US20130236891A1 - Method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells - Google Patents

Method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells Download PDF

Info

Publication number
US20130236891A1
US20130236891A1 US13/817,763 US201113817763A US2013236891A1 US 20130236891 A1 US20130236891 A1 US 20130236891A1 US 201113817763 A US201113817763 A US 201113817763A US 2013236891 A1 US2013236891 A1 US 2013236891A1
Authority
US
United States
Prior art keywords
rpn2
cancer
cells
stem cells
cancer stem
Prior art date
Legal status (The legal status 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 status listed.)
Abandoned
Application number
US13/817,763
Other languages
English (en)
Inventor
Takahiro Ochiya
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
NATIONAL CANCER CENTER
3D Matrix Ltd
Original Assignee
3D Matrix Ltd
National Cancer Center Research Institute
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 3D Matrix Ltd, National Cancer Center Research Institute filed Critical 3D Matrix Ltd
Assigned to 3-D MATRIX, LTD., NATIONAL CANCER CENTER RESEARCH INSTITUTE reassignment 3-D MATRIX, LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: OCHIYA, TAKAHIRO
Publication of US20130236891A1 publication Critical patent/US20130236891A1/en
Assigned to NATIONAL CANCER CENTER reassignment NATIONAL CANCER CENTER CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: NATIONAL CANCER CENTER RESEARCH INSTITUTE
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N15/1137—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 against enzymes
    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/99—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • C12Y204/99018—Dolichyl-diphosphooligosaccharide—protein glycotransferase (2.4.99.18)
    • 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
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • 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
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91091—Glycosyltransferases (2.4)
    • G01N2333/91097—Hexosyltransferases (general) (2.4.1)

