cancerous and non-cancerous cells can serve as an index of the putative therapeutic or prophylactic potential of a test substance in a test cell population from a selected subject in order to determine if the substance is a suitable inhibitor of cancer in the subject.
To identify an inhibitor of cancer that is appropriate for a specific subject, a test cell population from the subject is exposed to a candidate therapeutic substance, and the expression of MCM7 gene is determined. In the context of the method of the present invention, test cell populations contain cancer cells expressing the MCM7 gene. Preferably, the test cell is a lung, esophageal, colorectal, liver, pancreatic, bladder and testicular epithelial cell.
Specifically, a test cell population may be incubated in the presence of a candidate therapeutic substance and the expression of the MCM7 gene in the test cell population may be measured and compared to one or more reference profiles, e.g., a cancerous reference expression profile or a non-cancerous reference expression profile.
A decrease in the expression of the MCM7 gene in a test cell population relative to a reference cell population containing cancer indicates that the substance has therapeutic potential. Alternatively, a similarity in the expression of the MCM7 gene in a test cell population relative to a reference cell population not containing cancer indicates that the substance has therapeutic potential.
V. Pharmaceutical compositions for treating or preventing cancer
The substances identified by any of the screening methods of the present invention, as well as antisense nucleic acids and double-stranded molecules (e.g., siRNA) against the MCM7 gene, and antibodies against the MCM7 polypeptide that inhibit or suppress the expression of the MCM7 gene, or the biological activity of the MCM7 polypeptide are expected to inhibit or disrupt cancer cell proliferation and thus find utility in the context of pharmaceutical formulations. Thus, the present invention contemplates pharmaceutical compositions formulated for the treatment and/or prevention of cancer, wherein the compositions include substances identified by any of the screening methods of the present invention, antisense nucleic acids or double-stranded molecules against the MCM7 gene, or antibodies against the MCM7 polypeptide. The present compositions can be used for treating and/or preventing cancer, in particular, cancers such as lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, especially lung cancer and bladder cancer.
In the context of the present invention, the term "composition" is used to refer to a product including that include the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such terms, when used in relation to the modifier "pharmaceutical" (as in "pharmaceutical composition"), are intended to encompass products including a product that includes the active ingredient(s), and any inert ingredient(s) that make up the carrier, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, in the context of the present invention, the term "pharmaceutical composition" refers to any product made by admixing a molecule or compound of the present invention and a pharmaceutically or physiologically acceptable carrier.
The phrase "pharmaceutically acceptable carrier" or "physiologically acceptable carrier", as used herein, means a pharmaceutically or physiologically acceptable material, composition, substance or vehicle, including but not limited to, a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
The term "active ingredient" herein refers to a substance in composition that is biologically or physiologically active. Particularly, in the context of pharmaceutical composition, the term "active ingredient" refers to a substance that shows an objective pharmacological effect. For example, in case of pharmaceutical compositions for use in the treatment or prevention of cancer, active ingredients in the agents or compositions may lead to at least one biological or physiologically action on cancer cells and/or tissues directly or indirectly. Preferably, such action may include reducing or inhibiting cancer cell growth, damaging or killing cancer cells and/or tissues, and so on. Before being formulated, the "active ingredient" may also be referred to as "bulk", "drug substance" or "technical product".
Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. As used herein, "pharmaceutical formulations" include formulations for human and veterinary use. Thus, the compositions may be used as pharmaceuticals for humans and other mammals, such as mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees.
In the context of the present invention, suitable pharmaceutical formulations for the active ingredients of the present invention detailed below (including substances identified via screening methods, antisense nucleic acids, double-stranded molecules, antibodies, etc.) include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation. Other formulations include implantable devices and adhesive patches that release a therapeutic agent. When desired, the above-described formulations may be adapted to give sustained release of the active ingredient. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference. The preferred route of administration is intravenous delivery. The formulations are optionally packaged in discrete dosage units.
Pharmaceutical formulations suitable for oral administration include capsules, microcapsules, cachets and tablets, each containing a predetermined amount of active ingredient. Suitable formulations also include powders, elixirs, granules, solutions, suspensions and emulsions. The active ingredient is optionally administered as a bolus electuary or paste. Alternatively, according to needs, the pharmaceutical composition may be administered non-orally, in the form of injections of sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid. For example, the active ingredients of the present invention can be mixed with pharmaceutically acceptable carriers or media, specifically, sterilized water, physiological saline, plant-oils, emulsifiers, suspending substances, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and such, in a unit dose form required for generally accepted drug implementation. The amount of active ingredient contained in such a preparation makes a suitable dosage within the indicated range acquirable.
Examples of additives that can be admixed into tablets and capsules include, but are not limited to, binders, such as gelatin, corn starch, tragacanth gum and arabic gum; excipients, such as crystalline cellulose; swelling agents, such as corn starch, gelatin and alginic acid; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose or saccharin; and flavoring agents, such as peppermint, Gaultheria adenothrix oil and cherry. A tablet may be made by compression or molding, optionally with one or more formulational ingredients. Compressed tablets may be prepared by compressing in a suitable machine in which the active ingredients in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made via molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be coated according to methods well known in the art. The tablets may optionally be formulated so as to provide slow or controlled release of the active ingredient in vivo. A package of tablets may contain one tablet to be taken on each of the month.
Furthermore, when the unit-dosage form is a capsule, a liquid carrier, such as oil, can be further included in addition to the above ingredients.
Oral fluid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle prior to use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) or preservatives.
Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use. Alternatively, the formulations may be presented for continuous infusion. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Moreover, sterile composites for injection can be formulated following normal drug implementations using vehicles, such as distilled water, suitable for injection. Physiological saline, glucose, and other isotonic liquids, including adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used as aqueous solutions for injection. These can be used in conjunction with suitable solubilizers, such as alcohol, for example, ethanol; polyalcohols, such as propylene glycol and polyethylene glycol; and non-ionic surfactants, such as Polysorbate 80 (TM) and HCO-50.
Sesame oil or soybean oil can be used as an oleaginous liquid, which may be used in conjunction with benzyl benzoate or benzyl alcohol as a solubilizer, and may be formulated with a buffer, such as phosphate buffer and sodium acetate buffer; a pain-killer, such as procaine hydrochloride; a stabilizer, such as benzyl alcohol and phenol; and/or an anti-oxidant. A prepared injection may be filled into a suitable ampoule.
Formulations for rectal administration include suppositories with standard carriers such as cocoa butter or polyethylene glycol. Formulations for topical administration in the mouth, for example, buccally or sublingually, include lozenges, which contain the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles including the active ingredient in a base such as gelatin, glycerin, sucrose or acacia. For intra-nasal administration of an active ingredient, a liquid spray or dispersible powder or in the form of drops may be used. Drops may be formulated with an aqueous or non-aqueous base also including one or more dispersing agents, solubilizing agents or suspending agents.
For administration by inhalation the compositions are conveniently delivered from an insufflator, nebulizer, pressurized packs or other convenient means of delivering an aerosol spray. Pressurized packs may include a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichiorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.
Alternatively, for administration by inhalation or insufflation, the compositions may take the form of a dry powder composition, for example, a powder mix of an active ingredient and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflators.
Other formulations include implantable devices and adhesive patches that release a therapeutic agent.
When desired, the above-described formulations, adapted to give sustained release of the active ingredient, may be employed. The pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants or preservatives.
It should be understood that, in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question; for example, those suitable for oral administration may include flavoring agents.
Preferred unit dosage formulations are those containing an effective dose, as recited under the item of 'VI. Method for treating or preventing cancer' (infra), of each active ingredients of the present invention or an appropriate fraction thereof.
V-1. Pharmaceutical compositions containing screened substances
The present invention provides compositions for treating or preventing cancers including any of the substances selected by the above-described screening methods of the present invention.
An substances screened by the method of the present invention can be directly administered or can be formulated into a dosage form according to any conventional pharmaceutical preparation method detailed above.
V-2. Pharmaceutical compositions including double-stranded molecules
Double-stranded molecules (e.g., siRNA) against the MCM7 gene can be used to reduce the expression level of the MCM7 gene. Herein, the term "double-stranded molecule" refers to a nucleic acid molecule that inhibits expression of a target gene including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)) as described in "Definitions". In the context of the present invention, double-stranded molecules include a portion of the sense nucleic acid sequence and the complementary anti-sense nucleic acid sequence of the MCM7 gene. The double-stranded molecule is constructed so that it includes both a portion of the sense and complementary antisense sequences of the target gene (i.e., the MCM7 gene), and may also be a single construct taking a hairpin structure, wherein the sense and antisense strands are linked via a single-strand.
The double-stranded molecule serves as a guide for identifying homologous sequences in mRNA for the RISC complex, when the double-stranded molecule is introduced into cells. The identified target RNA is cleaved and degraded by the nuclease activity of Dicer, through which the double-stranded molecule eventually decreases or inhibits production (expression) of the polypeptide encoded by the RNA. Thus, a double-stranded molecule of the present invention can be defined by its ability to generate a single-strand that specifically hybridizes to the mRNA of the MCM7 gene under stringent conditions. Herein, the portion of the mRNA that hybridizes with the single-strand generated from the double-stranded molecule is referred to as "target sequence" or "target nucleic acid" or "target nucleotide". In the present invention, nucleotide sequence of the "target sequence" can be shown using not only the RNA sequence of the mRNA, but also the DNA sequence of cDNA synthesized from the mRNA.
In the context of the present invention, a double-stranded molecule is preferably less than 500, 200, 100, 50, or 25 base pairs in length. More preferably, a double stranded molecule is 19-25 base pairs in length. Exemplary target sequences of double-stranded molecules against the MCM7 gene include the nucleotide sequences of SEQ ID NO: 13 and 15. Accordingly, for example, the pharmaceutical composition of the present invention may include a double-stranded RNA molecule (i.e., siRNA) including the nucleotide sequence 5'- GGCUAAUGGAGAUGUCAA -3' (for SEQ ID NO: 13), and 5'- GAAAGAAGAUGUGAAUGA -3' (for SEQ ID NO: 15)