Definitions

  • the invention relates to a method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells through targeting the RPN2 gene.
  • cancer stem cells or tumor-initiating cells.
  • cancer stem cells When cancer stem cells are targeted, it is expected to be able to treat and prevent the development, metastasis, and recurrence of cancer.
  • markers defining cancer stem cells are unknown because the molecular basis of cancer stem cell phenotype remains largely unclear.
  • no method has been established for treatment or prevention through targeting cancer stem cells.
  • RPN2 ribophorin II
  • OST oligosaccharide transferase
  • the objective of the invention is to provide a method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells.
  • the inventors demonstrate that RPN2 was highly expressed in the cancer stem cell fraction of breast cancer cells and that RPN2 knockdown inhibited the ability of colonization and invasion of cancer stem cells in vitro. Further analysis demonstrated that RPN2 knockdown reduced tumor formation and suppressed the ability of metastasis in vivo. Global proteomics analysis demonstrated that RPN2 knockdown altered the expression of 14-3-3 ⁇ , which is known to regulate the TGF- ⁇ /Smad pathway. Thus, the inventors provide genetic and biological evidence that RPN2 can be important in maintaining the phenotype of cancer stem cells and that RPN2 can be a promising target for cancer stem cell therapy.
  • the present invention relates to
  • a pharmaceutical composition comprising an RPN2 inhibitor for the treatment or prevention of cancer containing or derived from cancer stem cells; [2] a pharmaceutical composition according to [1], wherein the cancer stem cells carry a mutated p53 gene; [3] a pharmaceutical composition according to [1] or [2], wherein the RPN2 inhibitor is an siRNA against the RPN2 gene; [4] a method for detecting cancer stem cells, comprising determining the presence or level of RPN2 expression; and [5] a method according to [4] further comprising detecting a mutated p53 gene.
  • the invention provides a method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells through targeting the RPN2 gene.
  • FIG. 1 (A) provides unequal division of CD44 + CD24 ⁇ cancer stem cells.
  • FIG. 1 (B) provides RPN2 expression analysis by RT-PCR.
  • FIG. 2 provides effects of RPN2 knockdown on the number of CD44 + CD24 ⁇ cancer stem cells.
  • FIG. 3 (A) provides effects of RPN2 knockdown on colony forming activity.
  • FIG. 3 (B) provides Effects of RPN2 knockdown on the number of colonies formed.
  • FIG. 4 provides effects of RPN2 knockdown on tumorigenicity.
  • FIG. 5 provides effects of RPN2 knockdown on tumor metastasis.
  • FIG. 6 provides effects of RPN2 knockdown on lethality.
  • FIG. 7 (A) provides Effects of RPN2 knockdown on mutated p53 expression.
  • FIG. 7 (B) provides effects of RPN2 knockdown on E-cadherin expression.
  • FIG. 8 provides effects of RPN2 knockdown on 14-3-3 ⁇ expression.
  • FIG. 9 provides immunohistochemical staining of tumors formed by transplanting MM231-LN cells into an animal.
  • FIG. 10 provides RPN2 and mutated p53 expressions in breast cancer tissue of a human patient with breast cancer.
  • FIG. 11 provides tumor apoptosis examined by TUNEL assay. Tumor apoptosis of breast cancer was demonstrated to be strongly induced by the intratumoral delivery of RPN2-siRNA/A 6 K on day 3 after administration.
  • FIG. 12 provides RPN2 knockdown analysis in canine breast cancer.
  • the invention provides a pharmaceutical composition containing an RPN2 inhibitor for the treatment or prevention of cancer containing or derived from cancer stem cells.
  • the invention provides a method for the treatment or prevention of cancer containing or derived from cancer stem cells, comprising administering the pharmaceutical composition of the invention to a subject.
  • cancer stem cells refers to cancer cells having pluripotency and self-renewal ability (also referred to herein as self-renewal and differentiation ability).
  • Cancer cells include any cancer cells, such as breast, stomach, colorectal, lung, prostate, and hematopoietic cancer cells.
  • cancer stem cells may be resistant to anticancer agents (drug resistance) and have an ability to invade surrounding tissues and/or metastasize to distant sites in the body (invasion and metastasis ability). It is considered that the development, metastasis, and recurrence of cancer could be treated and prevented by targeting cancer stem cells.
  • the invention is useful for such treatment and prevention of cancer through targeting cancer stem cells.
  • the cancer stem cells have an increased expression level of ribophorin II (RPN2).
  • RPN2 is one of the components (subunit) of the oligosaccharide transferase (OST) complex, which exists in the rough endoplasmic reticulum and functions to add an N-linked sugar chain to a nascent polypeptide chain.
  • Human RPN2 is a basic membrane protein consisting of 631 amino acids. The sources of the RPN2 include, but are not limited to, for example, animals, preferably mammals, more preferably primates, and even more preferably humans.
  • RPN2 gene is a gene that encodes RPN2. The base sequence of the human RPN2 gene is shown in SEQ ID NO: 1.
  • shRNA short hairpin RNA
  • cancer stem cells can have self-renewal and differentiation ability, drug resistance, and invasion and metastasis ability.
  • RPN2 involved in all of these will serve as a marker of cancer stem cells.
  • targeting RPN2 may allow the treatment and prevention of cancers containing or derived from cancer stem cells.
  • cancer stem cells carry a mutated p53 gene.
  • P53 is a tumor suppressor gene product. Mutations in the p53 gene have been found in many human cancers (Adorn°, M. et al., Cell, 137: 87-98 (2009); Wang, S. P. et al., Nat. Cell Biol., 11: 694-704 (2009); Muller, P. A. et al., Cell, 139: 1327-1341 (2009); Morton, J. P. et al., Proc. Natl. Acad. Sci. USA, 107:246-251 (2010)). Mutations in the p53 gene preferably include, but are not limited to, substituted point mutations (missense mutations), which cause no frameshift mutation, or deletion mutations (codon deletion mutations).
  • Cancer cells exhibit a phenomenon referred to as epithelial-mesenchymal transition (EMT).
  • EMT epithelial-mesenchymal transition
  • the expression of E-cadherin involved in cell adhesion is reduced upon EMT, resulting in the invasion or metastasis of cancer cells.
  • RPN2 reduces 14-3-3 ⁇ expression to stabilize mutated p53 and cause EMT in cancer cells, resulting in the invasion or metastasis of cancer cells.
  • Mutated P53 is involved in the self-renewal and differentiation and invasion and metastasis abilities of cancer cells. Many studies have been conducted on p53. However, no report has been published on the successful treatment or prevention of cancer through targeting p53. RPN2 is involved in self-renewal and differentiation and invasion and metastasis abilities, besides drug resistance. Thus, inhibiting RPN2 not only eliminates the drug resistance of cancer cells, but also inhibits the self-renewal and differentiation and invasion and metastasis abilities of cancer cells through inhibiting the effects of mutated p53 upstream. Detecting the presence of mutated p53, as well as RPN2 expression, allows more accurate identification of cancer cells having mutated p53, for which inhibiting RPN2 is effective. In addition, inhibiting RPN2 allows more effective treatment and prevention of cancers associated with mutated p53 than targeting the mutated p53.
  • RPN2 inhibitor refers to any substance that inhibits RPN2 gene expression or the effects of RPN2 gene product. RPN2 is little expressed in normal tissues, excluding the placenta. Thus, RPN2 inhibitors have no substantial effects on cells other than cancer cells in subjects, excluding pregnant women, and are useful as specific therapeutics for cancer without adverse effects. Subjects preferably include, but are not limited to, for example, animals, mammals, more preferably primates, and even more preferably humans.
  • substances that inhibit RPN2 gene transcription those that bind to or degrade RPN2 transcripts, and those that bind to the RPN2 protein can be used as RPN2 inhibitors.
  • substances that bind to the RPN2 protein include an anti-RPN2 antibody or fragments thereof (Fab, F(ab′)2, etc.), and other components that bind to RPN2 in the oligosaccharide transferase (OST) complex.
  • substances that bind to or degrade RPN2 transcripts include antisense RNA, ribozymes, small interfering RNA (siRNA), and micro RNA (miRNA) against the RPN2 gene.
  • RNA interference refers to a phenomenon in which gene expression is suppressed by a double-stranded (ds) RNA molecule in a sequence-specific manner.
  • ds double-stranded
  • RNA interference results from target mRNA cleavage by siRNA, gene silencing through heterochromatin formation in a target DNA region by siRNA, and translational and transcriptional repression and mRNA degradation by miRNA.
  • siRNA is preferably used in the present invention because its sequence can be designed based on the target RPN2 gene sequence and can be artificially synthesized.
  • siRNA can be obtained by any method known in the art.
  • siRNA can be chemically synthesized by the phosphoramidite method, which is also employed for the chemical synthesis of DNA, through the sequential condensation reaction of a single base at a time towards the 5′ to 3′ end.
  • the hydroxyl groups of the 2′ ends of individual ribonucleotides are protected to prevent the degradation by RNase during synthesis.
  • protecting groups include 2′-O-t-butyldimethylsilyl (2′-tBDMS), 2′-O-(triisopropylsilyloxy)methyl (2′-TOM), and 5′-silyl-2′-acetoxyethoxy (2′-ACE) groups.
  • siRNA against the RPN2 gene has a sequence corresponding to a predetermined sequence of the RPN2 gene, i.e., a sequence corresponding to a part of a target mRNA sequence.
  • dsRNA sequence A
  • SEQ ID NOS: 2 sense strand
  • 3 antisense strand
  • siRNA siRNA against the RPN2 gene
  • This dsRNA has 2-base overhangs at the 3′ ends of each strand.