as the sense strand.
In order to enhance the inhibition activity of the double-stranded molecule, 3' overhangs can be added to the 3'end of the target sequence in the sense and/or antisense strand. The number of nucleotides to be added is at least 2, generally 2 to 10, preferably 2 to 5. The added nucleotides form a single strand at the 3'end of the sense and/or antisense strand of the double-stranded molecule. The nucleotides to be added is preferably "u" or "t", but are not limited to.
A loop sequence composed of an arbitrary nucleotide sequence can be located between the sense and antisense strands in order to form a hairpin loop structure. Thus, the double-stranded molecule contained in the pharmaceutical composition of the present invention may take the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence, [B] is an intervening single-strand and [A'] is the antisense strand containing a complementary sequence to the target sequence. Herein, the polynucleotide strand which includes a sequence corresponding to a target sequence, may be referred to as "sense strand". In preferred embodiments, [A] is the sense strand; [B] is a single stranded polynucleotide composed of 3 to 23 nucleotides; and [A'] is a polynucleotide strand which includes the antisense strand containing a complementary sequence of a target sequence, specifically hybridizing to an mRNA or a cDNA of the MCM7 gene (i.e., a sequence hybridizing to the target sequence of the sense strand [A]). Herein, the polynucleotide strand which includes a complementary sequence to a target sequence, specifically hybridizing to an mRNA or a cDNA of the MCM7 gene may be referred to as "antisense strand". The region [A] hybridizes to [A'], and then a loop composed of the region [B] is formed. The loop sequence may be preferably 3 to 23 nucleotides in length. Exemplary loop sequences include, but are not limited to, the following sequences (www.ambion.com/techlib/tb/tb_506.html):
CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002, 418: 435-8.
UUCG: Lee NS et al., Nature Biotechnology 2002, 20:500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003, 100(4):1639-44.
UUCAAGAGA: Dykxhoorn DM et al., Nature Reviews Molecular Cell Biology 2003, 4:457-67.
'UUCAAGAGA ("ttcaagaga" in DNA)' is a particularly suitable loop sequence. Furthermore, loop sequence composed of 23 nucleotides also provides an active siRNA (Jacque JM et al., Nature 2002, 418:435-8).
Exemplary hairpin siRNA suitable for the MCM7 gene include:
5'- GGCUAAUGGAGAUGUCAA -[b]-UUGACAUCUCCAUUAGCC -3'
(for target sequence of SEQ ID NO: 13); and
5'- GAAAGAAGAUGUGAAUGA-[b]- UCAUUCACAUCUUCUUUC-3'
(for target sequence of SEQ ID NO: 15).
Other nucleotide sequences of suitable double-stranded molecules for the present invention can be designed using an siRNA design computer program available from the Ambion website (www.ambion.com/techlib/ misc/siRNA_finder.html). The computer program selects nucleotide sequences for double-stranded molecule synthesis based on the following protocol.
Selection of Target Sites for double-stranded molecules:
1. Beginning with the AUG start codon of the object transcript, scan downstream for AA dinucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential target sites. Tuschl et al. Genes Cev 1999, 13(24):3191-7 don't recommend designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 nucleotides) as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex.
2. Compare the potential target sites to the human genome database and eliminate from consideration any target sequences with significant homology to other coding sequences. The homology search can be performed using BLAST (Altschul SF et al., Nucleic Acids Res 1997, 25:3389-402; J Mol Biol 1990, 215:403-10.), which can be found on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/.
3. Select qualifying target sequences for synthesis. At Ambion, preferably several target sequences can be selected along the length of the gene to evaluate.
The method of preparing the double-stranded molecule can use any chemical synthetic method known in the art. According to the chemical synthesis method, sense and antisense single-stranded polynucleotides are separately synthesized and then annealed together via an appropriate method to obtain a double-stranded molecule. Alternatively, a double-stranded molecule or siRNA molecule of the present invention may also be synthesized with in vitro translation. In this embodiment, DNA encoding a nucleotide sequence that comprises the target sequence and antisense thereof is transcribed into the double-stranded molecule in vitro. In one embodiment for the annealing, the synthesized single-stranded polynucleotides are mixed in a molar ratio of at least about 3:7, for example, about 4:6, for example, substantially equimolar amount (i.e., a molar ratio of about 5:5). Next, the mixture is heated to a temperature at which double-stranded molecules dissociate and then is gradually cooled down. The annealed double-stranded polynucleotide can be purified by usually employed methods known in the art. Example of purification methods include methods utilizing agarose gel electrophoresis or wherein remaining single-stranded polynucleotides are optionally removed by, e.g., degradation with appropriate enzyme.
The regulatory sequences flanking target sequences can be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. The double-stranded molecules can be transcribed intracellularly by cloning MCM7 gene template into a vector containing, e.g., an RNA pol III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.
Standard techniques are known in the art for introducing a double-stranded molecule into cells. For example, a double-stranded molecule can be directly introduced into the cells in a form that is capable of binding to the mRNA transcripts. In these embodiments, the double-stranded molecules are typically modified as described below for antisense molecules. Other modifications are also available, for example, cholesterol-conjugated double-stranded molecule has shown improved pharmacological properties (Song et al., Nature Med 2003, 9:347-51). These conventionally used techniques may also be applied for the double-stranded molecules contained in the present compositions.
Alternatively, a DNA encoding the double-stranded molecule may be carried in a vector (hereinafter, also referred to as 'siRNA vector') and the double-stranded molecule may be contained in the present composition in the form of vector which enables expression of the double-stranded molecule in vivo. Such vectors may be produced, for example, by cloning a portion of the target sequence sufficient to inhibit the in vivo expression of the target gene into an expression vector having operatively-linked regulatory sequences (e.g., a RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter) flanking the sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nature Biotechnology 2002, 20: 500-5). For example, an RNA molecule that is antisense to mRNA of the target gene is transcribed by a first promoter (e.g., a promoter sequence 3' of the cloned DNA) and an RNA molecule that is the sense strand for the mRNA of the target gene is transcribed by a second promoter (e.g., a promoter sequence 5' of the cloned DNA). The sense and antisense strands hybridize in vivo to generate the double-stranded molecule construct for silencing the expression of the target gene. Alternatively, the sense and antisense strands may be transcribed together with the help of one promoter. In this case, the sense and antisense strands may be linked via a polynucleotide sequence to form a single-stranded construct having secondary structure, e.g., hairpin.
Thus, the present pharmaceutical composition for treating or preventing cancer may include either the double-stranded molecule (e.g., siRNA) or a vector expressing the double-stranded molecule in vivo. In particular, the present invention provides pharmaceutical compositions for treating or preventing cancer that include a double-stranded molecule that inhibits the expression of the MCM7 gene, or a vector expressing the double-stranded molecule in vivo.
Further, the present invention also provides pharmaceutical compositions for inhibiting cancer cell proliferation, such composition including a double-stranded molecule which inhibits the expression of the MCM7 gene, or a vector expressing the double-stranded molecule in vivo.
For introducing the double-stranded molecule vector into the cell, transfection-enhancing agent can be used. FuGENE6 (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are useful as the transfection-enhancing agent. Therefore, the present pharmaceutical composition may further include such transfection-enhancing agents.
In another embodiment, the present invention also provides the use of the double-stranded nucleic acid molecules of the present invention or vector encoding thereof in manufacturing a pharmaceutical composition for treating a cancer expressing the MCM7 gene. For example, the present invention relates to a use of double-stranded nucleic acid molecule that inhibits the expression of MCM7 gene in a cell that over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NOs: 13 or 15 for manufacturing a pharmaceutical composition for treating a cancer expressing the MCM7 gene.
The double-stranded nucleic acid molecules of the present invention find utility in the treatment of a cancer expressing the MCM7 gene. Accordingly, the present invention provides a method or process for manufacturing a pharmaceutical composition for treating a cancer expressing the MCM7 gene, wherein the method or process includes step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the expression of MCM7 gene in a cell, which over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NOs: 13 or 15 as active ingredients.
In another embodiment, the present invention provides a method or process for manufacturing a pharmaceutical composition for treating a cancer expressing the MCM7 gene, wherein the method or process includes step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double-stranded nucleic acid molecule inhibiting the expression of MCM7 gene in a cell, which over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets to a sequence of SEQ ID NOs: 13 or 15.
V-3. Pharmaceutical compositions including antisense nucleic acids
Antisense nucleic acids targeting the MCM7 gene can be used to reduce the expression level of the gene that is up-regulated in cancerous cells including lung cancer cells, esophageal cancer cells, colorectal cancer cells, liver cancer cells, pancreatic cancer cells, bladder cancer cells, testicular cancer cells, acute myeloid leukemia cells, Osteosarcoma cells and soft tissue tumor cells.
Such antisense nucleic acids are useful for the treatment of cancer, in particular lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor,
and thus are also encompassed by the present invention. An antisense nucleic acid acts by binding to the nucleotide sequence of the MCM7 gene, or mRNAs corresponding thereto, thereby inhibiting the transcription or translation of the gene, promoting the degradation of the mRNAs, and/or inhibiting the expression of the protein encoded by the gene.
Thus, as a result, an antisense nucleic acid inhibits the MCM7 protein to function in the cancerous cell. Herein, the phrase "antisense nucleic acids" refers to nucleotides that specifically hybridize to a target sequence and includes not only nucleotides that are entirely complementary to the target sequence but also that include mismatches of one or more nucleotides. For example, the antisense nucleic acids of the present invention include polynucleotides that have a homology of at least 70% or higher, preferably of at least 80% or higher, more preferably of at least 90% or higher, even more preferably of at least 95% or higher (up to 99% and 100% homology) over a span of at least 15 continuous nucleotides of the MCM7 gene or the complementary sequence thereof. Algorithms known in the art can be used to determine such homology.
Antisense nucleic acids of the present invention act on cells producing proteins encoded by the MCM7 gene by binding to the DNA or mRNA of the gene, inhibiting their transcription or translation, promoting the degradation of the mRNA, and inhibiting the expression of the protein, finally inhibiting the protein to function.
Antisense nucleic acids of the present invention can be made into an external preparation, such as a liniment or a poultice, by admixing it with a suitable base material which is inactive against the nucleic acids.
The antisense nucleic acids of the present invention can also be formulated into tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops and freeze-drying agents by adding excipients, isotonic agents, solubilizers, stabilizers, preservatives, pain-killers, and such. An antisense-mounting medium can also be used to increase durability and membrane-permeability. Examples include, but are not limited to, liposomes, poly-L-lysine, lipids, cholesterol, lipofectin, or derivatives of these. Such formulations can be prepared in accordance with conventional techniques familiar to those of skill in the art.