  • the double-stranded region is 19 bases in length.
  • SEQ ID NOS: 4-25 show the sequences of the sense and antisense strands of the siRNAs (sequences B-L) against the RPN2 gene, disclosed in Patent literature 1. These pairs (dsRNAs) can be used as RPN2 inhibitors in the present invention.
  • miRNA is a small RNA molecule that encodes no protein.
  • miRNA is transcribed into nucleotides of several hundred to several thousand bases and eventually undergoes processing into nucleotide dimers of 19-24 bases to suppress the gene expression through the translational repression, degradation, and transcriptional regulation of mRNA having a nucleotide sequence complementary to the miRNA.
  • RPN2 expression is also regulated by multiple miRNAs.
  • Such miRNAs can be artificially synthesized to be used in the present invention as RPN2 inhibitors in order to suppress RPN2 gene expression.
  • Known miRNA sequences that may inhibit RPN2 gene expression can be retrieved from public databases (e.g., TargetScan Release3.1).
  • the pharmaceutical composition of the present invention may be administered through either systemic or local administration.
  • the route of administration may be any route, such as intravenous, subcutaneous, intraperitoneal, intramuscular, and intranasal routes.
  • the pharmaceutical composition of the present invention may further contain any ingredients used in the field of drug formulation, such as excipients, diluents, and stabilizers.
  • an RPN2 inhibitor is a protein like an antibody
  • the pharmaceutical composition may further contain ingredients commonly used in the field of protein formulation.
  • an RPN2 inhibitor is a nucleic acid like siRNA, any substance (e.g., liposome) for introducing a nucleic acid may be contained.
  • the transfection agent containing a peptide surfactant described in WO 2010/024262, can be suitably used for the present invention, because it shows low toxicity, high efficiency for a target gene to reach an affected area, and high efficiency of target gene suppression and, therefore, can be systemically administered.
  • the amount of an RPN2 inhibitor, contained in the pharmaceutical composition of the present invention varies with administration methods, tumor types and sizes, patient's conditions, and concomitant drugs, and can be appropriately determined by those skilled in the art.
  • an siRNA is used as an RPN2 inhibitor
  • the amount is desirably 1-10 nmol/kg for local administration and 2-50 nmol/kg for systemic administration.
  • the present invention provides a method of detecting cancer stem cells, comprising determining the presence or level of RPN2 expression.
  • RPN2 exists in the cytoplasm.
  • an extract containing RNA and proteins is prepared from cells or tissues obtained from a subject.
  • transcripts RPN2 mRNA
  • translation products RPN2 protein
  • any methods known in the art such as Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR)
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • any methods known in the art for example, immunological methods using an anti-RPN2 antibody (Western blotting and ELISA), can be employed.
  • RPN2 is little expressed in normal tissues, excluding the placenta.
  • the presence of RPN2 expression or high-level RPN2 expression indicates the involvement of RPN2 in cancer.
  • treatments with the pharmaceutical composition of the present invention, containing an RPN2 inhibitor are effective.
  • the detection method of the present invention further comprises detecting a mutated p53 gene.
  • a mutated p53 gene can be detected by any detection and analysis methods of nucleic acid using hybridization, electrophoresis, nucleic acid amplification, and sequencing known in the art. As described above, when a mutated p53 exists in cancer cells expressing RPN2, cancers associated with the mutated p53 can be effectively treated and prevented by inhibiting the RPN2. Thus, the detection method of the present invention is useful as a diagnosis method for determining effective treatment and prevention methods.
  • Human breast cancer cell line MCF7-ADR was divided into two cell fractions, CD44 + CD24 ⁇ and CD44 + CD24 + , and were cultured for seven days. Only the CD44 + CD24 ⁇ fraction had an unequal division, a property of cancer stem cells (FIG. 1 A).
  • RPN2 expression was analyzed by RT-PCR.
  • RPN2 expression in CD44 + CD24 ⁇ was increased about 20-fold as compared with that in CD44 + CD24 + (non-cancer stem cells) ( FIG. 1B ).
  • MCF7 Human breast cancer cells with RPN2 expression constitutively knocked down was generated using the shRPN2 vector.
  • MCF7 is the parent cell line of MCF7-ADR (drug-resistant cell line), i.e., hormone receptor-positive and drug-sensitive non-malignant breast cancer cells.
  • MM231-LN is a hormone receptor-negative, highly metastatic and highly malignant cell line.
  • CD44 + CD24 ⁇ cancer stem cells were counted.
  • the numbers of CD44 + CD24 ⁇ cancer stem cells were significantly reduced for MCF7-ADR and MM231-LN cells introduced with the shRPN2 vector, as compared with those introduced with a control vector (shNC) ( FIG. 2 ).
  • FIG. 3A upper
  • MM231-LN-shNC MM231-LN cells formed many colonies.
  • the colony-forming ability was significantly suppressed where the shRPN2 vector was introduced ( FIG. 3A , lower, MM231-LN-shRPN2).
  • FIG. 3B shows the number of colonies formed.
  • cancer stem cells Another characteristic of cancer stem cells is that even a small number of cells can form an established tumor when transplanted into an animal.
  • MCF7-ADR and MM231-LN two human breast cancer cell lines, MCF7-ADR and MM231-LN, with (shRPN2) or without (shNC)RPN2 knockdown.
  • the cells introduced with the shRPN2 vector were transplanted into the right side of the back of a mouse (6-week-old female NOD-Scid mouse), while those introduced with the shNC vector were transplanted into the left side.
  • the numbers of cells transplanted were 1 ⁇ 10 4 cells/site for MCF7-ADR and 1 ⁇ 10 2 cells/site for MM231-LN.
  • MM231-LN cell is a highly malignant and highly metastatic one. This cell metastasizes to the lymph nodes under the armpit and chest, resulting in 100% lethality, when transplanted into the mouse mammary gland.
  • cells introduced with the shRPN2 vector were compared with those introduced with the shNC vector. As a result, lymph node metastasis was significantly suppressed for the group of shRPN2 vector ( FIG. 5 ). The results are summarized in Table 2.
  • mice transplanted with 1 ⁇ 10 2 or 1 ⁇ 10 3 cells for the shNC group become lethal, while all the mice transplanted with 1 ⁇ 10 2 or 1 ⁇ 10 3 cells for the shRPN2 group survived ( FIG. 6 ).
  • the vertical and horizontal axes represent metastasis-free survival (%) and time (days), respectively.
  • FIG. 7B shows the results for shNC.
  • FIG. 7B (middle) indicates the results for shRPN2.
  • FIG. 7B (right) indicates E-cadherin-positive cell rates (%).
  • E-cadherin expression disappears when cancer cells exhibit epithelial-mesenchymal transition (EMT). It is said that cancer cells with reduced E-cadherin expression are more likely to metastasize. Knocking down RPN2 expression in cells with high RPN2 expression using shRPN2 increased the E-cadherin expression. This fact supports the fact that PRN2 induces EMT.
  • MM231-LN cells were transplanted into an animal.
  • the tumor formed was examined by immunohistochemical staining using three colors: DAPI for nuclear-specific staining (blue), anti-RPN2 antibody (green), and anti-mutated p53 antibody (red) ( FIG. 9 , left).
  • the staining patterns of RPN2-positive and mutated p53-positive cells were consistent.
  • RPN2 expression was examined by staining using the ABC method ( FIG. 9 , middle). In cancer cells with high RPN2 expression, strongly stained in FIG. 9 (middle), E-cadherin expression was reduced ( FIG. 9 , right).
  • tissue specimens obtained from two human patients with breast cancer were examined by fluorescent immunostaining using three colors: DAPI for nuclear-specific staining (blue), anti-RPN2 antibody (green), and anti-mutated p53 antibody (red).
  • the tissue specimens were obtained from primary tumors, and both of which were positive for lymph node metastasis. In both specimens, the staining patterns of RPN2- and mutated p53-positive cells were consistent. This supports the in vitro results in Example 9 ( FIG. 10 ).
  • siRNA against canine RPN2 was designed and mixed with the aqueous solution of nucleic acid transport carrier (a transport carrier containing a peptide surfactant (A 6 K: Patent Publication 2010-222338) was used as a nucleic acid transport carrier at a final concentration of 0.5%) to prepare an siRNA-carrier complex (final concentration: 1 mg/mL).
  • nucleic acid transport carrier a transport carrier containing a peptide surfactant (A 6 K: Patent Publication 2010-222338) was used as a nucleic acid transport carrier at a final concentration of 0.5%) to prepare an siRNA-carrier complex (final concentration: 1 mg/mL).
  • siRNA-carrier complex was locally administered to the tumor of spontaneous breast cancer of a dog (golden retriever weighing about 40 kg) twice every three days. The tumor was surgically resected on day 3 after the last administration. Saline or carrier (A 6 K) alone was administered as a control.
  • GTV gross tumor volume
  • the invention provides a method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells through targeting the RPN2 gene.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Oncology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hospice & Palliative Care (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
US13/817,763 2010-08-20 2011-01-24 Method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells Abandoned US20130236891A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2010184764 2010-08-20
JP2010-184764 2010-08-20
PCT/JP2011/064527 WO2012023345A1 (fr) 2010-08-20 2011-06-24 Procédé et composition pour le traitement, la prévention et le diagnostic d'un cancer contenant des cellules souches cancéreuses ou issu de celles-ci