The antisense nucleic acids of the present invention inhibit the expression of the MCM7 gene and thus find utility in suppressing the biological activity of the protein. In addition, expression-inhibitors, including antisense nucleic acids of the present invention, are also useful in that they can inhibit the biological activity of the MCM7 protein.
The antisense nucleic acids of present invention may encompass modified oligonucleotides. For example, thioated oligonucleotides may be used to confer nuclease resistance to an oligonucleotide.
V-4. Pharmaceutical compositions including antibodies
The function of a gene product of the MCM7 gene that is over-expressed in cancers, in particular lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, can be inhibited by administering a substance that binds to or otherwise inhibits the function of the gene products. An antibody against the MCM7 polypeptide can be mentioned as such a substance and can be used as the active ingredient of a pharmaceutical composition for treating or preventing cancer.
The present invention relates to the use of antibodies against a protein encoded by the MCM7 gene, or immunogenic fragments of such antibodies. As used herein, the term "antibody" refers to an immunoglobulin molecule having a specific structure, that interacts (i.e., binds) only with the antigen that was used for synthesizing the antibody (i.e., the gene product of an up-regulated marker) or with an antigen closely related thereto. Molecules including the antigen that was used for synthesizing the antibody and molecules including the epitope of the antigen recognized by the antibody can be mentioned as closely related antigens thereto.
Furthermore, an antibody suitable for use as a pharmaceutical composition may be an immunogenic fragment of an antibody or a modified antibody, so long as it binds to the protein encoded by the MCM7 gene (e.g., an immunologically active fragment of anti- MCM7 antibody). For instance, the antibody fragment may be Fab, F(ab')2, Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston JS et al., Proc Natl Acad Sci USA 1988, 85:5879-83). Such antibody fragments may be generated by treating an antibody with an enzyme, such as papain or pepsin. Alternatively, a gene encoding the antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co MS et al., J Immunol 1994, 152:2968-76; Better M et al., Methods Enzymol 1989, 178:476-96; Pluckthun A et al., Methods Enzymol 1989, 178:497-515; Lamoyi E, Methods Enzymol 1986, 121:652-63; Rousseaux J et al., Methods Enzymol 1986, 121:663-9; Bird RE et al., Trends Biotechnol 1991, 9:132-7).
Any antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG). The present invention includes such modified antibodies. The modified antibody can be obtained by chemically modifying an antibody. Such modification methods are conventional in the field.
Alternatively, an antibody of the present invention may be a chimeric antibody having a variable region derived from a non-human antibody against the MCM7 polypeptide and a constant region derived from a human antibody, or a humanized antibody, including a complementarity determining region (CDR) derived from a non-human antibody, a frame work region (FR) and a constant region derived from a human antibody. Such antibodies can be prepared by using known technologies. Humanization can be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (see e.g., Verhoeyen et al., Science 1988, 239:1534-6). Accordingly, such humanized antibodies are chimeric antibodies, wherein an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
Complete human antibodies including human variable regions in addition to human framework and constant regions can also be used. Such antibodies can be produced using various techniques known in the art. For example in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage (e.g., Hoogenboom et al., J Mol Biol 1992, 227:381-8). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, e.g., in US Pat. Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.
When the obtained antibody is to be administered to the human body (antibody treatment), a human antibody or a humanized antibody is preferable for reducing immunogenicity.
Antibodies obtained as above may be purified to homogeneity. For example, the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins. For example, the antibody may be separated and isolated by the appropriately selected and combined use of column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and others (Antibodies: A Laboratory Manual. Ed Harlow and D Lane, Cold Spring Harbor Laboratory (1988)), but are not limited thereto. A protein A column and protein G column can be used as the affinity column. Exemplary protein A columns to be used include, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).
Exemplary chromatography, with the exception of affinity includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). The chromatographic procedures can be carried out by liquid-phase chromatography, such as HPLC and FPLC.
VI. Methods for treating and/or preventing cancer
Cancer therapies directed to specific molecular alterations that occur in cancer cells have been validated through clinical development and regulatory approval of anti-tumor pharmaceuticals such as trastuzumab (Herceptin) for the treatment of advanced cancers, imatinib mesylate (Gleevec) for chronic myeloid leukemia, gefitinib (Iressa) for non-small cell lung cancer (NSCLC), and rituximab (anti-CD20 mAb) for B-cell lymphoma and mantle cell lymphoma (Ciardiello F et al., Clin Cancer Res 2001, 7:2958-70, Review; Slamon DJ et al., N Engl J Med 2001, 344:783-92; Rehwald U et al., Blood 2003, 101:420-4; Fang G et al., Blood 2000, 96:2246-53). These drugs are clinically effective and better tolerated than traditional anti-tumor agents because they target only transformed cells. Hence, such drugs not only improve survival and quality of life for cancer patients, but also validate the concept of molecularly targeted cancer therapy. Furthermore, targeted drugs can enhance the efficacy of standard chemotherapy when used in combination with it (Gianni L, Oncology 2002, 63 Suppl 1:47-56; Klejman A et al., Oncogene 2002, 21:5868-76). Therefore, future cancer treatments will probably involve combining conventional drugs with target-specific agents aimed at different characteristics of tumor cells such as angiogenesis and invasiveness.
These modulatory methods can be performed ex vivo or in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). The methods involve administering a protein or combination of proteins or a nucleic acid molecule or combination of nucleic acid molecules as therapy to counteract aberrant expression of the differentially expressed genes or aberrant activity of their gene products.
Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) expression levels or biological activities of genes and gene products, respectively, may be treated with therapeutics that antagonize (i.e., reduce or inhibit) activity of the over-expressed gene. Therapeutics that antagonize activity can be administered therapeutically or prophylactically.
Accordingly, therapeutics that may be utilized in the context of the present invention include, e.g., (i) a polypeptide of the over-expressed MCM7 gene or analogs, derivatives, fragments or homologs thereof; (ii) antibodies against the over-expressed gene or gene products; (iii) nucleic acids encoding the over-expressed gene; (iv) antisense nucleic acids or nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the nucleic acids of over-expressed gene); (v) double-stranded molecules (e.g., siRNA); or (vi) modulators (i.e., inhibitors, antagonists that alter the interaction between an over-expressed polypeptide and its binding partner). The dysfunctional antisense molecules are utilized to "knockout" endogenous function of a polypeptide by homologous recombination (see, e.g., Capecchi, Science 1989, 244: 1288 92).
Increased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a gene whose expression is altered). Methods that are well known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
Prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
Therapeutic methods of the present invention may include the step of administering an agent that modulates one or more of the activities of the MCM7 gene products. Examples of agent that modulate protein activity include, but are not limited to, nucleic acids, proteins, naturally occurring cognate ligands of such proteins, peptides, peptidomimetics, and other small molecule.
Thus, the present invention provides methods for treating or alleviating a symptom of cancer, or preventing cancer in a subject by decreasing the expression of the MCM7 gene or the activity of the gene product. The present method is particularly suited for treating and/or preventing lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, especially lung cancer and bladder cancer.
Suitable therapeutics can be administered prophylactically or therapeutically to a subject suffering from or at risk of (or susceptible to) developing cancers. Such subjects can be identified by using standard clinical methods or by detecting an aberrant expression level ("up-regulation" or "over-expression") of the MCM7 gene or aberrant activity of the gene product.
According to an aspect of the present invention, substances identified through screening methods of the present invention may be used for treating or preventing cancer. Methods well known to those skilled in the art may be used to administer the substances to patients, for example, as an intra-arterial, intravenous, or percutaneous injection or as an intranasal, transbronchial, intramuscular, or oral administration. If the substances are encodable by a DNA, the DNA can be inserted into a vector for gene therapy and the vector can be administered to a patient to perform the therapy.
The dosage and methods for administration vary according to the body-weight, age, sex, symptom, condition of the patient to be treated and the administration method; however, one skilled in the art can routinely select suitable dosage and administration method.
For example, although the dose of a substance that binds to an MCM7 polypeptide or regulates the activity of the polypeptide depends on the aforementioned various factors, the dose is generally about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day and more preferably about 1.0 mg to about 20 mg per day, when administered orally to a normal adult human (60 kg weight).
When administering the agent parenterally, in the form of an injection to a normal adult human (60 kg weight), although there are some differences according to the patient, target organ, symptoms and methods for administration, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg per day and more preferably about 0.1 to about 10 mg per day. In the case of other animals, the appropriate dosage amount may be routinely calculated by converting to 60 kg of body-weight.
Similarly, a pharmaceutical composition of the present invention may be used for treating or preventing cancer. Methods well known to those skilled in the art may be used to administer the compositions to patients, for example, as an intraarterial, intravenous, or percutaneous injection or as an intranasal, transbronchial, intramuscular, or oral administration.
For each of the aforementioned conditions, the compositions, e.g., polypeptides and organic compounds, can be administered orally or via injection at a dose ranging from about 0.1 to about 250 mg/kg per day. The dose range for adult humans is generally from about 5 mg to about 17.5 g/day, preferably about 5 mg to about 10 g/day, and most preferably about 100 mg to about 3 g/day. Tablets or other unit dosage forms of presentation provided in discrete units may conveniently contain an amount which is effective at such dosage or as a multiple of the same, for instance, units containing about 5 mg to about 500 mg, usually from about 100 mg to about 500 mg.
The dose employed will depend upon a number of factors, including the age, body weight and sex of the subject, the precise disorder being treated, and its severity. Also the route of administration may vary depending upon the condition and its severity. In any event, appropriate and optimum dosages may be routinely calculated by those skilled in the art, taking into consideration the above-mentioned factors.
For example, an antisense nucleic acid against the MCM7 gene can be given to the patient by direct application onto the ailing site or by injection into a blood vessel so that it will reach the site of ailment.
The dosage of an antisense nucleic acid derivatives of the present invention can be adjusted suitably according to the patient's condition and used in desired amounts. For example, a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered.
In the preferred embodiments, the method of the present invention includes the step of administering the double-stranded molecule against the MCM7 gene to a subject. The preferred examples of the double-stranded molecule to be administered are described under the item of " V-2. Pharmaceutical compositions including double-stranded molecules" and the following item of "VII. Double-stranded molecules and vectors encoding them".
It is understood that the double-stranded molecules of the present invention degrade the mRNA of the MCM7 gene in substoichiometric amounts. Without wishing to be bound by any theory, it is believed that the double-stranded molecule of the invention causes degradation of the target mRNA in a catalytic manner. Thus, compared to standard cancer therapies, significantly less a double-stranded molecule needs to be delivered at or near the site of cancer to exert therapeutic effect.
One skilled in the art can readily determine an effective amount of the double-stranded molecule of the present invention to be administered to a given subject, by taking into account factors such as body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is regional or systemic. Generally, an effective amount of the double-stranded molecule of the invention is an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM to about 50 nM, more preferably from about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of the double-stranded molecule can be administered. The precise dosage required for a particular circumstance may be readily and routinely determined by one of skill in the art.
The present methods can be used to inhibit the growth or metastasis of cancer; for example lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, especially lung cancer and bladder cancer.
For treating cancer, the double-stranded molecule of the present invention can also be administered to a subject in combination with a pharmaceutical agent different from the double-stranded molecule. Alternatively, the double-stranded molecule of the present invention can be administered to a subject in combination with another therapeutic method designed to treat cancer. For example, the double-stranded molecule of the present invention can be administered in combination with therapeutic methods currently employed for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents).
In the present methods, the double-stranded molecule can be administered to the subject either as a naked double-stranded molecule, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector which expresses the double-stranded molecule.
Suitable delivery reagents for administration in conjunction with the present a double-stranded molecule include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes. A preferred delivery reagent is a liposome.
Liposomes can aid in the delivery of the double-stranded molecule to a particular tissue, such as retinal or tumor tissue, and can also increase the blood half-life of the double-stranded molecule. Liposomes suitable for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example as described in Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467; and US Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369, the entire disclosures of which are herein incorporated by reference.
Preferably, the liposomes encapsulating the present double-stranded molecule comprises a ligand molecule that can deliver the liposome to the cancer site. Ligands which bind to receptors prevalent in tumor or vascular endothelial cells, such as monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens, are preferred.
Particularly preferably, the liposomes encapsulating the present double-stranded molecule are modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example, by having opsonization-inhibition moieties bound to the surface of the structure. In one embodiment, a liposome of the invention can comprise both opsonization-inhibition moieties and a ligand.
Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane. As used herein, an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids. These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); e.g., as described in US Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference. Liposomes modified with opsonization-inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes.
Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature. Thus, target tissue characterized by such microvasculature defects, for example, solid tumors, will efficiently accumulate these liposomes; see Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53. In addition, the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation in liver and spleen. Thus, liposomes of the invention that are modified with opsonization-inhibition moieties can deliver the present double-stranded molecule to tumor cells.
Opsonization-inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM1. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable. In addition, the opsonization-inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups. Preferably, the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".
The opsonization-inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques. For example, an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane. Similarly, a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH3 and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratio at 60 degrees C.
Vectors expressing a double-stranded molecule of the present invention are discussed in the following item. Such vectors expressing at least one double-stranded molecule of the invention can also be administered directly or in conjunction with a suitable delivery reagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes. Methods for delivering recombinant viral vectors, which express a double-stranded molecule of the invention, to an area of cancer in a patient are within the skill of the art.
A double-stranded molecule of the present invention can be administered to the subject by any means suitable for delivering the double-stranded molecule into cancer sites. For example, the double-stranded molecule can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.
Suitable enteral administration routes include oral, rectal, or intranasal delivery.
Suitable parenteral administration routes include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation. It is preferred that injections or infusions of the double-stranded molecule or vector be given at or near the site of cancer.
A double-stranded molecule of the present invention can be administered in a single dose or in multiple doses. Where the administration of the double-stranded molecule of the invention is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. Injection of the agent directly into the tissue is at or near the site of cancer preferred. Multiple injections of the agent into the tissue at or near the site of cancer are particularly preferred.
One skilled in the art can also readily determine an appropriate dosage regimen for administering the double-stranded molecule of the invention to a given subject. For example, the double-stranded molecule can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site. Alternatively, the double-stranded molecule can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, more preferably from about seven to about ten days. In a preferred dosage regimen, the double-stranded molecule is injected at or near the site of cancer once a day for seven days. Where a dosage regimen comprises multiple administrations, it is understood that the effective amount of a double-stranded molecule administered to the subject can comprise the total amount of a double-stranded molecule administered over the entire dosage regimen.
VII. Double-stranded molecules and vectors encoding them
Herein, an siRNA including either of the sequences of SEQ ID NOs: 13 or 15 is demonstrated to suppress cell growth or viability of cells expressing the MCM7 gene. Accordingly, double-stranded molecules including any of these sequences and vectors expressing the molecules are considered to serve as preferable pharmaceutics for treating or preventing diseases which involve the proliferation of MCM7 gene expressing cells, for example, cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor, more particularly lung cancer and bladder cancer.
Thus, one aspect of the present invention relates to the provision of double-stranded molecules that include a nucleotide sequence corresponding to the target sequence selected from the group consisting of SEQ ID NOs: 13 and 15 and vectors expressing the molecules. More specifically, the present invention provides a double-stranded molecule, when introduced into a cell expressing the MCM7 gene, inhibits expression of the gene, wherein the double-stranded molecule includes a sense strand and an antisense strand, wherein the sense strand includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13 and 15 as a target sequence, and the antisense strand includes a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule. Alternatively, the present invention provides a double-stranded molecule, when introduced into a cell expressing an MCM7 gene, inhibits expression of the gene, wherein the double-stranded molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 13 and 15, and the antisense strand comprises a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule.
The target sequence for the MCM7 gene included in the sense strand may be composed of a sequence of a portion of SEQ ID NO: 17 or 19 that is less than about 500, 400, 300, 200, 100, 75, 50 or 25 contiguous nucleotides. Preferably, the target sequence may be from about 19 to about 25 contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 17 or 19. The present invention is not limited thereto, but suitable target sequences include the nucleotide sequences selected from the group consisting of SEQ ID NOs: 13 and 15.
The double-stranded molecule of the present invention may be composed of two polynucleotide constructs, i.e., a polynucleotide including the sense strand and a polynucleotide including the antisense strand. Alternatively, the molecule may be composed of one polynucleotide construct; i.e., a polynucleotide including both the sense strand and the antisense strand, wherein the sense and antisense strands are linked via a single-stranded polynucleotide which enables hybridization of the target sequences within the sense and antisense strands by forming a hairpin structure. Herein, the single-stranded polynucleotide may also be referred to as "loop sequence" or "single-strand". The single-stranded polynucleotide linking the sense and antisense strands may consist of 3 to 23 nucleotides. See under the item of "V-2. Pharmaceutical compositions including double-stranded molecules" for more details on the double-stranded molecule of the present invention.
The double-stranded molecules of the present invention may contain one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule. The skilled person will be aware of other types of chemical modification which may be incorporated into the present molecules (WO03/070744; WO2005/045037). In one embodiment, modifications can be used to provide improved resistance to degradation or improved uptake. Examples of such modifications include, but are not limited to, phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base" nucleotides, 5'-C- methyl nucleotides, and inverted deoxybasic residue incorporation (US20060122137).
In another embodiment, modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule. Modifications include chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3' or 5' terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2-fluoro modified ribonucleotides and 2'-deoxy ribonucleotides (WO2004/029212). In another embodiment, modifications can be used to increased or decreased affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (WO2005/044976). For example, an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine. Additionally, an unmodified purine can be substituted with a 7-deaza, 7-alkyl, or 7-alkenyl purine. In another embodiment, when the double-stranded molecule is a double-stranded molecule with a 3' overhang, the 3'- terminal nucleotide overhanging nucleotides may be replaced by deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200). For further details, published documents such as US20060234970 are available. The present invention is not limited to these examples and any known chemical modifications may be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene.
Furthermore, the double-stranded molecules of the invention may include both DNA and RNA, e.g., dsD/R-NA or shD/R-NA. Specifically, a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability. Mixing of DNA and RNA, i.e., a hybrid type double-stranded molecule consisting of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule including both DNA and RNA on any or both of the single strands (polynucleotides), or the like may be formed for enhancing stability of the double-stranded molecule. The hybrid of a DNA strand and an RNA strand may be the hybrid in which either the sense strand is DNA and the antisense strand is RNA, or the opposite so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene. Preferably, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA. Also, the chimera type double-stranded molecule may be either the molecule that both of the sense and antisense strands are composed of DNA and RNA, or the molecule that any one of the sense and antisense strands is composed of DNA and RNA so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
In order to enhance stability of the double-stranded molecule, the molecule preferably contains as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule is required to be RNA within a range to induce sufficient inhibition of the expression. As a preferred example of the chimera type double-stranded molecule, an upstream partial region (i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands) of the double-stranded molecule is RNA. The upstream partial region means the 5' side (5'-end) of the sense strand and the 3' side (3'-end) of the antisense strand. That is, in more preferred embodiments, a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand consists of RNA. For instance, the chimera or hybrid type double-stranded molecule of the present invention include following combinations.
sense strand:
5'-[---DNA---]-3'
3'-(RNA)-[DNA]-5'
:antisense strand,
sense strand:
5'-(RNA)-[DNA]-3'
3'-(RNA)-[DNA]-5'
:antisense strand, and
sense strand:
5'-(RNA)-[DNA]-3'
3'-(---RNA---)-5'
:antisense strand.
The upstream partial region preferably is a domain composed of 9 to 13 nucleotides counted from the terminus of the target sequence or complementary sequence thereto within the sense or antisense strands of the double-stranded molecules. Moreover, preferred examples of such chimera type double-stranded molecules include those having a strand length of 19 to 21 nucleotides in which at least the upstream half region (5' side region for the sense strand and 3' side region for the antisense strand) of the polynucleotide is RNA and the other half is DNA. In such a chimera type double-stranded molecule, the effect to inhibit expression of the target gene is much higher when the entire antisense strand is RNA (US20050004064).
In the context of the present invention, the double-stranded molecule may form a hairpin, such as a short hairpin RNA (shRNA) and short hairpin composed of DNA and RNA (shD/R-NA). The shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNA or shD/R-NA includes the sense target sequence and the antisense target sequence on a single strand wherein the sequences are separated by a loop sequence. Generally, the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the target sequence of the dsRNA or dsD/R-NA.
The present invention further provides vectors that include a combination of polynucleotide having a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid includes nucleotide sequence of SEQ ID NOs: 13 or 15, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing the MCM7, inhibit expression of said gene. Preferably, the sense strand of the polynucleotide is an oligonucleotide of between about 19 and 25 nucleotides in length (e.g., contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 17 or 19). More preferably, the combination of polynucleotide includes a single nucleotide transcript having the sense strand and the antisense strand linked via a single-stranded nucleotide sequence. More preferably, the combination of polynucleotide has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3' wherein [A] is a nucleotide sequence including SEQ ID NO: 13 or 15; [B] is a nucleotide sequence composed of about 3 to about 23 nucleotide; and [A'] is a nucleotide sequence complementary to the target sequence.
Vectors of the present invention can be produced by conventional means, for example, by cloning MCM7 sequence into an expression vector so that regulatory sequences are operatively-linked to MCM7 sequence in a manner to allow expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5). For example, RNA molecule that is the antisense to mRNA is transcribed by a first promoter (e.g., a promoter sequence flanking to the 3' end of the cloned DNA) and RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA). The sense and antisense strands hybridize in vivo to generate a double-stranded molecule constructs for silencing of the gene. Alternatively, two vectors constructs respectively encoding the sense and antisense strands of the double-stranded molecule are utilized to respectively express the sense and anti-sense strands and then forming a double-stranded molecule construct. Furthermore, the cloned sequence may encode a construct having a secondary structure (e.g., hairpin); namely, a single transcript of a vector contains both the sense and complementary antisense sequences of the target gene.
The vectors of the present invention may also be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; US Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-based delivery technologies include "naked DNA", facilitated (bupivacaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., US Patent No. 5,922,687).
The vectors of the present invention include, for example, viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox (see, e.g., US Patent No. 4,722,848). This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the double-stranded molecule. Upon introduction into a cell expressing the target gene, the recombinant vaccinia virus expresses the molecule and thereby suppresses the proliferation of the cell. Another example of useable vector includes Bacille Calmette Guerin (BCG). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60. A wide variety of other vectors are useful for therapeutic administration and production of the double-stranded molecules; examples include adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14: 571-85.
Hereinafter, the present invention is described in more detail with reference to the Examples. However, the following materials, methods and examples only illustrate aspects of the invention and in no way are intended to limit the scope of the present invention. As such, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
EXAMPLE1:General Methods
Cell lines and lung tissue samples
Cancer cell lines used in this study were as follows: lung adenocarcinoma (ADC) NCI-H1781, NCI-H1373, LC319, A549 and PC-14; lung squamous cell carcinoma (SCC) SK-MES-1, NCI-H2170, NCI-H520, NCI-H1703 and RERF-LC-AI; lung large cell carcinoma (LCC) LX1; and small cell lung cancer (SCLC) SBC-3, SBC-5, DMS273 and DMS114; bladder cancer SW780 and RT4; liver cancer SNU475 and Huh7. All cell lines were grown in monolayers in appropriate media supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Sigma). All cells were maintained at 37 degrees C in humid air with 5% CO2 (all cell lines except for SW780), or without CO2 (SW780). Human small airway epithelial cells (SAEC) were grown in optimized medium from Cambrex Bioscience, Inc. Cells were transfected with FuGENE6 (ROCHE, Basel, Switzerland) according to the manufacturer's protocols. Primary non-SCLC (NSCLC) tissue samples as well as their corresponding normal tissues adjacent to resection margins from patients having no anticancer treatment before tumor resection had been obtained earlier with informed consent (Kato T, et al, Proc natl Acad Sci USA 1999;96:14783-8, Kikuchi T, et al, Oncoene 2003;22:2192-205, Taniwaki M, et al, Int J Oncol 2006;29:567-75). All tumors were staged on the basis of the pathologic tumor-node-metastasis classification of the International Union Against Cancer. A total of 20 frozen primary lung cancer tissues for RNA extraction were obtained as published earlier (Kato T, et al, Proc natl Acad Sci USA 1999;96:14783-8, Kikuchi T, et al, Oncoene 2003;22:2192-205, Taniwaki M, et al, Int J Oncol 2006;29:567-75). Formalin-fixed primary lung tumors and adjacent normal lung tissue samples used for immunostaining on tissue microarrays had been obtained from 331 patients undergoing curative surgery at Saitama Cancer Center (Saitama, Japan) (Ishikawa N, et al, Clin Cancer Res 2004;10:8363-70, Ishikawa N, et al, Cancer Res 2007;67:11601-11). To be eligible for this study, tumor samples were selected from patients who fulfilled all of the following criteria: (a) patients suffered primary NSCLC with histologically confirmed stage (only pT1 to pT3, pN0 to pN2, and pM0); (b) patients underwent curative surgery, but did not receive any preoperative treatment; (c) among them, NSCLC patients with positive lymph node metastasis (pN1, pN2) were treated with platinum-based adjuvant chemotherapies after surgical resection, whereas patients with pN0 did not receive adjuvant chemotherapies; and (d) patients whose clinical follow-up data were available. This study and the use of all clinical materials mentioned were approved by individual institutional ethics committees.
Bladder tissue samples and RNA preparation
Bladder tissue samples and RNA preparation were described previously (hayami S, et al, Int J Cancer 2010, Hayami S, et al, Mol Cancer 2010;9:59, Wallard MJ, et al, Br J Cancer 2006;94:569-77.). Briefly, 136 surgical specimens of primary urothelial carcinoma were collected, either at cystectomy or transurethral resection of bladder tumor (TURBT), and snap frozen in liquid nitrogen. 23 specimens of normal bladder urothelial tissue were collected from areas of macroscopically normal bladder urothelium in patients with no evidence of malignancy. Approximately 10,000 cells were microdissected from both stromal and epithelial/tumor compartments in each tissue. RNA was extracted using an RNeasy Micro Kit (QIAGEN, Crawley, UK). Areas of cancer or stroma containing significant inflammatory areas of tumor or stroma containing significant inflammatory cell infiltration were avoided to prevent contamination (Wallard MJ, et al, Br J Cancer 2006;94:569-77). Use of tissues for this study was approved by Cambridge shire Local Research Ethics Committee (Ref 03/018).
Expression profiling in cancer using cDNA microarrays
A genome-wide cDNA microarray with 36,864 cDNAs selected from UniGene database of the National Center for Biotechnology Information (NCBI) was established. This microarray system was constructed essentially as described previously (Kikuchi T, et al, Oncogene 2003;22:2192-205, Kitahara O, et al, Cancer Res 2001;61:3544-9, Nakamura T, et al, Oncogene 2004;23:2385-400). Briefly, the cDNAs were amplified by RT-PCR using poly (A)+ RNAs isolated from various human organs as templates; the lengths of the amplicons ranged from 200 to 1,100 bp, without any repetitive or poly (A) sequences. Many types of tumor and corresponding non-neoplastic tissues were prepared in 8-micrometer, as described previously (Kitahara O, et al, Cancer Res 2001;61:3544-9). A total of 30,000-40,000 cancer or noncancerous cells were collected selectively using the EZ cut system (SL Microtest GmbH, Germany) according to the manufacturer's protocol. Extraction of total RNA, T7-based amplification, and labeling of probes were performed as described previously ( Kitahara O, et al, Cancer Res 2001;61:3544-9). A measure of 2.5-microgram aliquots of twice-amplified RNA (aRNA) from each cancerous and noncancerous tissue was then labeled, respectively, with Cy3-dCTP or Cy5-dCTP.
Quantitative real-time PCR
136 bladder cancer and 23 normal bladder tissues were prepared in Cambridge Addenbrooke's Hospital. Specific primers for human GAPDH (housekeeping gene) and MCM7 were designed (primer sequences in Table 1). PCR reactions were performed using the LightCycler(registered trademark) 480 System (Roche Applied Science, Mannheim, Germany) following the manufacture's protocol.
Figure JPOXMLDOC01-appb-T000001
siRNA transfection and cell growth assay
siRNA oligonucleotide duplexes were purchased from SIGMA Genosys for targeting the human MCM7 transcripts. siEGFP and siNegative control (siNC), which consists of three different oligonucleotide duplexes, were used as control siRNAs. The siRNA sequences are described in Table 2. siRNA duplexes (100 nM final concentration) were transfected into lung and bladder cancer cell lines with Lipofectamine 2000 (Invitrogen) for 72 hours, and cell growth was examined using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan).
Figure JPOXMLDOC01-appb-T000002
Western blot analysis
Whole cell lysates were prepared from the cells with RIPA-like buffer, and total protein (10 microgram) was transferred to nitrocellulose membrane. The membrane was probed with anti-MCM7 antibody (141.2, Santa Cruz Biotechnology, Santa Cruz, CA). ACTB (I-19, Santa Cruz Biotechnology) was used to ensure equal loading and transfer of proteins. Protein bands were detected by incubating with horseradish peroxidase-conjugated antibodies (GE Healthcare, Little Chalfont, UK) and visualizing with Enhanced Chemiluminescence (GE Healthcare).
BrdU labeling and immuocytochemical analysis
BrdU labeling and immunocytochemistry were performed according to previously reported protocols (Hayami S, et al, Int J Cancer 2010, Hayami S, et al, Mol Cancer 2010;9:59.). A549, SBC5 and SW780 cells were incubated with appropriate media containing 2 microM BrdU (BD Biosciences, Franklin Lakes, NJ) for 20 min, and fixed and permeabilized with 100% methanol for 5 min at room temperature. The cells were washed with PBS, and then blocked by 3% BSA for 1 hour at 37 degrees C. Then, the cells were incubated with an anti-MCM7 antibody in 3% BSA overnight at 4 degrees C. After incubation with 1st antibody, the cells were reacted with Alexa Fluor 594-conjugated goat anti-mouse IgG for 1 hour at 37 degrees C in the blocking solution. They were then re-fixed, treated with 4 M HCl for 30 min at room temperature and incubated with FITC-conjugated anti-BrdU (BD Biosciences), diluted 1:300, for 1 hour at room temperature, followed by observation with confocal microscopy.
Immunohistochemical staining and tissue microarray
Immunohistochemical analysis was performed using a specific mouse-MCM7 antibody as described previously (Sato N, et al, Clin Cancer Res 2010;16:226-39). For clinical lung cancer tissues and liver tissue microarray, ENVISION+ kit/horseradish peroxidase (Dako, Glostrup, Denmark) was applied, whereas VECTASTAIN(registered trademark) ABC KIT (VECTOR LABORATORIES, Burlingame, CA) was used for bladder tissue microarray and normal tissue slides. Tumor tissue microarrays were constructed with 331 primary NSCLCs which had been obtained by a single institutional group (please see above) with an identical protocol to collect, fix, and preserve the tissues after resection (Callagy G, et al, Diagn Mol Pathol 2003;12:27-34, Callagy G, et al, J Pathol 2005;205:388-96, Chin SF, et al, Mol Pathol 2003;56:275-9.). Considering the histologic heterogeneity of individual tumors, a tissue area for sampling was selected based on visual alignment with the corresponding H&E-stained section on a slide. Three, four or five tissue cores (diameter, 0.6 mm; depth, 3-4 mm) taken from a donor tumor block were placed into a recipient paraffin block with a tissue microarrayer (Beecher Instruments, Sun Prairie, WI). A core of normal tissue was punched from each case, and 5-micrometer sections of the resulting microarray block were used for immunohistochemical analysis. Three independent investigators semiquantitatively assessed MCM7 positivity without prior knowledge of clinicopathologic data. Because the intensity of staining within each tumor tissue core was mostly homogeneous, the intensity of MCM7 staining was semiquantitatively evaluated using the following criteria: negative (no appreciable staining in tumor cells) and positive (brown staining appreciable in the nucleus of tumor cells). Cases were accepted as positive only if all reviewers independently defined them as such.
Statistical analysis
The Kruskal-Wallis test was used to examine the difference between several independent subgroups. Student's t-test or Mann-Whitney's U-test was used to analyze the difference between two independent subgroups. Survival curves were calculated from the date of surgery to the time of death related to NSCLC or to the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and for MCM7 expression; differences in survival times among patient subgroups were analyzed using the log-rank test. Univariate and multivariate analyses were done with the Cox proportional hazard regression model to determine associations between clinicopathological variables and cancer-related mortality. First, associations between death and possible prognostic factors were analyzed including age, gender, histology, pT classification and pN classification, taking into consideration one factor at a time. Second, multivariate Cox analysis was applied on backward (stepwise) procedures that always forced strong MCM7 expression into the model, along with any and all variables that satisfied an entry level of P < 0.05. As the model continued to add factors, independent factors did not exceed an exit level of P < 0.05.
EXAMPLE2:MCM7 expression is significantly high in lung cancer tissues and correlated with poor prognosis in NSCLC
It was previously reported that PRMT6, a type I arginine methytransferase, is involved in human carcinogenesis (Yoshimatsu M, et al, Int J Cancer 2010) and identified that MCM7 is a binding partner based on IP-MS analysis (data not shown). Intriguingly, quantitative real-time PCR showed that expression levels of MCM7 in 9 lung cancer tissues (6 NSCLC cases and 3 SCLC cases) were significantly higher than those in 11 normal tissues containing lung, brain, colon, esophagus, eye, liver, rectum, stomach, bladder and kidney (Fig. 1A).
Therefore, it was hypothesized that MCM7 can also be involved in human carcinogenesis. To validate these results, immunohistochemical analysis was conducted on tissue microarray containing tissue sections from 331 NSCLC patients, who had under gone surgical resection. Immunohistochemistry using an MCM7-specific antibody showed nuclear localization in cancer tissues, but nothing was detected in normal lung tissues (Fig. 1B). Importantly, specific MCM7 signals were not detected in normal brain, heart, lung, liver, pancreas, stomach, testis, kidney and bladder tissues (Fig. 1C), indicating that MCM7 may be specifically overexpressed in cancer tissues. Of 331 cases, MCM7 stained positively in 196 cases (61.1%) and negatively in 135 cases (38.9%; Table 3). Subsequently, the association of MCM7 expression with clinical outcomes,was analyzed and found that expression of MCM7 in NSCLC patients was significantly associated with male gender (P < 0.0001, Fisher's exact test; Table 3), non-ADC histology (P < 0.0001), presence of lymph node metastasis (pN1-2; P < 0.0001) and tumor-specific 5-year survival after the resection of primary tumors (P = 0.0055 by log-rank test; Fig. 1D). Then, an univariate analysis was applied to evaluate associations between patient prognosis and several factors including MCM7 expression, age, gender, histologic type (non-ADC versus ADC), pT stage (tumor size, T1 versus T2 + T3) and pN stage (node status, N0 versus N1+N2). All those parameters were significantly associated with poor prognosis (Table 4). However, multivariate analysis revealed that MCM7 status did not show the statistical significance as an independent prognostic factor for surgically treated NSCLC patients enrolled in this study, whereas age, pT and pN stages did so (Table 4). This result might be due to the preponderance of MCM7 expression up to pN stage.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
EXAMPLE3:MCM7 is overexpressed in bladder and various types of cancers
In addition to lung tissues, expression levels of MCM7 in bladder tissues were examined. Quantitative real-time PCR analysis using 23 normal bladder tissues, 124 bladder transitional cell carcinomas (TCCs) and 12 upper tract TCCs showed elevated mRNA levels of MCM7 in bladder and especially upper tract TCCs compared with normal bladder tissues (Fig. 2A). Although there were significant differences between normal bladder and bladder cancers of any pT stages, no significant differences were observed between each pT stages as well as tumor grades (Fig. 2B; data not shown). Subsequently, immunohistochemical analysis using a bladder tissue microarray revealed that MCM7 was up-regulated in bladder cancer tissues at the protein level (19/28, 67.9%; Fig. 2C). In the microarray expression analysis, elevated MCM7 expression in various types of cancers including lung, bladder and liver (Table 5) was confirmed, and tissue microarray immunohistochemical analysis showed the up-regulation of MCM7 in liver cancer tissues at the protein level (25/70, 35.7%; Fig. 2D).
Figure JPOXMLDOC01-appb-T000005
EXAMPLE4:MCM7 is required for cancer cell proliferation
In order to examine whether elevated expression of MCM7 plays a critical role in the proliferation of cancer cells, siRNA oligonucleotide duplexes were prepared to specifically suppress the expression of MCM7 (siMCM7#1, #2), and each of them were transfected into cancer cells. Firstly, the expression profile of MCM7 in normal and cancer cell lines was examined. Quantitative real-time PCR analysis revealed that MCM7 expression levels in cancer cells were significantly higher than those in normal human cells (Fig. 4). Knockdown of MCM7 in A549 cells and SBC5 cells was confirmed by immunoblotting and immunocytochemical analysis as shown in Fig. 3A and 3B. Immunocytochemical analysis also showed a decreased proportion of cells incorporating BrdU, which is an indicator of DNA synthesis (Fig. 3B) (Gratzner HG, et al, Science 1982;218:474-5.). Next, to confirm this abrogation on the growth of cancer cells, cell growth assays were performed. As shown in Fig. 3C, knockdown of MCM7 significantly suppressed the growth of cancer cells, with a similar result obtained for the bladder cancer cell line, SW780 (Fig. 5A-C). These data indicate that MCM7 plays an important role in the proliferation of lung and bladder cancers, and inactivation of this gene should be a promising therapeutic target in various types of cancer.
Discussion
Cancer-related death is on the rise in most countries, and it is a serious public health problem. Among all types of cancer, lung cancer is the leading cause of death from cancer in the United States and Japan, and the median survival of advanced NSCLC patients treated with standard chemotherapy still remains as short as about 8 months (Sawabata N, et al, Nihon Kokyuki Gakkai Zasshi 2010;48:333-44, Jemal A, et al, CA Cancer J Clin 2008;58:71-96, Schiller JH, et al, N Engl J Med 2002;346:92-8.). It is true that novel molecular-targeting agents such as cetuximab and bevacizumab have been developed and proven to be efficacious, but their adverse effects and limited application for some patients, engender a drive towards novel molecular-targeting agents (Mendelsohn J, et al, J Clin Oncol 2002;20:1S-13S, Hurwitz H, et al, N Engl J Med 2004;350:22335-42, Horn L, et al, Clin Cancer Res 2009;15:5040-8, Pirker R, et al, Lancet 2009;383:1525-31, Sandler A, et al, N Engl J Med 2006;355:2542-50.).
Six of the MCM proteins, MCM2-MCM7, form complexes that participate in initiation and elongation steps of DNA replication (Lei M, et al, J Cell Sci 2001;114:1447-54.). They share a conserved 200-amino acid nucleotide-binding region and form different subcomplexes (dimers, trimers and a hexamer) (Koonin EV, et al, Nucleic Acids Res 1993;21:2541-7.). MCM4-MCM6-MCM7 trimers and hexamers (MCM2-MCM7) have ATPase and DNA helicase activities in vitro ( Lei M, et al, J Cell Sci 2001;114:1447-54.). MCM proteins are associated with chromatin in late telophase and at the beginning of the G1 phase of the cell cycle (Dimitrova DS, et al, J Cell Sci 2002;115:51-9.). During S phase, MCM proteins are released from origins of replication after initiation of DNA replication and subsequently move with replication folks where they are thought to function as a DNA helicase. Mechanisms that assure the replication of DNA only once per cycle involve the release MCM proteins from chromatin after firing of the origins of replication and prevent the reloading of MCM proteins on chromatin until telophase. This evidence indicates that MCM proteins are one of the essential regulators in DNA replication, and indeed, it has already been reported that dysregulation of some MCM proteins are apparent in human disease, including cancer (Fujioka S, et al, Lung Cancer 2009;65:223-9, Ramnath N, et al, J Clin 2009;65:223-9, Ramnath N, et al, J Clin Oncol 2001;19:4259-66, Ishimi Y, et al, Eur J Biochem 2003;270:1089-101, Honeycutt KA, et al, Oncogene 2006;25:4027-32, Shohet JM, et al, Cancer Res 2002;62:1123-8, Ren B, et al, Oncogene 2006;25:1090-8.)In this study, it was demonstrated that in various cancer tissues, expression levels of MCM7 were significantly high at both RNA and protein levels whereas MCM7 expression in various normal tissues was hardly detected. MCM7 could therefore be a good indicator enabling us to predict prognosis of NSCLC patients and to conduct a more intensive follow-up according to MCM7 expression status of resected specimens. In addition, it was demonstrated a critical role for MCM7 in the growth regulation of cancer cells. Examined effects of heliquinomycin, an inhibitor of MCM4/6/7 DNA helicase, on the growth of cancer cells and it effectively suppressed the growth of cancer cells in a dose dependent manner (Fig. 6). Anti-cancer drugs targeting DNA helicases are now in development (Sharma S, et al, Curr Med Chem Anticancer Agents 2005;5:183-99.). Further validation of present results may affirm the importance of this protein as a promising target for anti-cancer therapy and as a prognostic marker of various cancers.
As described herein, gene-expression analysis of cancers using the genome-wide cDNA microarray resulted in the identification of specific genes having utility as a target for cancer prevention and therapy. Based on the differentially expression of MCM7 gene, the present invention provides a molecular diagnostic marker for diagnosing or detecting cancers as well as monitoring, determining and/or assessing the prognosis of cancer, in particular, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor.
The data provided herein add to a comprehensive understanding of cancers, facilitate development of novel diagnostic strategies, and provide clues for identification of a molecular target for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of tumorigenesis, and provides indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of cancers.
As demonstrated herein, cell growth is suppressed by double-stranded molecules that specifically target the MCM7 gene. Thus, these novel double-stranded molecules are useful as anti-cancer pharmaceuticals.
The expression of the MCM7 gene is markedly elevated in cancer, specifically lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor, as compared to normal organs. Accordingly, this gene can be conveniently used as a diagnostic marker for cancer, in particular, lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor , and the proteins encoded thereby find utility in diagnostic assays for cancer.
Furthermore, the methods described herein are also useful in diagnosis of cancer, including lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor
Moreover, the present invention provides new therapeutic approaches for treating cancer, particularly lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma or soft tissue tumor. The MCM7 gene is a useful target for the development of anti-cancer pharmaceuticals.
All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.
Furthermore, while the invention has been described in detail and with reference to specific embodiments thereof, it is to be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the invention. Thus, the invention is intended to be defined not by the above description, but by the following claims and their equivalents.

Claims (32)

  1. A method for detecting or diagnosing cancer or a predisposition for developing cancer in a subject, comprising the step of determining an expression level of an MCM7 gene in a subject-derived biological sample, wherein an increase in said expression level as compared to a normal control level of said gene indicates that said subject suffers from or is at a risk of developing cancer, wherein said expression level is determined by a method selected from a group consisting of:
    (a) detecting mRNA of an MCM7 gene;
    (b) detecting a protein encoded by an MCM7 gene; and
    (c) detecting a biological activity of a protein encoded by an MCM7 gene.
  2. The method of claim 1, wherein said MCM7 gene expression level is at least 10% greater than the normal control level.
  3. The method of claim 1 or 2, wherein the subject-derived biological sample is a biopsy, saliva, sputum, blood, serum, plasma, pleural effusion or urine sample.
  4. The method of any one of claims 1 to 3, wherein the biological activity of the protein encoded by the MCM7 gene is cell proliferative activity.
  5. A method for assessing prognosis of a subject with cancer, wherein the method comprises steps of:
    (a) detecting an expression level of MCM7 gene in a subject-derived biological sample;
    (b) comparing the detected expression level to a control level; and
    (c) determining prognosis of the subject based on the comparison of (b).
  6. The method of claim 5, wherein the control level is a good prognosis control level and an increase of the expression level compared to the control level indicates poor prognosis.
  7. The method of claim 6, wherein the MCM7 gene expression level is at least 10% greater than said control level.
  8. A method of screening for a candidate substance for either or both of treating and preventing cancer, wherein said method comprises steps of:
    (a) contacting a test substance with an MCM7 polypeptide or a fragment thereof;
    (b) detecting binding between the polypeptide or fragment and the test substance; and
    (c) selecting a test substance that binds to the polypeptide or fragment as a candidate substance for either or both of treating and preventing cancer.
  9. A method of screening for a candidate substance for either or both of treating and preventing cancer, wherein said method comprises steps of:
    (a) contacting a test substance with an MCM7 polypeptide or a fragment thereof;
    (b) detecting a biological activity of the polypeptide or fragment;
    (c) comparing the biological activity of the polypeptide or fragment with the biological activity detected in the absence of the test substance; and
    (d) selecting a test substance that suppresses the biological activity of the polypeptide as a candidate substance for either or both of treating and preventing cancer.
  10. The method of claim 9, wherein the biological activity of said MCM7 polypeptide or fragment is cell proliferative activity.
  11. A method of screening for a candidate substance for either or both of treating and preventing cancer, wherein said method comprises steps of:
    (a) contacting a test substance with a cell expressing an MCM7 gene;
    (b) detecting an expression level of the MCM7 gene;
    (c) comparing the expression level with the expression level detected in the absence of the test substance; and
    (d) selecting a test substance that reduces the expression level as a candidate substance for either or both of treating and preventing cancer.
  12. A method of screening for a candidate substance for either or both of treating and preventing cancer, wherein said method comprises steps of:
    (a) contacting a test substance with a cell introduced with a vector that comprises a transcriptional regulatory region of an MCM7 gene and a reporter gene expressed under control of the transcriptional regulatory region;
    (b) measuring an expression level or activity of said reporter gene;
    (c) comparing the expression level or activity with the expression level or activity detected in the absence of the test substance; and
    (d) selecting a test substance that reduces the expression level or activity as a candidate substance for either or both of treating and preventing cancer.
  13. A method of screening for a candidate substance for either or both of treating and preventing cancer, wherein said method comprises steps of:
    (a) contacting a test substance with an MCM protein complex ;
    (b) detecting a biological activity of the complex;
    (c) comparing the biological activity of the complex with the biological activity detected in the absence of the test substance; and
    (d) selecting a test substance that suppresses the biological activity of the complex as candidate substance for either or both of treating and preventing cancer.
  14. The method of claim 13, wherein the biological activity of said MCM protein complex is DNA helicase activity.
  15. An isolated double-stranded molecule that, when introduced into a cell expressing an MCM7 gene, inhibits expression of the gene, wherein said double-stranded molecule comprises a sense strand and an antisense strand, further wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 13 and 15, and the antisense strand comprises a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule.
  16. The double-stranded molecule of claim 15, wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.
  17. The double-stranded molecule of claim 15 or 16, wherein said double-stranded molecule is a single polynucleotide construct comprising the sense strand and the antisense strand linked via a single-strand.
  18. The double-stranded molecule of claim 17, wherein said double-stranded molecule has a general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A], wherein [A] is a sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 13 and 15, [B] is a single-strand and consists of 3 to 23 nucleotides, and [A'] is an antisense strand comprising a nucleotide sequence complementary to the target sequence selected from the group consisting of SEQ ID NO: 13 and 15.
  19. A vector encoding the double-stranded molecule of any one of claims 15 to 18.
  20. Vectors comprising each of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises a nucleotide sequence corresponding to SEQ ID NO: 13 or 15, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing MCM7 gene, inhibit the cell proliferation.
  21. A method of either or both of the treatment and prevention of cancer in a subject, wherein said method comprises the step of administering to said subject a pharmaceutically effective amount of a double-stranded molecule against an MCM7 gene or a vector encoding said double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell expressing MCM7 gene, inhibits the expression of the MCM7 gene.
  22. The method of claim 21, wherein the double-stranded molecule is that of any one of claims 15 to 18.
  23. The method of claim 21, wherein the vector is that of claim 19 or 20.
  24. The method of any one of claims 1 to 14 or 21 or 22, wherein the cancer is selected from the group consisting of lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  25. A composition formulated for either or both of the treatment and prevention of cancer, which comprises a pharmaceutically effective amount of a double-stranded molecule against an MCM7 gene or a vector encoding said double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell expressing MCM7 gene, inhibits the expression of the MCM7 gene, and a pharmaceutically acceptable carrier.
  26. The composition of claim 25, wherein the double-stranded molecule is that of any one of claims 15 to 18.
  27. The composition of claim 25, wherein the vector is that of claim 19 or 20.
  28. The composition of any one of claims 25 to 27, wherein the cancer is selected from the group consisting of lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
  29. A kit for diagnosing or detecting cancer or a predisposition therefor, or assessing or determining prognosis of a subject diagnosed with cancer, wherein said kit comprises a reagent for detecting a transcription or translation product of an MCM7 gene.
  30. The kit of claim 29, wherein the reagent comprises a nucleic acid that binds to a transcription product of MCM7 gene or an antibody that binds to a translation product of an MCM7 gene.
  31. A reagent for diagnosing or detecting cancer or a predisposition therefor, or assessing or determining prognosis of a subject diagnosed with cancer, said reagent comprising a nucleic acid that binds to a transcription product of MCM7 gene or an antibody that binds to a translation product of an MCM7 gene.
  32. The kit of claim 29 or 30, or the reagent of claim 31, wherein the cancer is selected from the group consisting of lung cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, testicular cancer, acute myeloid leukemia, osteosarcoma and soft tissue tumor.
PCT/JP2011/007285 2010-12-28 2011-12-27 Mcm7 as a target gene for cancer therapy and diagnosis WO2012090479A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201061427563P 2010-12-28 2010-12-28
US61/427,563 2010-12-28

Publications (1)

Publication Number Publication Date
WO2012090479A1 true WO2012090479A1 (en) 2012-07-05

Family

ID=46382615

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2011/007285 WO2012090479A1 (en) 2010-12-28 2011-12-27 Mcm7 as a target gene for cancer therapy and diagnosis

Country Status (1)

Country Link
WO (1) WO2012090479A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021037265A1 (en) * 2019-08-30 2021-03-04 恩智（广州）医药科技有限公司 Sirna capable of inhibiting expression of mcm7 gene, composition, and application thereof
US11391744B2 (en) 2015-06-08 2022-07-19 Arquer Diagnostic Limited Methods and kits
US11519916B2 (en) 2015-06-08 2022-12-06 Arquer Diagnostics Limited Methods for analysing a urine sample

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999021014A1 (en) * 1997-10-21 1999-04-29 Cancer Research Campaign Technology Limited Determination of cellular growth abnormality
WO2005095964A2 (en) * 2004-03-24 2005-10-13 Tripath Imaging, Inc. Methods and compositions for the detection of cervical disease
WO2005097189A1 (en) * 2004-04-09 2005-10-20 Genecare Research Institute Co., Ltd. Carcinoma cell-specific apoptosis inducing agent targeting gene relevant to chromosome stabilization
EP1916301A1 (en) * 2005-08-11 2008-04-30 Banyu Pharmaceutical Co., Ltd. Method for evaluating compound using molecule on the rb pathway as index and molecular diagnostic method
WO2009045115A1 (en) * 2007-10-05 2009-04-09 Pacific Edge Biotechnology Ltd Proliferation signature and prognosis for gastrointestinal cancer
WO2009146545A1 (en) * 2008-06-05 2009-12-10 University Health Network Compositions and methods for classifying lung cancer and prognosing lung cancer survival

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999021014A1 (en) * 1997-10-21 1999-04-29 Cancer Research Campaign Technology Limited Determination of cellular growth abnormality
WO2005095964A2 (en) * 2004-03-24 2005-10-13 Tripath Imaging, Inc. Methods and compositions for the detection of cervical disease
WO2005097189A1 (en) * 2004-04-09 2005-10-20 Genecare Research Institute Co., Ltd. Carcinoma cell-specific apoptosis inducing agent targeting gene relevant to chromosome stabilization
EP1916301A1 (en) * 2005-08-11 2008-04-30 Banyu Pharmaceutical Co., Ltd. Method for evaluating compound using molecule on the rb pathway as index and molecular diagnostic method
WO2009045115A1 (en) * 2007-10-05 2009-04-09 Pacific Edge Biotechnology Ltd Proliferation signature and prognosis for gastrointestinal cancer
WO2009146545A1 (en) * 2008-06-05 2009-12-10 University Health Network Compositions and methods for classifying lung cancer and prognosing lung cancer survival

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FUJIOKA S. ET AL.: "Expression of minichromosome maintenance 7 (MCM7) in small lung adenocarcinomas (pTl): Prognostic implication.", LUNG CANCER, vol. 65, 2009, pages 223 - 229 *
NISHIHARA K. ET AL.: "Minichromosome maintenance protein 7 in colorectal cancer: Implication of prognositic significance.", INTERNATIONAL JOURNAL OF ONCOLOGY, vol. 33, 2008, pages 245 - 251 *
SAYDAM O. ET AL.: "Comperative protein profiling reveals Minichromosome Maintenance (MCM) Proteins as novel potential tumor markers for meningiomas.", JOURNAL OF PROTEOME RESEARCH, vol. 9, 2010, pages 485 - 494 *
TOYOKAWA G. ET AL.: "Minichromosome Maintenance Protein 7 is a potential therapeutic target in human cancer and a novel prognostic marker of non-small cell lung cancer.", MOLECULAR CANCER, vol. 10, 2011, pages 65 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11391744B2 (en) 2015-06-08 2022-07-19 Arquer Diagnostic Limited Methods and kits
US11519916B2 (en) 2015-06-08 2022-12-06 Arquer Diagnostics Limited Methods for analysing a urine sample
WO2021037265A1 (en) * 2019-08-30 2021-03-04 恩智（广州）医药科技有限公司 Sirna capable of inhibiting expression of mcm7 gene, composition, and application thereof

Similar Documents

Publication Publication Date Title
WO2012144220A1 (en) Ezh2 as target gene for cancer therapy and diagnosis
WO2011096211A1 (en) Whsc1 and whsc1l1 for target genes of cancer therapy and diagnosis
WO2011096210A1 (en) Prmt1 and prmt6 for target genes of cancer therapy and diagnosis
JP5764822B2 (en) PRMT1 as a target gene for cancer treatment and diagnosis
WO2006085684A9 (en) Method of diagnosing bladder cancer
WO2010095364A1 (en) Jarid1b for target gene of cancer therapy and diagnosis
JP2012501167A (en) Pancreatic cancer-related gene TTLL4
WO2012090479A1 (en) Mcm7 as a target gene for cancer therapy and diagnosis
WO2011161960A1 (en) C1orf59 for target genes of cancer therapy and diagnosis
US20110262463A1 (en) Syngr4 for target genes of cancer therapy and diagnosis
US20110160288A1 (en) Oip5 as a target gene for cancer therapy and diagnosis
WO2011024433A1 (en) Lung and esophageal cancer related gene adamts18
WO2010023837A1 (en) Breast cancer related gene rqcd1
WO2010023854A1 (en) Cancer related gene, lgn/gpsm2
WO2011024428A1 (en) Breast cancer related gene c12orf32
JP2010500004A (en) Prostate cancer-related gene STYK1
WO2011021386A1 (en) Cstf2 for target genes of lung cancer therapy and diagnosis
WO2011018898A1 (en) Cdc45l as tumor marker and therapeutic target for cancer
US20130203625A1 (en) Suv420h1 and suv420h2 as target genes for cancer therapy and diagnosis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11854119

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11854119

Country of ref document: EP

Kind code of ref document: A1