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2011/064527 A-371-Of-International WO2012023345A1 (fr) 2010-08-20 2011-06-24 Procédé et composition pour le traitement, la prévention et le diagnostic d'un cancer contenant des cellules souches cancéreuses ou issu de celles-ci

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/410,480 Division US10337012B2 (en) 2010-08-20 2017-01-19 Method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells

Publications (1)

Publication Number Publication Date
US20130236891A1 true US20130236891A1 (en) 2013-09-12

Family

ID=45605006

Family Applications (2)

Application Number Title Priority Date Filing Date
US13/817,763 Abandoned US20130236891A1 (en) 2010-08-20 2011-01-24 Method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells
US15/410,480 Active US10337012B2 (en) 2010-08-20 2017-01-19 Method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells

Family Applications After (1)

Application Number Title Priority Date Filing Date
US15/410,480 Active US10337012B2 (en) 2010-08-20 2017-01-19 Method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells

Country Status (7)

Country Link
US (2) US20130236891A1 (fr)
EP (1) EP2606909B1 (fr)
JP (2) JP5891173B2 (fr)
KR (3) KR20140032334A (fr)
CN (1) CN103068403A (fr)
ES (1) ES2586116T3 (fr)
WO (1) WO2012023345A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10337012B2 (en) 2010-08-20 2019-07-02 3-D Matrix, Ltd. Method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells
US11324703B2 (en) 2017-12-15 2022-05-10 3-D Matrix, Ltd. Surfactant peptide nanostructures and uses thereof in drug delivery

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014207883A (ja) * 2013-03-27 2014-11-06 国立大学法人岡山大学 がん幹細胞及びその用途
US20200129042A1 (en) * 2017-05-25 2020-04-30 Nec Corporation Information processing apparatus, control method, and program
CN109486942B (zh) * 2018-12-26 2021-04-06 苏州大学 一种用于类风湿性关节炎诊断的生物标志物及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010024262A1 (fr) * 2008-08-27 2010-03-04 株式会社スリー・ディー・マトリックス Agent de transfection
US20100087507A1 (en) * 2006-06-16 2010-04-08 Takahiro Ochiya Use of rpn2 gene expression inhibitor
US20100186103A1 (en) * 2008-05-28 2010-07-22 University Of Massachusetts Isolation Of Novel AAV'S And Uses Thereof

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070178458A1 (en) * 2003-09-05 2007-08-02 O'brien Philippa Methods of diagnosis and prognosis of ovarian cancer II
WO2009009739A2 (fr) * 2007-07-12 2009-01-15 Theracrine, Inc. Procédés et compositions permettant de réduire le nombre de cellules souches en oncogenèse
EP2316196B1 (fr) 2008-08-22 2019-02-27 Marvell World Trade Ltd. Procédé et appareil d'intégration d'un protocole de temps précis et sécurité du contrôle d'accès aux médias dans des éléments de réseau
CN102308212A (zh) * 2008-12-04 2012-01-04 加利福尼亚大学董事会 用于确定前列腺癌诊断和预后的材料和方法
WO2011040613A1 (fr) * 2009-10-01 2011-04-07 独立行政法人国立がん研究センター Agent thérapeutique anti-tumoral
US20130236891A1 (en) 2010-08-20 2013-09-12 3-D Matrix, Ltd. Method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100087507A1 (en) * 2006-06-16 2010-04-08 Takahiro Ochiya Use of rpn2 gene expression inhibitor
US20100186103A1 (en) * 2008-05-28 2010-07-22 University Of Massachusetts Isolation Of Novel AAV'S And Uses Thereof
WO2010024262A1 (fr) * 2008-08-27 2010-03-04 株式会社スリー・ディー・マトリックス Agent de transfection
EP2322608A1 (fr) * 2008-08-27 2011-05-18 3-D Matrix, Ltd. Agent de transfection
US20110152203A1 (en) * 2008-08-27 2011-06-23 Daizo Yoshida Transfection agent
US9133484B2 (en) * 2008-08-27 2015-09-15 3-D Matrix, Ltd. Transfection agent

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
An English translation for Ochiya Salshin Igaku (June 25, 2010), Vol. 65, pp. 1343-1352, pages 1-15 *
English Translation of WO 20100242626, Yoshida et al., pages 1-25 *
Honma et al. Nature Medicine 14: 939-948, 2008 *
von Maltzahn et al. Langmuir 2003, 19, 4332-4337 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10337012B2 (en) 2010-08-20 2019-07-02 3-D Matrix, Ltd. Method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells
US11324703B2 (en) 2017-12-15 2022-05-10 3-D Matrix, Ltd. Surfactant peptide nanostructures and uses thereof in drug delivery

Also Published As

Publication number Publication date
EP2606909B1 (fr) 2016-05-11
JP6262707B2 (ja) 2018-01-17
EP2606909A1 (fr) 2013-06-26
KR20170131723A (ko) 2017-11-29
ES2586116T3 (es) 2016-10-11
US20170240902A1 (en) 2017-08-24
WO2012023345A1 (fr) 2012-02-23
KR20140032334A (ko) 2014-03-14
CN103068403A (zh) 2013-04-24
JP2016101167A (ja) 2016-06-02
EP2606909A4 (fr) 2014-08-13
KR20180116465A (ko) 2018-10-24
US10337012B2 (en) 2019-07-02
JPWO2012023345A1 (ja) 2013-10-28
JP5891173B2 (ja) 2016-03-22
KR101911659B1 (ko) 2018-10-24

Similar Documents

Publication Publication Date Title
US10337012B2 (en) Method and composition for the treatment, prevention, and diagnosis of cancer containing or derived from cancer stem cells
JP2022008404A (ja) Ｃ／ＥＢＰα小分子活性化ＲＮＡ組成物
US11976280B2 (en) SMAC/Diablo inhibitors useful for treating cancer
US7902166B2 (en) Compositions comprising inhibitors of RNA binding proteins and methods of producing and using same
Fu et al. Exosomal microRNA-25 released from cancer cells targets SIK1 to promote hepatocellular carcinoma tumorigenesis
US20150344887A1 (en) siRNA FOR INHIBITION OF USP15 EXPRESSION AND PHARMACEUTICAL COMPOSITION CONTAINING THE SAME
US8748592B1 (en) siRNA for inhibition of OTUB1 expression and pharmaceutical composition containing the same
JP5762103B2 (ja) 頭頸部癌及び食道癌用抗癌剤及び増強剤
EP2810655A1 (fr) Nouvel agent antitumoral et son procede de depistage
KR101525122B1 (ko) Ｕｂｂ 넉­다운에 의한 암의 예방 또는 치료
KR101197627B1 (ko) Ｈｐｖ 감염과 관련된 암의 치료용 조성물
JP5887413B2 (ja) 非小細胞肺癌におけるｔｍ４ｓｆ４の発現または活性を調節することによって癌細胞の放射線耐性ならびに増殖、転移および浸潤を低減させる方法
JP5567555B2 (ja) 頭頸部腫瘍増殖抑制剤
US10260067B2 (en) Enhancing dermal wound healing by downregulating microRNA-26a
EP3978018A1 (fr) Nouvel agent thérapeutique contre les cancers digestifs, et méthode de criblage associée
US20110097335A1 (en) Abc transporter protein expression inhibitor
KR102143701B1 (ko) nc886 및/또는 PKR 저해제를 포함하는 항암 보조제, 및 암 치료를 위한 약물의 정보 제공 방법
US20210324385A1 (en) Compositions and Methods for Treating Endometriosis
US9464291B2 (en) Methods and compositions for the treatment of cancer
KR20150114637A (ko) ＳｅｒｐｉｎＡ1 유전자의 발현을 저해하는 저해제를 유효성분으로 포함하는 위암 치료용 약학적 조성물
CN116212024A (zh) Ube2t抑制剂的用途及抗恶性胶质瘤的药物组合物
KR20200118931A (ko) 저산소 환경에서 유발되는 암의 전이 억제용 조성물

Legal Events

Date Code Title Description
AS Assignment

Owner name: NATIONAL CANCER CENTER RESEARCH INSTITUTE, JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:OCHIYA, TAKAHIRO;REEL/FRAME:029838/0505

Effective date: 20130208

Owner name: 3-D MATRIX, LTD., JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:OCHIYA, TAKAHIRO;REEL/FRAME:029838/0505

Effective date: 20130208

AS Assignment

Owner name: NATIONAL CANCER CENTER, JAPAN

Free format text: CHANGE OF NAME;ASSIGNOR:NATIONAL CANCER CENTER RESEARCH INSTITUTE;REEL/FRAME:039165/0040

Effective date: 20150401

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION