JP2012506236A - Screening method for anti-lung cancer compound or anti-esophageal cancer compound - Google Patents

Abstract

Disclosed herein are methods for determining ERK kinase activity against CDCA5 and methods for screening for modulators of this kinase activity. Also disclosed are methods and pharmaceutical compositions for preventing and / or treating lung cancer or esophageal cancer using or including such modulators.

Description

Technical field
Priority This application claims the benefit of, filed on October 24, 2008, US Provisional Patent Application No. 61 / 197,263. The entire disclosure of which is incorporated herein by reference.

  The present invention relates to lung cancer and esophageal cancer, and specifically to its diagnosis and treatment.

Lung cancer and esophageal cancer Airway gastrointestinal cancers, including lung, esophageal, and nasopharyngeal cancers, account for nearly a quarter of all cancer deaths in Japan. Lung cancer is the leading cause of cancer-related death in the world, and 1.3 million patients die in one year (Non-Patent Document 1). Two major histologically distinct lung cancers, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), have different pathophysiological and clinical features. NSCLC accounts for nearly 80% of lung cancer, while SCLC accounts for 20% of lung cancer (Non-Patent Documents 2-3). Despite applying surgical techniques in combination with various treatment modalities such as radiation therapy and chemotherapy, the overall 5-year survival rate for lung cancer remains about 15% (non-patented) Reference 4). Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignant tumors of the gastrointestinal tract, and the overall 5-year survival rate of lung cancer is only 15% (Non-patent Document 5). It has been reported that the incidence of esophageal cancer is highest in an area called “Asian esophageal cancer belt” spreading from the eastern coast of the Caspian Sea to central China (Non-patent Document 6). Many genetic changes involved in the onset and / or progression of lung cancer and esophageal cancer have been reported, but the exact molecular mechanism has not yet been clarified (Non-patent Document 7).

  Despite the use of modern surgical techniques in combination with various treatment modalities such as radiation therapy and chemotherapy, lung cancer and ESCC have been shown to have the poorest prognosis among malignant tumors. The 5-year survival rate of lung cancer patients including all stages remains at 15%, and the 5-year survival rate of ESCC patients is 10% to 16% (Non-patent Document 8). Therefore, improvements in therapeutic strategies including the development of molecular targeted drugs and antibodies and cancer vaccines are awaited. Increased understanding of the molecular basis of lung cancer has identified targeting strategies that inhibit specific key molecules in tumor growth and progression. For example, epidermal growth factor receptor (CDCA5) is generally overexpressed in NSCLC, and its expression often correlates with poor prognosis (Non-patent Document 9). Recently, two major classes of CDCA5 inhibitors have been developed: small molecules that act as tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib; and monoclonal antibodies to the extracellular domain of CDCA5, such as cetuximab. Although the above-mentioned targeted therapy is expected to improve the prognosis of NSCLC, the results are still not sufficient. Erlotinib showed a survival benefit compared to placebo, with a median survival of 6.7 months for erlotinib compared to 4.7 months for placebo (Non-patent Document 10). On the other hand, only gefitinib showed an excellent response rate and symptom management (Non-patent Document 11). In the case of cetuximab, the current phase 2 data is not mature enough to draw any definitive conclusion about the role of this drug in NSCLC (Non-Patent Document 13). Therefore, an effective therapeutic strategy including the development of molecular targeted drugs and antibodies and cancer vaccines is awaited.

cDNA microarray analysis Systematic analysis of the expression levels of thousands of genes in cDNA microarrays is an effective approach to identify molecules involved in the carcinogenic pathway. Some of these genes or their products are considered targets for the development of effective anti-cancer agents and tumor markers that are reliable indicators of disease. To isolate such molecules, whole-genome expression profiles of lung cancer and ESCC were analyzed using a pure population of tumor cells prepared by laser microdissection (14-19).

CDCA5 (5 related to cell division cycle)
CDCA5, a cell cycle regulatory protein, has been identified as a regulator of sister chromatid adhesion. This 35 kDa protein is degraded in the G1 phase through late-promoting complex (APC) -dependent ubiquitination. Previous studies have shown that CDCA5 interacts with cohesion on chromatin and functions to support sister chromatid adhesion during interphase. Sister chromatids usually segregate further in most G2 phase cells, indicating that CDCA5 is already required in the S phase to establish adhesion (Non-patent Document 20). So far, there is only one other protein that is particularly necessary for the establishment of adhesion: budding yeast acetyltransferase Eco1 / Ctf7 (Non-Patent Documents 21-23). In addition, homologs of this enzyme are also required for adhesion in Drosophila and human cells (Non-patent Documents 24-25), but it is not yet known whether these proteins function in the S phase. It is therefore interesting to address whether CDCA5 and Eco1 / Ctf7 homologs cooperate to establish adhesion in cancer cells.

  In order to effectively perform accurate chromosome segregation, sister chromatid adhesions must be established and disassembled at the appropriate time in the cell cycle. Activation of APCCdc20 has been previously reported to control adhesion separation by targeting the late inhibitor securin for degradation. This allows for separase-dependent cleavage of Scc1 / Rad21 and induces the late phase. The degradation of most cell cycle substrates of APC is logical in terms of these functions. Degradation prevents the activity from appearing out of time and promotes cell cycle progression in stages.

  The function of CDCA5 also overlaps with the function of other factors that regulate adhesion, and their activity is combined to ensure that the chromosome replicates and segregates (Non-patent Document 26). According to these microarray data, APC and CDC20 are highly expressed in lung cancer and esophageal cancer, but their expression in normal tissues is low. Furthermore, CDC20 was confirmed to be highly expressed in clinical small cell lung cancer using semi-quantitative RT-PCR and immunohistochemical analysis (Non-patent Document 27).

  These data are consistent with the conclusion that CDCA5 promotes cancer cell growth by cooperating with CDC20 to promote cell cycle progression, but evidence that this molecule can interact directly with CDCA5. There is no. This protein is localized in the nucleus of interphase cells, disperses from the chromatid during mitosis, and interacts with the cohesion complex in the late phase (Non-patent Document 28). CDCA5 was reported to be required for stable binding of cohesion to chromatids and sister chromatid adhesion in interphase (Non-patent Document 29). Despite these biological studies, there are no reports prior to the present invention regarding the importance of CDCA5 activation in human carcinogenesis and its use as a therapeutic target.

PCT / JP2008 / 066533

WHO Cancer World Health Organization. 2006 Morita T & Sugano H. Acta Pathol Jpn. 1990 Sep; 40 (9): 665-75 Simon GR, et al., Chest. 2003 Jan; 123 (1 Suppl): 259S-271S Parkin DM. Lancet Oncol. 2001 Sep; 2 (9): 533-43 Shimada H, et al., Surgery. 2003 May; 133 (5): 486-94 Mosavi-Jarrahi A & Mohagheghi MA. Asian Pac J Cancer Prev. 2006 Jul-Sep; 7 (3): 375-80 Sozzi G. Eur J Cancer. 2001 Oct; 37 Suppl 7: S63-73 Parkin Dm et al., CA Cancer J Clin 2005; 55: 74-108 Global cancer statistics, 2002 Brabender J, et al., Clin Cancer Res. 2001 Jul; 7 (7): 1850-5 Shepherd FA. Et al., N Engl J Med. 2005 Jul 14; 353 (2): 123-32 Giaccone G, et al., J Clin Oncol. 2004 Mar 1; 22 (5): 777-84 Baselga J. J Clin Oncol. 2004 Mar 1; 22 (5): 759-61 Azim HA & Ganti AK. Cancer Treat Rev. 2006 Dec; 32 (8): 630-6. Epub 2006 Oct 10 Kikuchi T, et al., Oncogene. 2003 Apr 10; 22 (14): 2192-205 Kakiuchi S, et al., Mol Cancer Res. 2003 May; 1 (7): 485-99 Kakiuchi S, et al., Hum Mol Genet. 2004 Dec 15; 13 (24): 3029-43. Epub 2004 Oct 20 Kikuchi T, et al., Int J Oncol. 2006 Apr; 28 (4): 799-805 Taniwaki M, et al., Int J Oncol. 2006 Sep; 29 (3): 567-75 Yamabuki T, et al., Int J Oncol. 2006 Jun; 28 (6): 1375-84 Schmitz J, et al., Curr Biol. 2007 Apr 3; 17 (7): 630-6. Epub 2007 Mar 8 Skibbens RV, et al., Genes Dev. 1999 Feb 1; 13 (3): 307-19 Toth A, et al., Genes Dev. 1999 Feb 1; 13 (3): 320-33 Ivanov D, et al., Curr Biol. 2002 Feb 19; 12 (4): 323-8 Williams BC, et al., Curr Biol. 2003 Dec 2; 13 (23): 2025-36 Hou F & Zou H. Mol Biol Cell. 2005 Aug; 16 (8): 3908-18. Epub 2005 Jun 15 Rankin S, et al., Mol Cell. 2005 Apr 15; 18 (2): 185-200 Taniwaki M, et al, Int J Oncol. 2006 Sep; 29 (3): 567-75 Rankin S, et al., Mol Cell. 2005 Apr 15; 18 (2): 185-200 Schmitz J, et al., Curr Biol. 2007 Apr 3; 17 (7): 630-6. Epub 2007 Mar 8

  The present invention provides a method for identifying an agent that decreases the kinase activity of ERK (extracellular signal-regulated kinase) against CDCA5. This agent is incubated with ERK polypeptide or a functional equivalent thereof and CDCA5 polypeptide or a functional equivalent thereof in the presence of ATP as a phosphate donor to phosphorylate the CDCA5 polypeptide. Detected by determining the level. A decrease in CDCA5 phosphorylation level compared to the control level indicates that the test agent is an inhibitor of ERK kinase activity. An agent that decreases ERK kinase activity against CDCA5 is useful for treating or preventing at least one symptom of lung cancer or esophageal cancer.

  The present invention also provides a kit for detecting ERK kinase activity against CDCA5. The reagents are preferably packaged together in the form of a kit. Reagents may be packaged in separate containers, eg, ERK polypeptide, CDCA5 polypeptide, reagent for detecting CDCA5 phosphorylation level at amino acid residue serine 79 or 209 of SEQ ID NO: 5, control Reagents (positive and / or negative controls) and agents for detecting phosphorylation levels may be included.

  Furthermore, the present invention provides a novel synthetic polypeptide, CDCA5 (S209A) and functional equivalents thereof. When this polypeptide is expressed, it can inhibit the growth of cancer cells.

  In a preferred embodiment, the CDCA5 (S209A) polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7. The application also provides an isolated protein encoded by at least a portion of the CDCA5 (S209A) polynucleotide sequence set forth in SEQ ID NO: 6.

  In another aspect, the present invention provides a method for either or both treatment and prevention of lung cancer or esophageal cancer in a subject. The method comprises administering a CDCA5 polypeptide variant having a dominant negative effect, a polynucleotide encoding the variant, or a vector comprising said polynucleotide.

  A still further object of the present invention is to provide, as an active ingredient, a pharmaceutically effective amount of a CDCA5 polypeptide variant having a dominant negative effect, a polynucleotide encoding the variant, or a vector comprising the polynucleotide, and pharmaceutically It is to provide a composition for treating or preventing lung cancer or esophageal cancer comprising an acceptable carrier.

  Various aspects and uses of the present invention will become apparent to those skilled in the art in view of the following brief description of the drawings and detailed description of the invention and preferred embodiments thereof.

CDCA5 expression in lung tumors. A, CDCA5 gene expression in lung cancer tissues examined by semi-quantitative RT-PCR. B, CDCA5 gene expression in lung cancer cell lines examined by semi-quantitative RT-PCR and Western blotting. CDCA5 expression in lung tumors. C, CDCA5 protein expression in lung cancer cell lines examined by Western blot. D, intracellular localization of endogenous CDCA5 protein in lung cancer LC319 cells. Cells were stained with rabbit polyclonal anti-CDCA5 antibody and DAPI. CDCA5 expression in normal tissues and the relationship between CDCA5 expression and poor prognosis in NSCLC patients. A, Northern blot analysis of CDCA5 transcripts in various normal human tissues. B. Expression of CDCA5 protein in 5 normal tissues (liver, kidney, heart, lung, and testis) and lung adenocarcinoma. CDCA5 expression in normal tissues and the relationship between CDCA5 expression and poor prognosis in NSCLC patients. C, Immunohistochemical staining pattern of CDCA5 protein in representative lung adenocarcinoma using anti-CDCA5 polyclonal antibody on tissue microarray (upper X100; lower X200). D, Kaplan-Meier analysis of CDCA5 expression and tumor-specific survival on tissue microarrays. CDCA5 growth-promoting effect. A, oligonucleotide siRNA (si- # 1 and-# 2) specific for CDCA5 or control siRNA (si-LUC and si) confirmed by semi-quantitative RT-PCR analysis (top) and Western blot (bottom). -Knockdown of CDCA5 expression in A549 and LC319 cells by -CNT). B, Colony formation assay of A549 and LC319 cells transfected with siRNA. CDCA5 growth-promoting effect. C, Viability of A549 and LC319 cells in response to siRNA as examined by MTT assay. All assays were performed 3 times independently in 3 wells. D, proliferation of COS-7 cells stably expressing exogenous CDCA5 was enhanced in vitro. The cell viability of the two stable clones (COS-7-CDCA5- # A and-# B) and the two control clones (COS-7-mock- # A and-# B) were measured on day 1. Quantified by MTT assay on days 3, 5, and 7. All assays were performed 3 times independently in 3 wells. In vitro CDCA5 phosphorylation by ERK protein kinase. A, In vitro phosphorylation of recombinant human CDCA5 (rhCDCA5) by recombinant ERK2 (rhERK2). B, R-250 staining of rhCDCA5 phosphorylated by rhERK in vitro for MALDI-TOF mass spectrometry to identify phosphorylation sites on CDCA5. Identification of ERK-dependent phosphorylation site of CDCA5 in cultured cells. A, Endogenous CDCA5 was phosphorylated by ERK in HeLa cells after stimulation with EGF with or without the MEK inhibitor U0126. B, Untagged CDCA5 expression vector was transfected into HeLa cells. After the cells were starved for 20 hours without FBS, EGF stimulation was performed for 5 minutes, 10 minutes, 20 minutes, 30 minutes. 10 uM U0126 treatment was performed for 1 hour. Identification of ERK-dependent phosphorylation site of CDCA5 in cultured cells. A vector expressing A, ERK1 or ERK2 and an untagged CDCA5 expression vector were cotransfected into HeLa cells. After HeLa cells were starved for 20 hours without FBS, the cells were stimulated with 50 ng / ml EGF for 5 minutes, 10 minutes, 20 minutes, and 30 minutes, respectively. Prior to EGF stimulation, 10 uM U0126 treatment was performed for 1 hour. Identification of ERK-dependent phosphorylation site of CDCA5 in cultured cells. B, Starved HeLa cells co-transfected with a vector expressing ERK2 and a vector expressing CDCA5 were stimulated with 50 ng / ml EGF for 20 minutes. Treatment with 10 uM U0126 inhibitor was performed for 1 hour prior to EGF stimulation. After immunoprecipitation of untagged CDCA5 protein with anti-CDCA5 antibody, the sample was subjected to SDS-PAGE. Colloid CBB staining was performed overnight. A band corresponding to CDCA5 protein was excised for MS analysis. Western blot using anti-CDCA5 antibody showed that CDCA5 protein was expressed in each sample. Identification of ERK-dependent phosphorylation site of CDCA5 in cultured cells. C, MS analysis result of untreated sample. Identification of ERK-dependent phosphorylation site of CDCA5 in cultured cells. D, MS analysis result of EGF stimulation sample. Identification of ERK-dependent phosphorylation site of CDCA5 in cultured cells. E, MS analysis results of EGF-stimulated samples after U0126 treatment. Identification of ERK-dependent phosphorylation site of CDCA5 in cultured cells. A vector expressing F, ERK2, and a vector expressing an untagged wild type, and a vector expressing a CDCA5 alanine substitute were co-transfected into HeLa cells. Cells were stimulated with EGF after being starved. Function of ERK-dependent phosphorylation sites in cancer cells. A, Western blot using an anti-CDCA5 antibody showed that CDCA5 was expressed in lung cancer cell lines. Proliferation assays were performed using A549 and LC319 lung cancer cell lines transfected with mock, wild type, and CDCA5 alanine replacement vectors. MTT assay was performed on the 6th day after transfection. Function of ERK-dependent phosphorylation sites in cancer cells. Proliferation assays were performed using LC319 and A549 lung cancer cell lines transfected with B, mock, wild type, and CDCA5-phosphate mimetic mutant vectors. MTT assay was performed on the 6th day after transfection.

Aspect description
Definitions As used herein, the terms “a”, “an”, and “the” mean “at least one” unless otherwise specified.

  The terms “isolated” and “purified” as used for a substance (eg, a polypeptide, antibody, polynucleotide, etc.) refer to the substance at least one substance that may be included in a natural source. Not included. Thus, an isolated or purified protein (eg, antibody) is substantially free of cellular material, eg, carbohydrates, lipids, or other contaminating proteins derived from the cell or tissue source from which the protein was obtained. A protein that is absent or substantially free of chemical precursors or other chemicals when chemically synthesized. The term “substantially free of cellular material” includes preparations of a polypeptide that is separated from cellular components of the cell from which the polypeptide is isolated or recombinantly produced.

  Thus, a polypeptide that is substantially free of cellular material has less than about 30%, 20%, 10%, or 5% (by dry weight) of a heterologous protein (also referred to herein as a “contaminating protein”). Non-polypeptide preparations. When the polypeptide is produced recombinantly, in some embodiments, it is also substantially free of media, which has a contaminated media that is less than about 20%, 10%, or 5% of the volume of the protein preparation. Polypeptide preparations are included. When the polypeptide is produced by chemical synthesis, in some embodiments, it is substantially free of chemical precursors or other chemicals, including about 30%, 20%, 10% of the volume of the protein preparation. Polypeptide preparations having less than 5% (by dry weight) of chemical precursors or other chemicals involved in protein synthesis are included. The fact that a particular protein preparation contains an isolated or purified polypeptide may, for example, indicate the appearance of a single band after sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis of the protein preparation and the gel Of Coomassie brilliant blue and the like. In one aspect, the protein of the invention is isolated or purified.

  An “isolated” or “purified” nucleic acid molecule, eg, a cDNA molecule, can be substantially free of other cellular material or media when produced by recombinant techniques, or chemically synthesized Can be substantially free of chemical precursors or other chemicals. In one aspect, a nucleic acid molecule encoding a protein of the invention is isolated or purified.

  The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are modified or unnatural residues, eg, artificial chemical mimetics of the corresponding natural amino acids, as well as natural amino acid polymers Is done.

  The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Natural amino acids are those encoded by the genetic code, as well as those that are post-translationally modified in cells (eg, hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine). The phrase “amino acid analog” has the same basic chemical structure as a natural amino acid (hydrogen, carboxy group, amino group, and α-carbon bonded to an R group), but has a modified R group or modified backbone (eg, Homoserine, norleucine, methionine, sulfoxide, methionine methylsulfonium). The phrase “amino acid mimetic” refers to a chemical compound that has a different structure but functions similarly to a common amino acid.

  Amino acids may be referred to herein by either the commonly known three letter symbols or the one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

  The terms “polynucleotide”, “oligonucleotide”, “nucleotide”, “nucleic acid”, and “nucleic acid molecule” are used interchangeably unless otherwise specified, and are generally accepted single letter symbols as well as amino acids. Mentioned by. As well as amino acids, it includes both natural or non-natural nucleic acid polymers. A polynucleotide, oligonucleotide, nucleotide, nucleic acid, or nucleic acid molecule may consist of DNA, RNA, or a combination thereof.

  The present invention relates to cancer therapy (treatment) and prevention. In the context of the present invention, therapy for cancer or prevention of cancer development includes any of the following stages: inhibition of cancer cell growth, regression of cancer, and suppression of cancer development. Reducing mortality and morbidity in individuals with cancer, reducing blood tumor marker levels, alleviating detectable symptoms associated with cancer, etc. are also included in cancer therapy or prevention. Such therapeutic and prophylactic effects are preferably statistically significant. For example, at the time of observation, the therapeutic or prophylactic effect of the pharmaceutical composition for cell proliferative diseases is at a significance level of 5% or less compared to the control without administration. For example, Student's t-test, Mann-Whitney U-test or ANOVA can be used for statistical analysis.

  Further, in the context of the present invention, the term “prevention” includes any activity that reduces the mortality or morbidity burden of the disease. Prevention can occur at primary, secondary and tertiary prevention levels. Whereas primary prevention avoids the development of the disease, secondary and tertiary levels of prevention prevent disease progression and restore function and reduce disease-related complications. Includes activities aimed at preventing the appearance of symptoms as well as reducing the adverse effects of already established diseases.

  In the context of the present invention, an “effective” treatment is a treatment that results in a decrease in the size, prevalence, or metastatic potential of lung or esophageal cancer in a subject. “Effective” when the treatment is applied prophylactically means that the treatment delays or prevents the development of cancer or reduces the clinical symptoms of the cancer. Assessment of lung cancer or esophageal cancer can be performed using standard clinical protocols. Furthermore, the effectiveness of treatment can be determined in connection with any known method for the diagnosis of lung cancer or esophageal cancer. For example, lung cancer or esophageal cancer is diagnosed routinely by histopathology or by identifying symptomatic abnormalities.

  Additional definitions are included in the following text as appropriate.

CDCA5
The nucleotide sequence of the human CDCA5 gene is shown in SEQ ID NO: 4, and is also available as GenBank accession number NM_080668 or BC011000. As used herein, the phrase “CDCA5 gene” includes, but is not limited to, the human CDCA5 gene and the CDCA5 gene of other animals including non-human primates, mice, rats, dogs, cats, horses, and cows. Not including allelic variants and genes found in other animals corresponding to the CDCA5 gene.

  The amino acid sequence encoded by the human CDCA5 gene is shown in SEQ ID NO: 5 and is also available as GenBank accession number AAH11000. In the present invention, the polypeptide encoded by the CDCA5 gene is referred to as “CDCA5” and is sometimes referred to as “CDCA5 polypeptide” or “CDCA5 protein”.

CDCA5 (S209A)
The nucleotide sequence of the CDCA5 mutant gene is shown in SEQ ID NO: 6. The amino acid sequence encoded by the CDCA5 mutant gene is shown in SEQ ID NO: 7. As used herein, the phrase “CDCA5 variant” includes CDCA5 (S209A). “S209A” indicates that the serine at position 209 in the CDCA5 amino acid sequence is substituted with alanine.

Epidermal growth factor (EGF)
The amino acid sequence encoded by the human EGF gene is available as GenBank accession number NP_001954. EGF (polypeptide) is believed to exist as a membrane-bound molecule that yields a 53 amino acid peptide that can be proteolytically cleaved to bind to the appropriate receptor. This 53 amino acid fragment is shown in SEQ ID NO: 3. Both EGF fragments and full-length EGF are referred to as “EGF”. As used herein, the phrase “EGF gene” includes, but is not limited to, the human EGF gene and EGF genes of other animals including non-human primates, mice, rats, dogs, cats, horses, and cows. .

ERK
In the present invention, ERK1 and ERK2 are both referred to as “ERK”. The amino acid sequence encoded by the human ERK1 gene is shown in SEQ ID NO: 1, but is not limited thereto. ERK1 is believed to contain three isoforms, shown as GenBank accession numbers NP_002737.2, NP_001035145.1, and NP_0011303361.1. Accordingly, the phrase “ERK1” herein includes these three isoforms. On the other hand, the amino acid sequence encoded by the human ERK2 gene is shown in SEQ ID NO: 2 and is also available as GenBank accession number NP_002736.3. As used herein, the phrase “ERK gene” includes, but is not limited to, human ERK genes and ERK genes of other animals including non-human primates, mice, rats, dogs, cats, horses, and cows. Not. In the present specification, “ERK” is sometimes referred to as “ERK polypeptide” or “ERK protein”.

Functional Equivalent Polypeptide The polypeptide of the present invention has an amino acid sequence, molecular weight, isoelectric point, presence or absence of a sugar chain, depending on the cell or host used to produce the polypeptide of the present invention or the purification method used. Or the form may be different. Nevertheless, so long as it has a function equivalent to that of the original protein of the present invention, it is within the scope of the present invention.

  Thus, according to one aspect of the invention, CDCA5, CDCA5 (S209A), EGF, and ERK also include functional equivalents. As used herein, a “functional equivalent” of a protein is a polypeptide having a biological activity equivalent to that protein. That is, any polypeptide that retains at least one biological activity of the original protein can be used as a functional equivalent in the present invention. For example, a functional equivalent of CDCA5 retains the activity of promoting cell growth of CDCA5. Furthermore, the biological activity of CDCA5 includes binding activity to ERK and / or activity phosphorylated by ERK, whereas the exemplary biological activity of CDCA5 (S209A) Ability to inhibit the growth of cancer cells. Furthermore, exemplary biological activity of EGF includes binding activity to EGFR, and exemplary biological activity of ERK is kinase activity against CDCA5.

  Methods for obtaining or preparing a protein functionally equivalent to a particular protein are well known to those skilled in the art and include conventional methods for introducing mutations into proteins. For example, those skilled in the art can prepare proteins functionally equivalent to human proteins by introducing appropriate mutations into human protein amino acid sequences via site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995), Gene 152, 271-5; Zoller, MJ, and Smith, M. (1983), Methods Enzymol. 100, 468-500; Kramer, W. et al. (1984), Nucleic Acids Res. 12, 9441-56; Kramer W, and Fritz HJ. (1987) Methods. Enzymol. 154, 350-67; Kunkel, TA (1985), Proc. Natl. Acad. Sci. USA. 82, 488- 92; Kunkel (1991), Methods Enzymol. 204, 125-39).

  Alternatively, a functionally equivalent protein for a specific protein can be obtained by a hybridization method using a gene of a specific protein or a polynucleotide fragment thereof as a probe. In the context of the present invention, hybridization conditions for isolating a DNA encoding a polypeptide functionally equivalent to the original protein can be routinely selected by those skilled in the art. For example, hybridization may be performed by performing prehybridization at 68 ° C. for 30 minutes or more using “Rapid-hyb buffer” (Amersham LIFE SCIENCE), adding a labeled probe, and heating at 68 ° C. for 1 hour or more. Good. The following washing process can be performed, for example, under low stringent conditions. Exemplary low stringency conditions may include 42 ° C., 2 × SSC, 0.1% SDS, preferably 50 ° C., 2 × SSC, 0.1% SDS. Preferably, high stringency conditions are often used. Exemplary high stringency conditions are 20 minutes at room temperature, 3 washes with 2 × SSC, 0.01% SDS, then 3 washes with 1 × SSC, 0.1% SDS for 20 minutes at 37 ° C., and 50 Two washings with 1 × SSC, 0.1% SDS may be included for 20 minutes at 0 ° C. However, several factors, such as temperature and salt concentration, can affect the stringency of hybridization, and one of ordinary skill in the art can appropriately select the factors to achieve the required stringency. .

  The phrase “stringent (hybridization) conditions” means that a nucleic acid molecule hybridizes to its target sequence, typically in a complex mixture of nucleic acids, but to a detectable extent to other sequences. It refers to the condition not to soy. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Extensive guidelines for nucleic acid hybridization are 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 ° C. to 10 ° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probe complementary to the target hybridizes to the target sequence in equilibrium (because of the excess target sequence, Tm 50% of the probes are occupied at equilibrium). Stringent conditions may be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least twice background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions include: 50% formamide, 5 × SSC, and 1% SDS, 42 ° C. incubation, or 5 × SSC, 1% SDS, 65 ° C., 0 ° Wash at 50 ° C. in 2 × SSC and 0.1% SDS.

  In general, it is known that modification of one or more amino acids in a protein does not affect the function of the protein. Indeed, a mutated or modified protein, i.e. a protein having an amino acid sequence modified by substitution, deletion, insertion, and / or addition of one or more amino acid residues to a particular amino acid sequence (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)). Amino acid mutations can occur naturally. Thus, one of skill in the art will have a protein with a similar function by individual additions, deletions, insertions or substitutions to the amino acid sequence, or changes in the protein that change one amino acid or a small percentage of amino acids. It will be appreciated that what is considered a “conservative modification” is acceptable in the context of the present invention.

  As long as the activity of the protein is maintained, the number of amino acid mutations is not particularly limited. However, in general, it is preferred to change 5% or less of the amino acid sequence. Accordingly, in a preferred embodiment, the number of amino acids mutated in such variants is generally 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, more preferably 6 amino acids or less, and even more. Preferably it is 3 amino acids or less. Functional equivalents of the proteins of the invention include substitutions, deletions, additions of one or more amino acids, eg 1-5 amino acids, eg up to 5% of the amino acids, relative to the natural amino acid sequence of the protein Or inserted. For example, with respect to the CACR5 protein, amino acids at serine-79 and serine-209 of SEQ ID NO: 5 that are susceptible to ERK phosphorylation must be conserved in order to retain its activity. With respect to ERK, it is preferred that the kinase domain is conserved in the amino acid sequence of the mutant protein so that the protein maintains kinase activity against CDCA5.

The amino acid residue to be mutated is preferably mutated to another amino acid whose amino acid side chain properties are conserved (a process known as conservative amino acid substitution). Examples of amino acid side chain properties 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 groups having the following functional groups or common characteristics: aliphatic side chains (G, A, V, L, I, P); side chains containing hydroxyl groups (S, T Y); side chain containing sulfur atom (C, M); side chain containing carboxylic acid and amide (D, N, E, Q); side chain containing base (R, K, H); and aromatic Is a side chain (H, F, Y, W) containing Conservative substitution tables that yield amino acids with similar functions are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for each other:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), glutamic acid (E);
3) Asparagine (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, for example, Creighton, Proteins 1984).

  Such conservatively modified polypeptides are included in the protein of the present invention. However, the present invention is not limited to these, and the protein includes non-conservative modifications as long as at least one biological activity of the protein is retained. Furthermore, modified proteins do not exclude proteins encoded by polymorphic variants, interspecies homologs, and alleles of these proteins.

  Furthermore, the genes of the present invention include polynucleotides that encode such functional equivalents of proteins. In addition to hybridization, a polypeptide that is functionally equivalent to a protein is encoded using a primer synthesized based on the above sequence information using a gene amplification method, for example, a polymerase chain reaction (PCR) method. The polynucleotide can be isolated. A polynucleotide functionally equivalent to a human gene and a polypeptide functionally equivalent to a human protein usually have a high homology with the original nucleotide or amino acid sequence. “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 according to the algorithm of “Wilbur and Lipman, Proc Natl Acad Sci USA 80: 726-30 (1983)”.

Production of polypeptides Further, the present invention provides methods for producing the polypeptides of the present invention. The polypeptide may be prepared by culturing host cells having an expression vector containing a gene encoding the polypeptide. A method of amplifying the copy number of a gene in a cell at the same time as stably expressing the gene may be used according to needs. For example, a vector containing a complementary DHFR gene (eg, pCHOI) may be introduced into CHO cells lacking the nucleic acid synthesis pathway and then amplified with methotrexate (MTX). Furthermore, in the case of transient expression of a gene, a method of transforming a COS cell containing an SV40T antigen-expressing gene on a chromosome with a vector (such as pcD) containing an SV40 origin of replication can be used.

  The polypeptide of the present invention obtained as described above can be isolated from the inside or outside of a host cell (for example, a medium) and purified as a substantially pure and homogeneous polypeptide. The term “substantially pure” as used herein with respect to a particular polypeptide means that the polypeptide is substantially free of other biological macromolecules. A substantially pure polypeptide is at least 75% (eg, at least 80%, 85%, 95%, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The method for polypeptide isolation and purification is not limited to any particular method. In fact, any standard method can be used. For example, to isolate and purify polypeptides, column chromatography, filters, ultrafiltration, salt precipitation, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing Dialysis, and recrystallization can be appropriately selected and combined.

  Examples of chromatography include, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual Ed. Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). These chromatography may be performed by liquid chromatography such as HPLC and FPLC. Thus, the present invention provides a highly purified polypeptide prepared by the above method.

  The polypeptides of the present invention may optionally be modified or partially deleted by treatment with an appropriate protein modifying enzyme before or after purification. Useful protein modifying enzymes include, but are not limited to, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, glucosidase and the like.

Vectors and host cells The present invention also provides vectors and host cells into which a polynucleotide of the present invention has been introduced. The vectors of the present invention are useful for keeping the polynucleotides of the present invention, particularly DNA, in host cells, in order to express the polypeptides of the present invention or to administer the polynucleotides of the present invention in gene therapy. It is.

  If E. coli is the host cell and the vector is to be amplified and produced in large quantities in E. coli (eg, JM109, DH5α, HB101, or XL1Blue), the vector will be “ori” amplified in E. coli and transformed. Must have a marker gene for selecting E. coli (for example, a drug resistance gene selected by drugs such as ampicillin, tetracycline, kanamycin, chloramphenicol). For example, M13 series vector, pUC series vector, pBR322, pBluescript, pCR-Script, etc. can be used. Furthermore, similar to the vectors described above, pGEM-T, pDIRECT, and pT7 can also be used for subcloning and extracting cDNA. Expression vectors are particularly useful when vectors are used to produce the proteins of the invention. For example, an expression vector expressed in E. coli must have the above characteristics in order to be amplified in E. coli. When E. coli such as JM109, DH5α, HB101, or XL1Blue is used as the host cell, the vector can be a promoter that can efficiently express the desired gene in E. coli, such as the lacZ promoter (Ward et al., Nature 341 : 544-6 (1989); FASEB J 6: 2422-7 (1992)), araB promoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter, etc. In this regard, instead of the vectors described above, for example, pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP, and pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase). Can be used). Furthermore, the vector may contain a signal sequence for polypeptide secretion. An exemplary signal sequence that directs the polypeptide to be secreted into the E. coli periplasm is the pelB signal sequence (Lei et al., J Bacteriol 169: 4379 (1987)). Means for introducing the vector into the target host cell include, for example, the calcium chloride method and the electroporation method.

  In order to produce the polypeptides of the present invention, in addition to E. coli, for example, expression vectors derived from mammals (eg, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res 18 (17): 5322 (1990)) , PEF, pCDM8), expression vectors derived from insect cells (eg, “Bac-to-BAC baculovirus expression system” (GIBCO BRL), pBacPAK8), plant-derived expression vectors (eg, pMH1, pMH2), animals Expression vectors derived from viruses (eg, pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (eg, pZIpneo), expression vectors derived from yeast (eg, “Pichia Expression Kit” (Invitrogen), pNV11, S -Q01), and Bacillus subtilis (ogen), pNV11, SP-Q01), and expression vectors derived from Bacillus subtilis (Bacillus subtilis) (e.g., can be used pPL608, pKTH50).

  In order to express the vector in animal cells such as CHO cells, COS cells, or NIH3T3 cells, the vector may be a promoter required for expression in such cells, such as the SV40 promoter (Mulligan et al., Nature 277: 108 ( 1979)), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), CMV promoter and the like, and preferably a marker gene for selecting a transformant (eg, drug (For example, a drug resistance gene selected by neomycin, G418). Examples of known vectors having these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

ERK kinase activity on CDCA5 Selective phosphorylation of CDCA5 by ERK is demonstrated herein. As a result, in another aspect, the present invention provides a method for measuring the kinase activity of ERK or its functional equivalent to CDCA5. Such a method is as follows:
a. Incubating ERK or a functional equivalent thereof with CDCA5 or a functional equivalent under conditions suitable for CDCA5 phosphorylation by ERK, comprising:
The functional equivalent of ERK is:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, and ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2 wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is SEQ ID NO: 1 or 2 Selected from the group consisting of polypeptides having a kinase activity equivalent to a polypeptide consisting of the amino acid sequence of
The functional equivalent of CDCA5 is:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5,
ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5, wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is derived from the amino acid sequence of SEQ ID NO: 5 A polypeptide having a kinase activity equivalent to that of the polypeptide, and iii. A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, provided that the resulting polypeptide is SEQ ID NO: 5 A process selected from the group consisting of polypeptides having a biological activity equivalent to a polypeptide consisting of an amino acid sequence;
b. Detecting the phosphorylation level of CDCA5 (ie, phosphorylation at amino acid residue serine 79 and / or 209 of SEQ ID NO: 5); and c. A step of determining ERK kinase activity by correlating with the phosphorylation level of CDCA5 detected in step (b) may be included. In the context of the present invention, conditions suitable for CDCA5 phosphorylation, or conditions suitable for determining the kinase activity of ERK or a functional equivalent thereof, are CDCA5 (or a functional thereof) in the presence of a phosphate donor. Equivalents) and ERK (or functional equivalents thereof) can be obtained by incubation. In the present invention, CECA5 or ERK may be provided as a cell extract, and a preferred phosphate donor is ATP. In the present specification, ATP may be labeled as necessary. For example, in a hot assay, radiolabeled ATP may be used. The phosphorylation reaction of CDCA5 was carried out by using CDCA5 and ERK for 20 minutes at 30 ° C. in a kinase assay buffer (eg, 50 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM EGTA, 2 mM DTT, 0.01% Briji 35, 1 mM ATP). It may be performed by incubation. The reaction can be stopped by adding Laemmli sample buffer.

  In a cold assay, phospho-CDCA5 levels (also referred to as “CDCA5 phosphorylation levels”) can be detected after incubation. In the present specification, “phospho-CDCA5” indicates phosphorylated CDCA5. Prior to detecting phosphorylated CDCA5, CDCA5 can be separated from other elements of CDCA5-expressing cells or cell lysates. For example, gel electrophoresis (eg, SDS-PAGE) may be used to separate CDCA5. Alternatively, CDCA5 may be captured by bringing CDCA5 into contact with a carrier linked to an anti-CDCA5 antibody.

When a labeled phosphate donor is used, phospho-CDCA5 levels can be detected by following the label. Alternatively, phospho-CDCA5 levels can be detected using an antibody that recognizes phosphorylated CDCA5 (eg, by Western blot assay or ELISA). In particular, the antibody specifically recognizes CDCA5 phospho-Ser79 or Ser209. On the other hand, when radiolabeled ATP (eg ³² P-ATP) is used as the phosphate donor (hot assay), the radioactivity of the isolated CDCA5 correlates with phospho-CDCA5 levels, and thus scintillation The level can be detected using a counter. Alternatively, other methods for detecting phospho-CDCA5 levels include, but are not limited to, mass spectrometry, such as MALDI-TOF-MS.

A variety of low-throughput and high-throughput enzyme assay formats are known in the art and can be readily adapted to detect or measure CDCA5 phosphorylation levels by ERK. For high throughput assays, CDCA5 is preferably immobilized on a solid support such as multiwell plates, slides, and chips. After the reaction, phosphate-CDCA5 can be detected on the solid support. To detect phospho-CDCA5, for example, an antibody that binds to phospho-CDCA5 can be used. For example, the phosphorylation site of CDCA5 by ERK is Ser79 or Ser209, and phosphorylation of SerCA or Ser209 of CDCA5 by ERK can be detected using an antibody specific for phospho-CDCA5 (Ser79 or Ser209). ^{Or, P 32} labeled ATP may be used as a phosphate donor. In this case, phospho -CDCA5 can be tracked by radioactive ^{P 32.} To facilitate such an assay, the solid support may be coated with streptavidin and CDCA5 may be labeled with biotin. One skilled in the art can determine other suitable assay formats depending on the desired throughput capability.

  In another embodiment, conditions suitable for CDCA5 phosphorylation by ERK may be obtained by culturing cells that express the polypeptide. For example, a transformed cell having an expression vector containing a polynucleotide encoding a polypeptide may be used.

  In the context of the present invention, the kinase activity of ERK in a biological sample can be assessed. For example, the biological sample of the present invention may include cancer tissue obtained from a patient, or a cancer cell line. The kinase activity of ERK in such biological samples serves as a reliable marker to indicate lung or esophageal cancer or to assess or determine patient prognosis.

  Furthermore, the present invention provides a reagent for measuring ERK kinase activity against CDCA5. An example of such a reagent includes CDCA5 which can be used with a phosphate donor. In the present invention, a kit for measuring the kinase activity of ERK against CDCA5 is also provided. Such a kit may comprise a reagent of the invention and a detection agent for detecting phospho-CDCA5 levels. Preferred detection agents include antibodies that specifically recognize phosphorylated CDCA5 from non-phosphorylated CDCA5. For example, in the present invention, a preferred antibody recognizes CDCA5 in which Ser79 or Ser209 is phosphorylated.

Screening Method The present invention also relates to the finding that ERK has kinase activity against CDCA5. The phosphorylation sites of CDCA5 by ERK are Ser79 and Ser209, and this phosphorylation is EGF-dependent. To this end, one aspect of the invention involves identifying a test compound that modulates or modulates ERK-mediated CDCA5 phosphorylation. As used herein, modulating or modulating means that the level of CDCA5 phosphorylation by ERK is altered, eg, decreased, decreased, inhibited, or in some cases increased or enhanced, by the function of the test compound. It shows that.

Thus, the present invention provides a novel method for identifying compounds that modulate ERK kinase activity against CDCA5. For example, the present invention provides methods for identifying agents that modulate ERK kinase activity against CDCA5. The method is as follows:
a. Incubating ERK or a functional equivalent thereof with CDCA5 or a functional equivalent thereof in the presence of a test compound under conditions suitable for CDCA5 phosphorylation by ERK, comprising:
The functional equivalent of ERK is:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, and ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2 wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is SEQ ID NO: 1 or 2 Selected from the group consisting of polypeptides having a kinase activity equivalent to a polypeptide consisting of the amino acid sequence of
The functional equivalent of CDCA5 is:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5,
ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5, wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is an amino acid sequence of SEQ ID NO: 5 A polypeptide having a kinase activity equivalent to a polypeptide comprising: iii. A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, provided that the resulting polypeptide is SEQ ID NO: 5 A process selected from the group consisting of polypeptides having a biological activity equivalent to a polypeptide consisting of an amino acid sequence;
b. Detecting CDCA5 phosphorylation at serine 79 or 209 amino acid residues of SEQ ID NO: 5, and determining the phosphorylation level of CDCA5; and c. Comparing the level determined in step (b) with the level determined in the absence of the test agent; and d. Selecting a test agent that has a reduced level of phosphorylation of CDCA5 compared to the level determined in the absence of the test agent in step (c).

Agents identified by the methods of the present invention constitute candidate compounds that can delay or stop the progression of lung or esophageal cancer, for example, by inhibiting CDCA5 phosphorylation via ERK. Therefore, the present invention provides a method for screening a compound that modulates ERK kinase activity against CDCA using cells expressing ERK and CDCA5, or a method for screening a drug for treating or preventing lung cancer or esophageal cancer. provide. The method comprises the following steps:
a. A cell expressing CDCA5 or a functional equivalent thereof and ERK or a functional equivalent thereof (ie, the cell is a polynucleotide encoding CDCA5 or a functional equivalent thereof, and a polynucleotide encoding ERK or a functional equivalent thereof) Containing and translating nucleotides),
The functional equivalent of CDCA5 is:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5,
ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5, wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is an amino acid sequence of SEQ ID NO: 5 A polypeptide having a kinase activity equivalent to a polypeptide comprising: iii. A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, provided that the resulting polypeptide is SEQ ID NO: 5 Selected from the group consisting of polypeptides having a biological activity equivalent to a polypeptide consisting of an amino acid sequence,
The functional equivalent of ERK is:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, and ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2 wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is SEQ ID NO: 1 or 2 A step of contacting a cell with a test agent in the presence of EGF, which is selected from the group consisting of polypeptides having a kinase activity equivalent to a polypeptide consisting of the amino acid sequence of:
b. Detecting CDCA5 phosphorylation at amino acid residue serine 79 or 209 of SEQ ID NO: 5, and determining the phosphorylation level of CDCA5; and c. Comparing the level determined in step (b) with the level determined in the absence of the test agent; and d. Selecting a test agent that has a reduced level of phosphorylation of CDCA5 compared to the level determined in the absence of the test agent in step (c).

  For example, the phosphorylation site of CDCA5 by ERK is Ser79 or Ser209. The method comprises contacting ERK having a kinase activity against CDCA5 or a functional equivalent thereof, CDCA5 or a functional equivalent thereof easily phosphorylated by ERK with one or more candidate compounds, and phospho-CDCA5. This is done by assaying the level. For example, a functional equivalent of CDCA5 that is susceptible to phosphorylation by ERK may include at least one of Ser79 or Ser209, which is a phosphorylation site of CDCA5 via ERK. Compounds that modulate CDCA5 phosphorylation by ERK or functional equivalents thereof are thus identified.

  Includes cell extracts, cell culture supernatants, fermented microbial products, marine organism extracts, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low molecular compounds and natural compounds. Any non-limiting test compound can be used in the screening method of the present invention. Test compounds of the present invention include (1) biological libraries, (2) spatially addressable parallel solid phase libraries or liquid phase libraries, (3) synthetic library methods that require deconvolution, Using any of a number of approaches in combinatorial library methods known in the art, including (4) “one bead one compound” library method, and (5) a synthetic library method using affinity chromatography selection. It can also be obtained. Biological library methods using affinity chromatography selection are limited to peptide libraries, but the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule library (Lam (1997 ) Anticancer Drug Des. 12: 145-67). Examples of methods for synthesizing molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al. (1993) Science 261: 1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; Gallop et al. (1994) J. Med. Chem 37: 1233-51.). A library of compounds may be provided in solution (see Houghten (1992) Bio / Techniques 13: 412-21.), Or beads (Lam (1991) Nature 354: 82-4.), Chips (Fodor (1993) Nature 364: 555-6.), Bacteria (US Pat. No. 5,223,409), spores (US Pat. Nos. 5,571,698; 5,403,484), and No. 5,223,409), plasmid (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-9.), Or phage (Scott and Smith (1990) Science 249: 386- 90 .; Devlin (1990) Science 249: 404-6 .; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378-78 .; Felici (1991) J. Mol. Biol. 222: 301-10 .; U.S. Patent Application No. 2002103360).

  Furthermore, a compound in which a part of the structure of a compound screened as inhibiting ERK kinase activity against CDCA5 or phosphorylation of CDCA5 by ERK is converted by addition, deletion, and / or substitution is also included in the present invention. It is included in the compound which can be obtained by the screening method.

  According to the present invention, it has been clarified that cell proliferation decreases when CDCA5 phosphorylation by ERK is suppressed. Therefore, a candidate compound having the ability to treat or prevent cancer can be identified by screening a candidate compound that inhibits CDCA5 phosphorylation by ERK or the kinase activity of ERK against CDCA5. The ability of these candidate compounds to treat or prevent cancer may be examined by secondary screening and / or further screening to identify cancer therapeutic agents.

  In the present invention, the therapeutic effect of a test compound can be correlated with ERK CDCA5 phosphorylation or ERK kinase activity. For example, if a test compound suppresses or inhibits CDCA5 phosphorylation by ERK or kinase activity of ERK as compared to the level detected in the absence of the test compound, the test compound is a candidate compound having a therapeutic effect. Can be specified or selected. Alternatively, a test compound has a significant therapeutic effect if it does not inhibit or inhibit CDCA5 phosphorylation or ERK kinase activity by ERK compared to the level detected in the absence of the test compound. You can specify that you do not.

One aspect of the invention provides a kit for detecting the ability of a test compound to modulate ERK kinase activity against CDCA5. Such a kit has the following components:
a. CDCA5 (polypeptide) or a functional equivalent thereof, wherein the functional equivalent of CDCA5 is:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5,
ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5, wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is derived from the amino acid sequence of SEQ ID NO: 5 A polypeptide having a kinase activity equivalent to that of the polypeptide, and iii. A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, provided that the resulting polypeptide is SEQ ID NO: 5 A polypeptide having biological activity equivalent to a polypeptide consisting of an amino acid sequence,
CDCA5 (polypeptide) or a functional equivalent thereof selected from the group consisting of:
b. ERK (polypeptide) or a functional equivalent thereof, wherein the functional equivalent of ERK is:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, and ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2 wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is SEQ ID NO: 1 or 2 ERK (polypeptide) or a functional equivalent thereof selected from the group consisting of polypeptides having a kinase activity equivalent to a polypeptide consisting of the amino acid sequence of
c. A reagent for determining the phosphorylation level of CDCA5, ie, a reagent for detecting CDCA5 phosphorylation at, for example, amino acid residue serine 79 or 209 of SEQ ID NO: 5; and d. ATP and a suitable buffer may be included.

Furthermore, the present invention also provides a kit for detecting the ability of a test compound to modulate ERK kinase activity against CDCA5. Such a kit has the following components:
a. CDCA5 or a functional equivalent thereof, and a cell expressing ERK or a functional equivalent thereof, ie, a polynucleotide encoding CDCA5 or a functional equivalent thereof, and a polynucleotide encoding ERK or a functional equivalent thereof A cell that contains and transcribes,
The functional equivalent of CDCA5 is:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5,
ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5, wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is an amino acid sequence of SEQ ID NO: 5 A polypeptide having a kinase activity equivalent to a polypeptide comprising: iii. A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, provided that the resulting polypeptide is SEQ ID NO: 5 Selected from the group consisting of polypeptides having a biological activity equivalent to a polypeptide consisting of an amino acid sequence,
The functional equivalent of ERK is:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, and ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2 wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is SEQ ID NO: 1 or 2 A cell selected from the group consisting of polypeptides having a kinase activity equivalent to a polypeptide consisting of the amino acid sequence of:
b. EGF, and c. A reagent for determining the phosphorylation level of CDCA5 may be included, for example, a reagent for detecting CDCA5 phosphorylation at amino acid residue serine 79 or 209 of SEQ ID NO: 5.

  As a reagent for determining the phosphorylation level of CDCA5, for example, an antibody that detects CDCA5 phosphorylation, in particular, CDCA5 phosphorylation at amino acid residue serine 79 or 209 of SEQ ID NO: 5 can be used. Furthermore, when a labeled ATP is included in the kit as a phosphate donor, a reagent for detecting the ATP label can be used. Each component of the kit may be placed in a separate container with a label indicating the contents of the container. Further, if desired, the kit may include instructions for performing the assay (eg, written, tape, VCR, CD-ROM, etc.).

Dominant negative protein that inhibits ERK kinase activity against CDCA5 It is a novel finding demonstrated by the present invention that CDCA5 mutants inhibit cancer cell growth. Such mutants are considered to have a dominant negative effect. In the context of the present invention, “having a dominant negative effect” means that the polypeptide inhibits CDK5 phosphorylation by ERK and subsequently suppresses cell proliferation in vivo (ie, with the polypeptide of SEQ ID NO: 7). Similar activity). A polypeptide having such a dominant negative effect is also referred to herein as “dominant negative protein”, “dominant negative mutant”, or “dominant negative CDCA5”.

  The activity of the variant polypeptide of the present invention may be lower than that of SEQ ID NO: 7, equivalent to that of SEQ ID NO: 7, or significantly higher than that of SEQ ID NO: 7. . For example, the phosphorylation level of CDCA5 by ERK is about 90%, 80%, 70%, 60 at least compared to the absence of the mutant polypeptide due to the presence of the mutant polypeptide of the present invention. %, 50%, 40%, 30%, 25%, 20%, 10%, 5%, 1%, or less (eg, 0%).

Preferably, the CDCA5 variant having a dominant negative effect may comprise an amino acid sequence in which at least one ERK-dependent phosphorylation site on CDCA5 is substituted with an amino acid residue other than serine. In the present invention, the ERK-dependent phosphorylation site on CDCA5 may be selected from the group consisting of Ser-79 and Ser-209 of CDCA5 (SEQ ID NO: 5). Accordingly, either or both of Ser-79 and Ser-209 may be substituted with an amino acid residue other than serine. For example, Ser-79 and / or Ser-209 of CDCA5 (SEQ ID NO: 5) may be substituted with alanine. Specifically, the present invention provides the following:
a. A polypeptide comprising the amino acid sequence of SEQ ID NO: 7,
b. A biological activity equivalent to that of a protein comprising the amino acid sequence of SEQ ID NO: 7, comprising the amino acid sequence of SEQ ID NO: 7, wherein one or more amino acids are substituted, deleted, inserted and / or added. Having a polypeptide, and c. A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 7 Relates to a substantially pure polypeptide selected from the group consisting of polypeptides having a biological activity equivalent to.

In the present invention, it was revealed that a polypeptide consisting of the amino acid sequence SEQ ID NO: 7 has a biological activity that inhibits ERK-mediated CDCA5 phosphorylation and cell proliferation. Furthermore, a polypeptide having a biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7 is also included in the present invention. For example, the invention provides the following polypeptides:
b. Biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7, comprising the amino acid sequence of SEQ ID NO: 7, wherein one or more amino acids are substituted, deleted, inserted and / or added A polypeptide having, and c. A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6, and is equivalent to a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 Polypeptides having pharmacological activity may be included.

  Methods and conditions for obtaining such modified polypeptides are detailed above. Furthermore, a method for evaluating whether a polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7 is also provided by the present invention (supra). Only a few amino acids or only a small percentage of amino acids can be modified (added, deleted, inserted, or substituted) to retain the necessary dominant negative effect of the CDCA5 variant consisting of the amino acid sequence of SEQ ID NO: 7. preferable. The number of residues to be modified is generally 20 amino acids or less, preferably 15 amino acids or less, more preferably 10 amino acids or less, and even more preferably 1 to 5 amino acids. Alternatively, the percentage of amino acids to be modified is preferably 20% or less, more preferably 15% or less, more preferably 10%, and even more preferably 1 to 5%. In addition to the above-described modification of the mutant polypeptide of the present invention, the mutant polypeptide of the present invention may be further linked to another substance as long as it retains a dominant negative effect. Substances that can be used for such linking include peptides, lipids, sugars and sugar chains, acetyl groups, natural and synthetic polymers, and the like. These types of modifications may be made to confer additional functions (eg, targeting and delivery functions) or to stabilize mutant polypeptides.

  For example, it is known in the art to introduce various amino acid mimetics or unnatural amino acids that are particularly useful to increase the in vivo stability of the polypeptide. This idea can also be incorporated into the mutant polypeptides of the present invention. Polypeptide stability can be assayed in a number of ways. For example, peptidases and various biological media, such as human plasma and serum, have been used to test stability (eg, Coos Verhoef et al. (1986) Eur. J. Drug Metab. Pharmacokin. 11: 291-302). For example, the activity of a polypeptide to inhibit ERK-mediated CDCA5 phosphorylation can be examined by determining the kinase activity of ERK in the presence of the polypeptide. Specifically, ERK kinase activity against CDCA5 can be determined by incubating the polypeptide with ERK and CDCA5 under conditions suitable for CDCA5 phosphorylation and detecting phospho-CDCA5 levels. The level of phosphorylation of CDCA5 may be detected as detailed above or using other well-known methods for detecting polypeptide phosphorylation. For example, phosphorylation levels can be detected by an antibody that recognizes phosphorylation at a site on the polypeptide. The phosphorylation site of CDCA5 by ERK is Ser79 or Ser209.

  Alternatively, in addition to phosphorylation of CDCA5 by ERK, the CDCA5 mutant also has an activity of inhibiting or suppressing cell proliferation. Therefore, an organism equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7 is correlated with this activity. It may be examined whether a polypeptide has a pharmacological activity (dominant negative effect). Such a dominant negative effect of the mutant polypeptide can be detected by a dominant negative assay using cells expressing both CDCA5 and ERK. Specifically, the cell viability can be detected after the polypeptide of the present invention is introduced into the cell.

  A polypeptide that reduces cell viability compared to cell viability measured in the absence of the polypeptide can be considered to have a dominant negative effect. In the present invention, the activity of a polypeptide that inhibits cell proliferation can also be compared with the activity of a polypeptide having the amino acid sequence of SEQ ID NO: 7 that inhibits cell proliferation.

  As long as CDCA5 and ERK are expressed, usable cells are not limited. Examples include, but are not limited to, cells from clinical samples and NSCLC cell lines such as A549 (ATCC CCL-185) and LC319 (Aichi Cancer Center).

  Cell proliferation may be measured by methods known in the art, for example using the MTT cell proliferation assay. Briefly, cell-counting kit-8 solution (DOJINDO) is added to each dish at a concentration of 1/10 volume and the plate is incubated at 37 ° C. for an additional 2 hours. The absorbance is then measured at 450 nm as a reference using a Microplate Reader 550 (BIO-RAD, Hercules, CA).

  Introduction of the polypeptide of the present invention into a cell may be performed by transfecting a vector designed to express the polypeptide in the cell. Alternatively, a polypeptide to be investigated for activity may be introduced into a living cell using a cell membrane permeable substance described below. Alternatively, the introduction may be performed by modifying the polypeptide so that it penetrates into the cell and incubating the modified polypeptide with the cell. Such polypeptide modifications include linking with cell penetrating peptides, attachment on the surface of microparticles (eg, metal particles), and encapsulation in liposomes.

The mutant polypeptides of the present invention can be chemically synthesized based on the selected amino acid sequence. Conventional methods used in peptide chemistry can be used in the methods for synthesizing the mutant polypeptides of the present invention. In particular, the methods include those described in the following documents and Japanese patent publications:
Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press Inc., New York, 1976;
Peptide Synthesis, Maruzen (Inc.), 1975;
Fundamental and Experimental Peptide Synthesis, Maruzen (Inc.), 1985;
Development of Pharmaceuticals, Vol. 14: Peptide Synthesis, Hirokawa Shoten, 1991; and International Publication No. WO99 / 67288.

  The mutant polypeptides of the present invention can also be synthesized by known genetic engineering techniques. An example of genetic engineering technology is as follows. Specifically, a DNA encoding a desired peptide is introduced into an appropriate host cell to prepare a transformed cell. The mutant polypeptide of the present invention can be obtained by recovering the polypeptide produced by this transformed cell. Alternatively, the desired polypeptide can be synthesized in vitro using an in vitro translation system in which the elements necessary for protein synthesis are reconstituted.

When genetic engineering techniques are used, the mutant polypeptide of the present invention can be expressed as a fusion protein having peptides having different amino acid sequences. A vector that expresses the desired fusion protein is ligated with a polynucleotide that encodes a different peptide from the polynucleotide that encodes the mutant polypeptide of the invention, so that it is in the same reading frame, and then the resulting polynucleotide. It can be obtained by introducing a nucleotide into an expression vector. The fusion protein is expressed by transforming a suitable host with the resulting vector. Different peptides that are particularly useful for the formation of fusion proteins include the following peptides:
FLAG (Hopp et al., (1988) BioTechnology 6, 1204-10),
6xHis consisting of 6 His (histidine) residues,
10xHis,
Influenza hemagglutinin (HA),
Human c-myc fragment,
VSV-GP fragment,
p18 HIV fragment,
T7-tag,
HSV-tag,
E-tag,
SV40T antigen fragment,
lck tag,
α-tubulin fragment,
B-tag,
Protein C fragment,
GST (glutathione-S-transferase),
HA (influenza hemagglutinin),
An immunoglobulin constant region,
β-galactosidase, and MBP (maltose binding protein).

  The mutant polypeptide of the present invention can be obtained by treating the fusion protein thus produced with an appropriate protease and then recovering the desired polypeptide. To purify the polypeptide, the fusion protein can be pre-captured by affinity chromatography that binds to the fusion protein, and then the captured fusion protein can be treated with a protease. The desired polypeptide is separated from the affinity chromatography resin by protease treatment, and the high-purity desired polypeptide is recovered.

  Variant polypeptides of the present invention include modified polypeptides. In the present invention, the term “modified” refers to, for example, binding to other substances. Therefore, in the present invention, the mutant polypeptide of the present invention may further contain another substance such as a cell membrane permeable substance. Other materials include organic compounds such as peptides, lipids, saccharides, and various natural or synthetic polymers. The mutant polypeptide of the present invention may have any modification as long as it retains the desired activity of inhibiting ERK kinase activity against CDCA5. In some embodiments, the inhibitory polypeptide can directly compete with ERK binding to CDCA5. Modifications can also confer additional functions to the mutant polypeptides of the invention. Examples of additional functions include targetability, deliverability, and stabilization.

  Preferred examples of the modification of the mutant polypeptide in the present invention include, for example, introduction of a cell membrane permeable substance. Usually, the intracellular structure is blocked from the outside by the cell membrane. Therefore, it is difficult to efficiently introduce extracellular substances into cells. Cell membrane permeability can be imparted to the mutant polypeptide of the present invention by modifying the polypeptide with a cell membrane permeable substance. As a result, by contacting the mutant polypeptide of the present invention with a cell, the polypeptide can be delivered to the cell and act on the cell.

A “cell membrane permeable substance” refers to a substance that can penetrate the mammalian cell membrane to enter the cytoplasm. For example, certain liposomes fuse with the cell membrane and release the contents into the cell. On the other hand, a specific type of polypeptide penetrates the cell membrane of mammalian cells and enters the cells. In the present invention, a cytoplasmic membrane or the like is preferable as a substance for such a polypeptide having an activity of entering cells. In particular, the present invention includes variant polypeptides having the general formula:
[R]-[D] or [D]-[R];
Where
[R] is a cell membrane permeable substance (membrane transducing agent), and [D] is a fragment sequence containing SEQ ID NO: 7. In the above general formula, [R] and [D] may be directly connected or indirectly connected via a linker. Peptides and compounds having a plurality of functional groups and the like can be used as a linker. Specifically, for example, an amino acid sequence containing -GGG- can be used as a linker. Alternatively, a cell membrane permeable substance and a polypeptide containing a selected sequence may be bound to the surface of the microparticle. [R] can be linked to any position of [D]. Specifically, [R] may be linked to the N-terminus or C-terminus of [D], or may be linked to the side chain of the amino acid constituting [D]. Furthermore, a plurality of [R] molecules may be linked to one molecule of [D]. In this case, the [R] molecule can be introduced at different positions on the [D] molecule. Alternatively, [D] may be modified by linking a number of [R]. For example, various natural polypeptides or artificially synthesized polypeptides having cell membrane permeability have been reported (Joliot A. & Prochiantz A., Nat Cell Biol. 2004; 6: 189-96). All of these known cell membrane permeable substances can be used to modify the mutant polypeptides in the present invention.

The membrane introduction agent can be selected from the group listed below.
[Poly-arginine], Matsushita, M. et al, J Neurosci. 21, 6000-7 (2003);
[Tat / RKKRRQRRR] (SEQ ID NO: 6) Frankel, A. et al, Cell 55, 1189-93 (1988) and Green, M. & Loewenstein, PM Cell 55, 1179-88 (1988);
[Penetratin / RQIKIWFQNRRMKWKK] (SEQ ID NO: 7), Derossi, D. et al, J. Biol. Chem. 269, 10444-50 (1994);
[Bufolin II / TRSSRAGLQFPVGRVHRLLRK] (SEQ ID NO: 8) Park, CB et al. Proc. Natl Acad. Sci. USA 97, 8245-50 (2000);
[Transport / GWTLNSAGYLLLGKINLKALALAAKIL] (SEQ ID NO: 9) Pooga, M. et al. FASEB J. 12, 67-77 (1998);
[MAP (model amphiphilic peptide) / KLALKLALKALKALKALA] (SEQ ID NO: 10), Oehlke, J. et al. Biochim. Biophys. Acta. 1414, 127-39 (1998);
[K-FGF / AAVALLPAVLLALLAP] (SEQ ID NO: 11), Lin, YZ et al. J. Biol. Chem. 270, 14255-14258 (1995);
[Ku70 / VPMLK] (SEQ ID NO: 12), Sawada, M. et al. Nature Cell Biol. 5, 352-7 (2003);
[Ku70 / PMLKE] (SEQ ID NO: 13), Sawada, M. et al. Nature Cell Biol. 5, 352-7 (2003);
[PRION / MANLGYWLLALFVTMWTDGLGLCKKKRPKP] (SEQ ID NO: 14), Lundberg, P. et al. Biochem. Biophys. Res. Commun. 299, 85-90 (2002);
[PVEC / LLIILRRRIRKQAHAHSK] (SEQ ID NO: 15), Elmquist, A. et al. Exp. Cell Res. 269, 237-44 (2001);
[Pep-1 / KETWWETWWTEQPQPKKRKV] (SEQ ID NO: 16), Morris, MC et al. Nature Biotechnol. 19, 1173-6 (2001);
[SynB1 / RGGRLSYSRRRRSTSTGR] (SEQ ID NO: 17), Rousselle, C. et al. Mol. Pharmacol. 57, 679-86 (2000);
[Pep-7 / SDLWEMMVSLACQY] (SEQ ID NO: 18), Gao, C. et al. Bioorg. Med. Chem. 10, 4057-65 (2002); and [HN-1 / TSPLNIHNGQKL] (SEQ ID NO: 19), Hong, FD & Clayman, GL Cancer Res. 60, 6551-6 (2000).

  In the present invention, the number of arginine residues constituting poly-arginine is not limited. In some preferred embodiments, 5-20 consecutive arginine residues may be exemplified. In a preferred embodiment, the number of arginine residues in poly-arginine is 11 (SEQ ID NO: 22).

  Furthermore, the present invention provides isolated polynucleotides encoding the dominant negative proteins of the present invention, and vectors and host cells containing the polynucleotides. Polynucleotides, vectors, and host cells have already been defined above.

Treatment and Prevention of Lung Cancer or Esophageal Cancer Using Dominant Negative Mutants The dominant negative mutants of CDCR5 disclosed herein can be used to treat or prevent lung cancer or esophageal cancer. . For example, the present invention relates to the treatment and treatment of lung cancer or esophageal cancer in a subject by administering a CDCA5 variant having a dominant negative effect, a polynucleotide encoding such a variant, or a vector comprising the polynucleotide. Provide a method for either or both prevention.

  In some preferred embodiments, the CDCA5 variant is linked to a membrane transducing agent for administration to a subject. Numerous peptide sequences have been characterized for their ability to enter living cells and can be used for this purpose in the present invention. Such membrane transduction agents (typically peptides) are defined by their ability to reach the cytoplasmic and / or nuclear compartments of living cells after internalization. Examples of proteins from which transduction agents can be obtained include HIV Tat transactivators 1 and 2, Drosophila melanogaster transcription factor antennapedia 3. Furthermore, non-natural peptides having introduction activity are used. These peptides are typically small peptides known for membrane interaction properties that have been tested for migration. A hydrophobic region within the secretory signal sequence of K-fibroblast growth factor (FGF), venom toxin mastoparan (transportan) 13, and buforin 114 (an amphibian antimicrobial peptide) have been shown to be useful as transducing agents. ing. For a review of introduction agents useful in the present invention, see Joliot et al. Nature Cell Biology 6: 189-96 (2004).

The CDCA5 variant used for treatment or prevention is preferably of the following general formula:
[R]-[D] or [D]-[R]
You may have. In the formula, as defined in the section “Dominant negative protein that inhibits ERK kinase activity against CDCA5”, [R] is a membrane-introducing agent, and [D] is an amino acid sequence of SEQ ID NO: 7. A polypeptide having

  In another aspect, the present invention provides a method for treating or preventing lung cancer or esophageal cancer in a subject comprising administering a vector expressing a CDCA5 variant of the present invention. In this embodiment, such a vector expresses CDCA5 in the cell, so that the CDCA5 variant can be introduced into the cell without using a membrane transducing agent. In order to express the vector in animal cells such as CHO, COS, or NIH3T3 cells, the vector may be a promoter required for expression in such cells, eg the SV40 promoter (Mulligan et al., Nature 277: 108 (1979)), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), CMV promoter, etc., preferably a marker gene for selecting a transformant (for example, a drug (For example, a drug resistance gene selected by neomycin, G418). Examples of known vectors having these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

Pharmaceutical Compositions The present invention further relates to lung cancer or esophagus comprising a pharmaceutically effective amount of a compound that reduces ERK kinase activity against CDCA5 or inhibits phosphorylation of CDCA5 by ERK, and a pharmaceutically acceptable carrier. Compositions for treating or preventing cancer are provided. Whether or not the compound of the present invention has the target activity can be determined, for example, according to the screening method of the present invention. For example, kinase activity against CDCA5 can be determined by incubating a compound of the invention under conditions suitable for CDCA5 phosphorylation and detecting phospho-CDCA5 levels. The phosphorylation site of CDCA5 by ERK can be Ser79 or Ser209.

Furthermore, the dominant negative polypeptide of the present invention suppresses ERK-dependent phosphorylation on CDCA5. Therefore, according to another aspect of the present invention, the pharmaceutical composition of the present invention comprising either the dominant negative polypeptide of the present invention or a polynucleotide encoding this polypeptide comprises ERK-dependent phosphorylation of CDCA5. Can be used to inhibit. Furthermore, the inventors have shown that ERK-dependent phosphorylation for CDCA5 is essential for the growth and / or survival of lung cancer cells or esophageal cells. Accordingly, a pharmaceutical composition of the invention consisting of any of the aforementioned polypeptides or polynucleotides can be used to treat or prevent lung cancer or esophageal cancer, particularly NSCLC. That is, in one embodiment of the present invention, the present invention provides:
[1] A composition for treating or preventing lung cancer or esophageal cancer, which is a pharmaceutically effective amount of a CDCA5 mutant having a dominant negative effect as an active ingredient, a polynucleotide encoding the mutant, or the A composition comprising a vector comprising a polynucleotide and a pharmaceutically acceptable carrier;
[2] The composition of [1], wherein the CDCA5 variant comprises an amino acid sequence in which at least one ERK-dependent phosphorylation site in CDCA5 is substituted with an amino acid residue other than the wild type.
[3] The composition of [2], wherein the ERK-dependent phosphorylation site is one or both of Ser-79 and Ser-209,
[4] The composition of [3], wherein the CDCA5 variant comprises the amino acid sequence of SEQ ID NO: 7;
[5] A CDCA5 variant having the general formula: [R]-[D], wherein [R] is a membrane-introducing agent and [D] is a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 The composition according to [4],
[6] The membrane introduction agent is as follows:
Poly-arginine;
Tat / RKKRRQRRR / SEQ ID NO: 8;
Penetratin / RQIKIWFQNRRMKWKK / SEQ ID NO: 9;
Buforin II / TRSSRAGLQFPVGRVHRRLRK / SEQ ID NO: 10;
Transportan / GWTLNSAGYLLLGKINLKALAALAKIL / SEQ ID NO: 11;
MAP (Model Amphiphilic Peptide) / KLALKLALKALKALKALA / SEQ ID NO: 12;
K-FGF / AAVALLPAVLLALLAP / SEQ ID NO: 13;
Ku70 / VPMLK / SEQ ID NO: 14;
Ku70 / PMLKE / SEQ ID NO: 15;
Prion / MANLGYWLLALFVTMWTDVGLCKKKRPKP / SEQ ID NO: 16;
pVEC / LLIILRRRIRKQAHAHSK / SEQ ID NO: 17;
Pep-1 / KETWWETWWTEWSQPKKRKV / SEQ ID NO: 18;
SynB1 / RGGRRLSYSRRRRSTSTGR / SEQ ID NO: 19;
Pep-7 / SDLWEMMVSLACQY / SEQ ID NO: 20; and HN-1 / TSPLNIHNGQKL / SEQ ID NO: 21
The composition of [5] selected from the group consisting of:

  Since the dominant negative polypeptide of the present invention functions to suppress the growth of lung cancer cells or esophageal cancer cells, the polypeptide in the pharmaceutical composition needs to be introduced into the cells to produce an effect. Thus, suitable polypeptides for pharmaceutical compositions include polypeptides that have been modified to penetrate into cells. Examples of such modifications include linking with cell penetrating peptides, attachment on the surface of microparticles (eg, metal particles), and encapsulation within liposomes, as detailed above. It is not limited to this.

  In another aspect, the invention also provides the use of a dominant negative polypeptide of the invention or a compound screened by the invention in the manufacture of a pharmaceutical composition for the treatment of cancers such as lung cancer and esophageal cancer. provide.

  Alternatively, the present invention further provides a dominant negative polypeptide of the present invention or a compound screened by the present invention for use in the treatment of cancers such as lung cancer and esophageal cancer. Alternatively, the present invention further provides a pharmaceutical for treating cancers such as lung cancer and esophageal cancer, comprising a step of formulating a pharmaceutically or physiologically acceptable carrier and a polypeptide or compound as an active ingredient. A method or process for manufacturing a pharmaceutical composition is provided. In another embodiment, the present invention also includes the step of mixing the active ingredient with a pharmaceutically or physiologically acceptable carrier, the active ingredient being screened by the dominant negative polypeptide of the present invention or the present invention. Also provided are methods or processes for making a pharmaceutical composition for treating cancer, such as lung cancer and esophageal cancer, which are compounds.

  The pharmaceutical compositions of the present invention also include mice, rats, guinea pigs, rabbits, cats, dogs, sheep, goats, pigs, cows, horses, monkeys, baboons, and chimpanzees, especially commercially important animals or livestock. Including but not limited to, it may also be used to treat and / or prevent disorders in humans and any other mammal.

  The pharmaceutical composition of the present invention contains a pharmaceutically effective amount of an active ingredient (a dominant negative polypeptide of the present invention, a polynucleotide encoding the polypeptide, or a compound isolated by the screening method of the present invention). A “pharmaceutically effective amount” of a compound (including proteins and polynucleotides) is an amount sufficient to treat and / or prevent a disorder of interest for which ERK-dependent phosphorylation for CDCA5 plays an important role. An example of a pharmaceutically effective amount may be the amount necessary to treat or prevent a disorder by reducing the level of ERK-dependent phosphorylation for CDCA5 when administered to a patient. A decrease in phosphorylation level is, for example, a decrease of at least about 5%, 10%, 20%, 30%, 40%, 50%, 75%, 80%, 90%, 95%, 99%, or 100% But you can. Alternatively, a pharmaceutically effective amount may be an amount that leads to a decrease in the size, prevalence, or metastatic potential of lung or esophageal cancer in a subject. Further, when the pharmaceutical composition of the present invention is applied prophylactically, the “pharmaceutically effective amount” may be an amount that delays or prevents the occurrence of lung cancer or esophageal cancer, or clinical practice of lung cancer or esophageal cancer. It may be an amount that reduces symptoms.

  To determine such a pharmaceutically effective amount of a polypeptide or polynucleotide of the present invention, using standard clinical protocols including histopathological diagnosis or chronic cough, hoarseness, hemoptysis, weight loss, Lung cancer or esophageal cancer can be assessed by confirming symptomatic abnormalities such as decreased appetite, shortness of breath, wheezing, repeated attacks of bronchitis or pneumonia, and chest pain.

The dose used will depend on many factors, including the age and sex of the subject, the exact disorder being treated, and its severity. In addition, the route of administration may vary depending on the condition and its severity. However, the determination of an effective dosage range for the pharmaceutical agents of the present invention is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein. A pharmaceutically or prophylactically effective amount (dose) of a dominant negative polypeptide of the invention, a polynucleotide encoding the polypeptide, or a compound isolated by the screening method of the invention is first determined in cell culture. It can be estimated from assays and / or animal models. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 50 as determined in cell culture _(dose showing 50% of the desired effect of the cells). Toxicity and therapeutic efficacy of a polypeptide, polynucleotide, or compound is also therapeutically effective in cell cultures or experimental animals, eg, LD ₅₀ (dose that causes 50% of the population to die) and ED ₅₀ (50% of the population). It can also be determined by standard pharmaceutical procedures for determining (dose). The dose ratio between toxic and therapeutic effects is the therapeutic index (ie, the ratio between LD ₅₀ and ED ₅₀ ). Polypeptides, polynucleotides, and compounds that exhibit high therapeutic indices are preferred. Data obtained from these cell culture assays and animal studies may be used in dosage range formulations for use in humans. The dosage of such polypeptides and polynucleotides can be within a range of circulating concentrations including ED ₅₀ with little or no toxicity. Within this range, dosage may vary depending on the unit dosage form used and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (eg, Fingl et al. (1975) in “The Pharmacological Basis of Therapeutics”, Ch. 1 (See p1). Dosage amount and interval may be adjusted individually to provide plasma levels of the active ingredient which are sufficient to maintain the desired effects.

  If necessary, the pharmaceutical composition of the present invention comprising a dominant negative polypeptide of the present invention, a polynucleotide encoding the polypeptide, or a compound isolated by the screening method of the present invention may optionally contain other Other therapeutic agents may be included as active ingredients as long as the therapeutic agent does not inhibit the in vivo dominant negative effect of the polypeptide of interest. For example, the formulation may include anti-inflammatory agents, analgesics, chemotherapeutic agents and the like. In addition to including other therapeutic substances in the medical agent itself, the medical agents of the present invention may also be administered sequentially or concurrently with one or more other pharmacological agents. The amount of medical and pharmacological agent depends, for example, on the type of pharmacological agent used, the disease being treated, and the dosage regimen and route of administration.

  In addition to the ingredients, particularly those mentioned herein, it is understood that the formulations of the present invention may include other agents conventional in the art in view of the type of formulation in question. Should be done.

  In one aspect of the present invention, the pharmaceutical composition of the present invention may be included in articles of manufacture and kits comprising materials useful for treating lung cancer or esophageal cancer, particularly NSCLC pathological conditions. . The article of manufacture may include a container of any pharmaceutical composition of the present invention with a label. Suitable containers include bottles, vials, and test tubes. The container may be formed from a variety of materials such as glass or plastic. The label on the container must indicate that the composition is used to treat or prevent one or more conditions of the disease. The label may also indicate directions such as administration.

  In addition to the container described above, the kit containing the pharmaceutical composition of the present invention may optionally further comprise a second container that exempts a pharmaceutically acceptable diluent. The kit may further include other materials desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

  The pharmaceutical composition can be placed in a packaging or dispenser device that can contain one or more unit dosage forms containing the active ingredients, if desired. The packaging may include metal foil or plastic foil, such as blister packaging. The packaging or dispenser device may be accompanied by instructions for administration.

(1) A pharmaceutical composition comprising a polypeptide as an active ingredient:
The present invention provides a pharmaceutical composition comprising the dominant negative polypeptide of the present invention as an active ingredient. Since the polypeptide of the present invention exerts the function of suppressing the growth of CDCA5-expressing cells, the polypeptide in the pharmaceutical composition needs to be introduced into the cell to produce an effect. Thus, suitable polypeptides for pharmaceutical compositions include polypeptides that have been modified to penetrate into cells. Examples of such modifications include, but are not limited to, conjugation with cell penetrating peptides, attachment of microparticles (eg, metal particles) onto the surface, and encapsulation within liposomes. The use of cell penetrating peptides is particularly preferred for the pharmaceutical compounds of the invention.

  To treat and / or prevent cancers such as lung cancer and esophageal cancer, the dominant negative polypeptide of the present invention may be administered directly to a patient as a pharmaceutical composition or formulated according to conventional pharmaceutical methods. May be. For example, the polypeptide may be administered orally, rectally, nasally, topically (including buccal and sublingual), vaginal or parenteral (intramuscular, subcutaneous, intravenous) as needed. Administration, including intratumoral administration) or in a form suitable for administration by inhalation gas infusion. Accordingly, the present invention includes pharmaceutical compositions comprising any pharmaceutically acceptable excipient or carrier in addition to the polypeptide. The phrase “pharmaceutically acceptable” indicates that the substance is inert and includes conventional substances used as drug diluents or vehicles. Suitable excipients and formulations thereof are described, for example, in Remington's Pharmaceutical Sciences, 16th ed. (1980) Mack Publishing Co., ed. Oslo et al.

  For example, excipients may be used to maintain the correct pH of the formulation. For optimal shelf life, the pH of the solution containing the polypeptide is preferably about 5 to about 8, more preferably about 7 to about 7.5. The formulation may also include a lyophilized powder or any other excipient suitable for the present invention.

  For aqueous preparations, an appropriate amount of a pharmaceutically acceptable salt is typically used in the formulation to make the formulation isotonic. Examples of pharmaceutically acceptable isotonic excipients include, but are not limited to, liquids such as saline, Ringer's solution, Hank's solution, and dextrose solution. Isotonic excipients are particularly important for injectable formulations.

  Pharmaceutical formulations suitable for oral administration include, but are not limited to, capsules, cachets and tablets each containing a predetermined amount of the active ingredient. Formulations also include drugs, liquids, gels, syrups, slurries, pills, powders, granules, solutions, suspensions, emulsions and the like. The active ingredient is optionally administered as a bolus, electuary or paste. Tablets and capsules for oral administration may contain conventional excipients such as binders, fillers, lubricants, disintegrants, and wetting agents. Suitable excipients are in particular sugars including bulking agents such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxy Propylmethyl-cellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof such as sodium alginate. A tablet may be made by compression or molding, optionally with one or more pharmaceutical ingredients. Compressed tablets mix an active ingredient in a freely flowable form such as a powder or granules, optionally with a binder, lubricant, inert diluent, lubricant, surfactant, or dispersant. And may be prepared by compression in a suitable machine. A wet tablet can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets can be coated according to methods well known in the art. Oral liquid preparations may take the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or dry products for reconstitution with water or other suitable vehicle before use. May be provided as Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), and preservatives. Depending on the formulation or dose of the medicinal agent in these preparations, an appropriate dosage within the indicated range can be obtained.

  Although the dosage can vary depending on the symptoms, an exemplary dose of a polypeptide or fragment thereof for treating or preventing lung cancer or esophageal cancer is given when administered to a normal adult (body weight 60 kg) About 0.1 mg to about 100 mg / day, preferably about 1.0 mg to about 50 mg / day, more preferably about 1.0 mg to about 20 mg / day.

  When administered parenterally in the form of injections to normal adults (body weight 60 kg), there are some differences depending on the disease state, patient symptoms, and administration method, but about 0.01 mg to about 30 mg / day, It is convenient to inject a dose preferably from about 0.1 to about 20 mg / day, more preferably from about 0.1 to about 10 mg / day. Even in the case of animals, an amount converted to 60 kg body weight can be administered.

  Formulations for parenteral administration include aqueous and non-aqueous sterile injectable solutions that may further include antioxidants, buffers, bacteriostats, and solutes that make the target recipient's blood and formulation isotonic. It is. Similarly, aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents are included. The formulation may be provided in unit-dose containers or multi-dose containers, such as sealed ampoules and vials, only added immediately prior to use of a sterile liquid carrier such as saline, water for injection. It may be stored in the necessary freeze-dried (freeze-dried) state. Alternatively, the formulation may be provided for continuous infusion. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

  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, buccal or sublingual, include sucrose and lozenges containing the active ingredient in a flavored base such as gum arabic or gum tragacanth and gelatin, glycerin, sucrose, or arabic A lozenge containing the active ingredient in a base such as rubber is included. For intranasal administration of the active ingredient, liquid sprays or dispersible powders or drops can be used. Drops may be formulated with an aqueous or non-aqueous base that also contains one or more dispersants, solubilizers, or suspending agents.

  For administration by inhalation, the composition is conveniently delivered from an insufflator, nebulizer, pressurized pack, or other convenient means of delivering an aerosol spray. Pressurized packs typically contain a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve that delivers a fixed amount. Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder composition, for example a powder mixture of the active ingredient with a suitable powder base such as lactose or starch. Powder compositions may be provided in unit dosage forms such as capsules, cartridges, gelatin or blister packs, from which powders can be administered with the aid of an inhaler or insufflator.

  Other formulations include implantable devices and adhesive patches that release therapeutic agents.

  If desired, the formulations described above adapted for sustained, controlled release or sustained release of the active ingredient in vivo may be used.

(2) A pharmaceutical composition comprising a polynucleotide as an active ingredient:
Furthermore, the present invention provides a pharmaceutical composition comprising, as an active ingredient, a polynucleotide encoding the dominant negative polypeptide of the present invention in an expressible form. As used herein, the phrase “in expressible form” means that once a polynucleotide is introduced into a cell, it will be expressed in vivo as a polypeptide having a dominant negative effect. In a preferred embodiment, the nucleic acid sequence of the polynucleotide of interest includes regulatory elements necessary for expression of the polynucleotide in the target cell. This polynucleotide may be provided for stable insertion into the genome of the target cell (eg, Thomas KR & Capecchi MR (1987) Cell 51: 503-12 for a description of homologous recombination cassette vectors). See).

  Delivery of the polynucleotide to the patient may be direct delivery where the patient is directly exposed to the vector carrying the polynucleotide, or cells are first transformed in vitro with the polynucleotide of interest and then transplanted into the patient. Indirect delivery may also be used. Each of these two approaches is known as in vivo gene therapy or ex vivo gene therapy.

  For a review of gene therapy, see Goldspiel et al., (1993) Clinical Pharmacy 12: 488-505; Wu and Wu (1991) Biotherapy 3: 87-95; Tolstoshev (1993) Ann. Rev. Pharmacol. Toxicol. 33: 573-96; see Mulligan (1993) Science 260: 926-32; Morgan & Anderson (1993) Ann. Rev. Biochem. 62: 191-217; and (1993) Trends in Biotechnology 11 (5): 155-215 I want to be. Available methods generally known in the technical field of recombinant DNA technology are described in Ausubel et al. (1993) Current Protocols in Molecular Biology, John Wiley & Sons, NY; and Krieger (1990) Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

(3) Pharmaceutical composition comprising a compound selected by the screening method of the present invention The present invention is for treating or preventing lung cancer or esophageal cancer comprising any compound selected by the screening method of the present invention. A composition is provided.

  Administration of compounds isolated by the methods of the present invention as pharmaceuticals for humans and other mammals such as mice, rats, guinea pigs, rabbits, cats, dogs, sheep, pigs, cows, monkeys, baboons, and chimpanzees If so, the isolated compounds can be administered directly or prepared into dosage forms using conventional pharmaceutical methods of preparation. For example, depending on needs, the drug may be orally administered as a sugar-coated tablet, capsule, elixir and microcapsule, or parenterally with sterile liquid or sterile suspension using water or any other pharmaceutically acceptable liquid. It can take the form of a suspension injection. For example, the compound may be administered in a pharmaceutically acceptable carrier or medium, in particular sterile water, physiological saline, vegetable oil, emulsifier, suspension, in unit dosage forms generally required for acceptable dosage administration. , Surfactants, stabilizers, fragrances, excipients, solvents, preservatives, binders and the like. When the amount of active ingredient in these formulations is used, an appropriate dosage within the indicated range can be obtained.

  Examples of additives that can be mixed to form tablets and capsules include, for example, binders such as gelatin, corn starch, gum tragacanth, and gum arabic, 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 flavors such as peppermint, galtheria adenothrix oil, and cherries. Where the unit dosage form is a capsule, a liquid carrier such as oil can also be included in the above components. Sterile compositions for injection can be formulated according to normal drug practice using vehicles such as distilled water used for injection.

  Other isotonic solutions containing physiological saline, glucose, and adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as an aqueous solution for injection. These can be used in combination with suitable solubilizers such as alcohols, especially polyalcohols such as ethanol, propylene glycol and polyethylene glycol, polysorbate 80 ™ and nonionic active agents such as HCO-50. Sesame oil and soybean oil are examples of suitable oily liquids and can be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer. These may be further formulated with a buffer such as a phosphate buffer or sodium acetate buffer, an analgesic such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The prepared injection solution may be filled into a suitable ampoule.

  In order to administer the pharmaceutical composition of the present invention to a patient, methods well known to those skilled in the art, such as intraarterial injection, intravenous injection, or transdermal injection, and intranasal administration, transbronchial administration, intramuscular administration, Or oral administration may be used. The dosage and method of administration can vary depending on the weight and age of the patient and the selected method of administration. However, one of ordinary skill in the art can routinely select an appropriate method of administration and dosage. If the compound can be encoded by DNA, the DNA can be inserted into a gene therapy vector and the vector administered to a patient for treatment. The dosage and method of administration can also vary depending on the patient's weight, age, and symptoms. However, those skilled in the art can optimally select them.

  For example, the dose of a compound that binds to ERK and modulates its activity or inhibits phosphorylation of CDCA5 depends on the symptoms, but when administered orally to a normal adult (body weight 60 kg), the appropriate dose is generally To about 100 mg / day, preferably about 1.0 mg to about 50 mg / day, more preferably about 1.0 mg to about 20 mg / day.

  In the case of parenteral administration, in the form of injection into a normal adult (body weight 60 kg), depending on the patient, target organ, symptom and administration method, it is about 0.01 mg to about 30 mg / day, preferably about 0.1 to 0.1 mg. Intravenous injection of about 20 mg / day, and more preferably about 0.1 to about 10 mg / day is convenient. In the case of other animals, it is possible to administer an amount converted to 60 kg body weight.

  The present invention includes pharmaceutical or therapeutic compositions comprising one or more therapeutic compounds described herein. The pharmaceutical formulation is oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous), or by inhalation or gas infusion. One suitable for administration may be included. The formulations are conveniently provided in individual dosage units as appropriate, and may be prepared by any method conventionally used in the pharmaceutical arts. All pharmaceutical methods herein include a liquid carrier or a finely divided solid carrier, optionally bringing both the active compound and a compound together, and if necessary, shaping the product into the desired formulation. Including.

  Pharmaceutical formulations suitable for oral administration can conveniently be presented as discrete units, such as capsules, cachets or tablets, each active as a powder or granule, as a solution, suspension, or emulsion. Contains a predetermined amount of ingredients. The active ingredient may also be provided as a bolus, electuary or paste and may be present in pure form, ie without a carrier. Tablets and capsules for oral administration may contain conventional excipients such as binders, bulking agents, lubricants, disintegrating agents, or wetting agents. A tablet may be made by compression or molding, optionally with one or more pharmaceutical ingredients. Compressed tablets are prepared by mixing the free flowing active ingredient, such as powders or granules, optionally with a binder, lubricant, inert diluent, lubricant, surfactant, or dispersant. You may prepare by compressing in a machine. A wet tablet may be made by 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. Oral liquid preparations exist, for example, in the form of aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or for constitution with water or other suitable vehicle prior to use. It may be provided as a dry product. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) or preservatives. Furthermore, the tablets may optionally be formulated for delayed or controlled release of the active ingredient therein.

  Formulations for parenteral administration include aqueous and non-aqueous sterile injectable solutions that may contain antioxidants, buffers, bacteriostats, and solutes that make the target recipient's blood and formulation isotonic, and suspensions. Aqueous and non-aqueous sterile suspensions that may contain turbidity and thickening agents are included. Formulations may be provided in unit-dose containers or multi-dose containers, such as sealed ampoules and vials, requiring only the addition of a sterile liquid carrier such as saline or water for injection just prior to use. May be stored in a freeze-dried state. Alternatively, the formulation may be provided for continuous infusion. Extemporaneous injection liquids and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

  Rectal preparations may be provided as suppositories with standard carriers such as cocoa butter or polyethylene glycol. Formulations for topical administration in the mouth, for example, buccal or sublingual, include lozenges containing the active ingredient in a flavored base such as sucrose and gum arabic or tragacanth, and gelatin and glycerin or sucrose and gum arabic. A lozenge containing the active ingredient in the base is included. For intranasal administration, the compounds of the invention can be used as liquid sprays or dispersible powders or in the form of drops. Drops may be formulated with an aqueous or non-aqueous base that also contains one or more dispersants, solubilizers, or suspending agents. Liquid sprays are conveniently delivered from pressurized packs.

  For administration by inhalation, the compounds of the invention are conveniently delivered from a convenient means of delivering an insufflator, nebulizer, pressurized pack or other aerosol spray. The pressurized pack may contain a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve that delivers a fixed amount.

  Alternatively, for administration by inhalation or insufflation, the compounds of the invention may take the form of a dry powder composition, for example a powder mixture of the compound and a suitable powder base such as lactose or starch. Powder compositions may be provided in unit dosage forms such as capsules, cartridges, gelatin or blister packs, from which powders can be administered with the aid of an inhaler or insufflator.

  If desired, the above formulations adapted to provide sustained release of the active ingredient may be used. The pharmaceutical compositions of the present invention may also contain other active ingredients such as antibacterial agents, immunosuppressive agents, or preservatives.

  In addition to the ingredients specifically described above, the formulations of the present invention may include other agents conventionally known in the art in view of the type of formulation concerned, for example, formulations suitable for oral administration are perfumes. It should be understood that may be included.

  Preferred unit dosage formulations are those containing an effective amount of the active ingredients described below or a suitable portion thereof.

  For each of the aforementioned conditions, the composition may be administered orally or by injection at a dose of about 0.1 to about 250 mg / kg / day. The dose range for human adults is usually 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. Presented tablets or other unit dosage forms provided in separate units conveniently comprise a unit comprising such a dose or an effective amount thereof, for example from about 5 mg to about 500 mg, usually from about 100 mg to about 500 mg. Can be included.

  The pharmaceutical composition is preferably administered orally or by injection (intravenous or subcutaneous) and the exact amount administered to the subject is the responsibility of the attending physician. However, the dose used will depend on many factors, including the age of the subject, sex, the particular disorder being treated, and its severity. In addition, the route of administration can vary depending on the condition and its severity.

Methods to assess cancer prognosis:
The present invention relates to a novel discovery that CDCA5 expression is significantly associated with poor patient prognosis. Therefore, the present invention detects the expression level of the CDCA5 gene in a biological sample of a patient having cancer, particularly lung cancer, compares the detected expression level with a control level, and has a poor prognosis (low survival rate). Provides a method for determining or assessing the prognosis of a patient by determining an increase in expression level relative to a control level.

  As used herein, the term “prognosis” refers to predictions about the probable outcome of the disease and the likelihood of recovery from the disease, as indicated by the nature and symptoms of the case. Thus, an unfavorable, bad, or poor prognosis is defined by a decrease in survival time or survival after treatment. Conversely, a good, favorable or good prognosis is defined by an increase in survival or survival after treatment.

  The term “evaluating prognosis” predicts, predicts, or correlates with a patient's future cancer outcomes (eg, malignancy, likelihood of cancer cure, survival, etc.) Refers to the ability to attach. For example, by obtaining the expression level of CDCA5 over time, it becomes possible to predict the outcome (for example, increase / decrease in malignancy, increase / decrease in cancer classification, cancer cure likelihood, survival rate, etc.).

  In the context of the present invention, the phrase “assessing (or determining) prognosis” is intended to include prediction and likelihood analysis of cancer progression, particularly cancer recurrence, metastatic spread, and disease recurrence. The The method of assessing prognosis of the present invention is clinical in making conclusions about therapeutic interventions, diagnostic criteria such as disease staging, and disease monitoring and monitoring for neoplastic disease metastasis or recurrence. It is intended to be used.

  The biological sample derived from the patient used in the method may be any sample derived from the subject to be evaluated as long as the CDCA5 gene can be detected in the sample. Preferably, the biological sample is a lung cell (a cell obtained from the lung). Furthermore, the biological sample may include body fluids such as sputum, blood, serum, or plasma. Further, the sample may be cells purified from tissue. The biological sample may be obtained from the patient at various times including before, during and / or after treatment.

  According to the present invention, the higher the expression level of the CDCA5 gene measured in a biological sample derived from a patient, the poorer and worse the prognosis for post-treatment remission, recovery, and / or survival. It was shown that the likelihood of clinical outcome is high. Thus, according to the method, the “control level” used for comparison was detected before any type of treatment, for example in an individual or population of individuals who showed a good or good prognosis of cancer after treatment. This may be the case for the expression level of the CDCA5 gene, also referred to herein as the “good prognosis control level”. Alternatively, a “control level” can be the level of expression of the CDCA5 gene detected prior to any type of treatment in an individual or population of individuals who have shown a poor or poor prognosis of cancer after treatment, as used herein. It will also be called “poor prognosis control level”. A “control level” is a single expression pattern or multiple expression patterns derived from a single reference population. Thus, the control level can be determined based on the expression level of the CDCA5 gene detected before any type of treatment in a population of patients with cancer or a known disease state (good prognosis or poor prognosis). . Preferably, the cancer is lung cancer. It is preferable to use a standard value for the expression level of the CDCA5 gene in a group of patients with known disease states. The standard value can be obtained by any method known in the art. For example, average +/- 2 D. Or mean +/- 3 D. Can be used as standard values.

  Control levels are achieved by using previously collected and stored samples from cancer patients (control or control group) with known disease state (good or poor prognosis) prior to any type of treatment. It can be determined simultaneously with the test biological sample.

  Alternatively, the control level can be determined by statistical methods based on the results obtained by analyzing the expression level of the CDCA5 gene in samples collected and stored so far from the control group. Furthermore, the control level may be a database of expression patterns from previously tested cells.

  Furthermore, according to one aspect of the present invention, the expression level of the CDCA5 gene in a biological sample can be compared to a plurality of control levels determined from a plurality of reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type comparable to that of a biological sample derived from the patient.

  According to the present invention, the similarity between the expression level of the CDCA5 gene and the good prognosis control level indicates a more favorable prognosis for the patient, and an increase in the expression level relative to the good prognosis control level indicates remission, recovery, survival, and / or Or poor prognosis with poor clinical outcome. On the other hand, a reduction in the expression level of the CDCA5 gene relative to the poor prognosis control level indicates a more favorable prognosis for the patient, and the similarity between the expression level and the poor prognosis control level is indicative of post-treatment remission, recovery, survival and / or clinical outcome. Shows poor prognosis.

  The expression level of the CDCA5 gene in the biological sample is greater than 1.0, 1.5, 2.0, 5.0, 10.0, or more than the control level It can be considered that it has changed in different cases.

  Differences in expression levels between test biological sample levels and control levels can be normalized to controls, eg, housekeeping genes. For example, polynucleotides whose expression levels are known not to differ between cancer cells and non-cancer cells, including those encoding β-actin, glyceraldehyde-3-phosphate dehydrogenase, and ribosomal protein P1 Can be used to normalize the expression level of the CDCA5 gene.

  Expression levels may be determined by detecting gene transcripts in a biological sample obtained from a patient using techniques well known in the art. Gene transcripts detected by the methods of the present invention include both transcripts and translation products, eg, mRNA and protein.

  For example, a transcript of the CDCA5 gene can be detected by hybridization using a CDCA5 gene probe to the gene transcript, eg, Northern blot hybridization analysis. Detection may be performed on a chip or array. In order to detect the expression level of a plurality of genes including the CDCA5 gene, it is preferable to use an array. As another example, an amplification-based detection method using primers specific for the CDCA5 gene, such as reverse transcription-based polymerase chain reaction (RT-PCR) may be utilized for detection (see Examples). ). Probes or primers specific for the CDCA5 gene can be designed and prepared using conventional techniques by reference to the entire sequence of the CDCA5 gene (SEQ ID NO: 4). For example, the primer (SEQ ID NO: 25-28) used in the Examples can be used for detection by RT-PCR, but the present invention is not limited thereto.

  In particular, the probe or primer used in the method of the present invention hybridizes with the mRNA of the CDCA5 gene under stringent, moderate stringent or low stringent conditions. As used herein, the phrase “stringent conditions” refers to conditions under which a probe or primer will hybridize to its target sequence but not to other sequences. Sure. Stringent conditions are sequence-dependent and will be different in various circumstances. Specific hybridization of long sequences is observed at higher temperatures than short sequences. Generally, the temperature of stringent conditions is selected to be about 5 ° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Tm is the temperature at which 50% of the probe complementary to the target sequence hybridizes to the target sequence in equilibrium (at a defined ionic strength, pH and nucleic acid concentration). Generally, there is an excess of target sequence in Tm, so 50% of the probes are occupied in equilibrium. Typically, stringent conditions are such that the salt concentration is pH 7.0-8.3 and less than about 1.0M sodium ion, typically about 0.01-1.0M sodium ion (or other salt). And the temperature is at least about 30 ° C. for short probes or primers (eg, 10-50 nucleotides) and at least about 60 ° C. for long probes or primers. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.

  Alternatively, a translation product may be detected for the evaluation of the present invention. For example, the amount of CDCA5 protein may be determined. Methods for determining the amount of protein as a translation product include immunoassay methods using an antibody that specifically recognizes the CDCA5 protein. The antibody may be a monoclonal antibody or a polyclonal antibody. In addition, any fragment or modification of the antibody (eg, chimeric antibody, scFv, Fab, F (ab ′) 2, Fv, etc.) can be used for detection as long as the fragment retains the ability to bind to the CDCA5 protein. Also good. Methods for preparing these types of antibodies for protein detection are well known in the art, and any method of preparing such antibodies and their equivalents can be used in the present invention.

  As another method for detecting the expression level of the CDCA5 gene based on the translation product of the CDCA5 gene, the staining intensity may be observed by immunohistochemical analysis using an antibody against the CDCA5 protein. That is, when strong staining is observed, the presence of CDCA5 protein is increased, and at the same time, the expression level of CDCA5 gene is high.

  Furthermore, the CDCA5 protein is known to have cell proliferation activity. Therefore, the expression level of the CDCA5 gene can be determined using such cell proliferation activity as an index. For example, cell growth of a biological sample by preparing and culturing cells expressing CDCA5 and then detecting the growth rate or measuring the cell cycle or colony forming ability, in the presence of the biological sample. Activity can be determined.

  Furthermore, in order to improve the accuracy of evaluation, in addition to the expression level of the CDCA5 gene, the expression level of other lung cancer-related genes, for example, genes that are known to be differentially expressed in lung cancer may be determined. it can. Examples of such other lung cancer-related genes include the genes described in WO2004 / 031413 and WO2005 / 090603. The contents of which are incorporated herein by reference.

  Or, according to the present invention, intermediate results may be obtained in addition to other test results to assess the prognosis of a subject. Such intermediate results can help a physician, nurse, or other practitioner to assess, determine, or infer the subject's prognosis. Additional information that can be considered in combination with intermediate results obtained by the present invention to assess prognosis includes the subject's clinical symptoms and physical condition. Patients to be assessed for cancer prognosis according to this method are preferably mammals and include humans, non-human primates, mice, rats, dogs, cats, horses, and cows.

Kits for diagnosing cancer or evaluating cancer prognosis :
The present invention provides a kit for diagnosing cancer or a kit for evaluating the prognosis of cancer. Preferably, the cancer is lung cancer. Specifically, the kit includes at least one reagent for detecting the expression of the CDCA5 gene in a biological sample derived from a patient, the reagent comprising:
(A) a reagent for detecting mRNA of the CDCA5 gene;
It may be selected from the group consisting of (b) a reagent for detecting CDCA5 protein; and (c) a reagent for detecting the biological activity of CDCA5 protein.

  Suitable reagents for detecting mRNA of the CDCA5 gene include nucleic acids that specifically bind to or specify CDCA5 mRNA, eg, oligonucleotides having a complementary sequence to a portion of CDCA5 mRNA. . These types of oligonucleotides are exemplified by primers and probes specific for CDCA5 mRNA. These types of oligonucleotides may be prepared based on methods well known in the art. If necessary, the reagent for detecting CDCA5 mRNA may be immobilized on a solid matrix. Furthermore, a plurality of reagents for detecting CDCA5 mRNA may be included in the kit.

  On the other hand, suitable reagents for detecting CDCA5 protein include antibodies to CDCA5 protein. The antibody may be a monoclonal antibody or a polyclonal antibody. Furthermore, any fragment or modification of the antibody (eg, chimeric antibody, scFv, Fab, F (ab ′) 2, Fv, etc.) should be used as a reagent as long as the fragment retains the ability to bind to the CDCA5 protein. Can do. Methods for preparing these types of antibodies for protein detection are well known in the art, and any method of preparing such antibodies and their equivalents can be used in the present invention. In addition, the antibody may be labeled with a signal generating molecule by direct binding or by indirect labeling methods. Labels and methods for labeling antibodies, and labels and methods for detecting binding of antibodies to their targets are well known in the art and any label and method should be used for the present invention Can do. Furthermore, a plurality of reagents for detecting the CDCA5 protein may be included in the kit.

  Furthermore, biological activity can be determined, for example, by measuring cell proliferative activity by the expressed CDCA5 protein in a biological sample. For example, culturing cells in the presence of a biological sample from a patient and then detecting the growth rate, or measuring the cell cycle or colony forming ability, thereby increasing the cell proliferation activity of the biological sample. Can be determined. If necessary, a reagent for detecting CDCA5 mRNA may be immobilized on a solid matrix. In addition, multiple reagents for detecting the biological activity of the CDCA5 protein may be included in the kit.

  The kit may include a plurality of the reagents described above. In addition, the kit includes a solid matrix and reagents for binding probes to the CDCA5 gene or antibodies to the CDCA5 protein, media and containers for culturing cells, positive and negative control reagents, and antibodies to the CDCA5 protein A secondary antibody for detecting may be included. For example, a tissue sample obtained from a patient with a good or poor prognosis can serve as a useful control reagent. The kit of the present invention further includes a buffer and diluent, a filter, a needle, a syringe, and a package insert including instructions for use (eg, written, tape, CD-ROM, etc.) from a commercial and user perspective. Other materials may be included as desired. These reagents and the like can be contained in a container provided with a label. Suitable containers include bottles, vials, and test tubes. The container can be formed from a variety of materials, such as glass or plastic.

  As one aspect of the present invention, when the reagent is a probe for CDCA5 mRNA, the reagent may be immobilized on a solid matrix such as a porous strip to form at least one detection site. The measurement region or detection region of the porous strip may include a plurality of sites each containing a nucleic acid (probe). The test strip may also include sites for negative and / or positive controls. Alternatively, the control site may be located on a strip that is separate from the test strip. Optionally, different detection sites may contain different amounts of immobilized nucleic acid, i.e., a large amount of nucleic acid at the first detection site and a small amount of nucleic acid at subsequent sites. When a test sample is added, the number of sites exhibiting a detectable signal can provide a quantitative indicator of the amount of CDCA5 mRNA present in the sample. The detection site may be configured in any suitably detectable shape and is typically in the shape of a bar or dot across the width of the test strip.

  The kit of the present invention may further comprise a positive control sample or a CDCA5 standard sample. Positive control samples of the present invention may be prepared by collecting CDCA5 positive blood samples, and these CDCA5 levels are then assayed. Alternatively, purified CDCA5 protein or polynucleotide may be added to serum without CDCA5 to form a positive sample or CDCA5 standard. In the present invention, purified KDD1 may be a recombinant protein. The CDCA5 level of the positive control sample is, for example, above the cutoff value.

Materials and Methods Cell Lines and Clinical Samples The 23 human lung cancer cell lines used in this study include 19 NSCLC (A427, A549, NCI-H1373, LC319, PC-14, PC-3, PC-9, NCI). -H1666, NCI-H1781, NCI-H647, NCI-H226, NCI-H1703, NCI-H520, LU61, RERF-LC-AI, SK-MES-1, EBC-1, LX1, and NCI-H2170) and 4 Types of small cell lung cancer (SCLC: DMS114, DMS273, SBC-3, and SBC-5) were included. The human esophageal cancer cell lines used in this study were as follows: nine SCC cell lines (TE1, TE2, TE3, TE4, TE5, TE6, TE8, TE9, and TE10) and one adenocarcinoma (ADC) Cell line (TE7) (Nishihira T et al. J Cancer Res Clin Oncol 1993; 119: 441-49). All cells were grown in monolayers in appropriate medium supplemented with 10% fetal calf serum (FCS) and maintained at 37 ° C. in a humidified atmosphere containing 5% CO ₂ . The cell panel used in this study also included human airway epithelial cells, SAEC (Cambrex Bio Science Inc., East Rutherford, NJ). Primary NSCLC and ESCC samples were obtained as previously reported elsewhere. All tumors were staged based on the pTNM pathologic classification of UICC (International Union Against Cancer) (Sobin L, Wittekind C. Anonymous. New York: Wiley-Liss. 2002). A total of 262 NSCLC (156 adenocarcinomas (ADC), 88 squamous cell carcinomas (SCC), 2 adenosquamous cell carcinomas (ASC), 16 cases) for immunostaining in formalin fixed tissue microarray Large cell carcinoma (LCC); 88 female patients and 174 male patients; median age 65.0 years in the range 26-84 years; 111 pT1, 125 pT2, 26 pT3 tumor size; 204 pN0, 24 pN1 , 34 pN2 nodules) and adjacent normal lung tissue samples were also obtained from patients who had surgery at Hokkaido University and its related hospitals (Sapporo, Japan). This study and the use of all mentioned clinical materials were approved by individual institutional ethics committees.

Semi-quantitative RT-PCR
Appropriate dilutions of each single-stranded cDNA were prepared from mRNA of clinical lung cancer and clinical esophageal cancer samples, and β-actin (ACTB) expression level was measured as a quantitative control. The primer set for amplification was as follows: For ACTB, ACTB-F (5′-GAGGTGATATAGCATTGCTTTCG-3 ′; SEQ ID NO: 23) and ACTB-R (5′-CAAGTCAGTGTACAGGTAGAG-3 ′; SEQ ID NO: 24), for CDCA5, CDCA5-F (5′-CGCCAGAGACTTGGAAATGT-3 ′; SEQ ID NO: 25) and CDCA5-R (5′-GTTTCTGTTTCTCGGGTGGT-3 ′; SEQ ID NO: 26) . All reactions were performed on a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, Calif.) At 95 ° C. for 5 minutes followed by 95 ° C. for 30 seconds, 56 ° C. for 30 seconds, and 72 ° C. for 60 seconds. It was accompanied by 22 cycles (for ACTB) or 30 cycles (for CDCA5).

Immunocytochemistry analysis COS-7 cells were transiently transfected with c-Myc-tagged CDCA5 (pcDNA3.1 / myc-His-CDCA5) on coverslips (Becton Dickinson Labware, Franklin Lakes, NJ). . After 48 hours, cells were fixed with 4% paraformaldehyde and then permeabilized with PBS (-) containing 0.1% Triton X-100 for 3 minutes at room temperature. Nonspecific binding was blocked with Casblock (ZYMED, San Francisco, Calif.) For 10 minutes at room temperature. Cells were then incubated with primary antibody (rabbit polyclonal anti-c-Myc antibody, Santa Cruz Biotechnology, Santa Cruz, CA) diluted in PBS containing 3% BSA for 60 minutes at room temperature. After washing with PBS, cells were stained with Alexa488-conjugated anti-rabbit secondary antibody (Molecular Probes) diluted 1: 1,000 for 60 minutes at room temperature. After another wash with PBS (−), each specimen was mounted using Vectashield (Vector Laboratories, Inc., Burlingame, Calif.) Containing 4 ′, 6-diamidino-2-phenylindole, and Spectral Conflict Scanning. Visualization using Systems (TSC SP2 AOBS; Leica Microsystems, Wetzlar, Germany).

Northern blot analysis Human multi-tissue blot (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, leukocyte, stomach, thyroid, spinal cord, lymph node, trachea , adrenal, bone marrow, 23 types of normal tissues; BD Biosciences Clontech, Palo Alto, CA and) were hybridized with ³² P-labeled PCR product of CDCA5. Partial length cDNA of CDCA5 was prepared by RT-PCR using primers: CDCA5-F1 (GCTTGTAAAAGTCCTCGGAAAGTT; SEQ ID NO: 27) and CDCA5-R1 (ATCTCAACTCTCATCATCATCTGT; SEQ ID NO: 28). Prehybridization, hybridization, and washing were performed according to the supplier's recommendations. Blots were autoradiographed for 7 days at -80 ° C using an intensifying screen.

Anti-CDCA5 antibody pET28 vector (Novagen) and primers: CDCA5-F3 (5′-CCCGGAATTCATGTCTGGGAGGGCAACGCG-3 ′; SEQ ID NO: 36) and CDCA5-R3 (5′-CCGCTCGAGTTCACACCAGGATG-3TC) Thus, a plasmid expressing full-length CDCA5 containing a His-tagged epitope at the N-terminus was prepared. Recombinant protein was expressed in the BL21 codon plus strain (Stratagene) of Escherichia coli and purified using Ni-NTA (QIAGEN) according to the supplier's protocol. The protein was inoculated into rabbits and immune serum was purified on an affinity column according to standard methodology. Affinity purified anti-CDCA5 antibody was used for Western blotting and immunocytochemistry and immunohistochemistry studies. Western blots using lysates from cell lines transfected with CDCA5 expression vectors and lysates from lung cancer cell lines that endogenously express CDCA5 or airway epithelial cells that do not express CDCA5. The antibody was confirmed to be specific for CDCA5.

Western Blot Cells Lysis Buffer; 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 0.5% Deoxycholate-Na, 0.1% SDS, and protease Dissolved in inhibitors (Protease Inhibitor Cocktail Set III; Calbiochem). The ECL Western blot analysis system (GE Healthcare Bio-sciences) was used as previously described (Kato T, Daigo Y, Hayama S, et al. Cancer Res 2005; 65: 5638-46).

Immunocytochemical analysis Cultured cells were washed twice with PBS (−), fixed with 4% paraformaldehyde solution for 30 minutes at 37 ° C., and then for 3 minutes with PBS containing 0.1% Triton X-100 (− ) To make it permeable. Prior to the primary antibody reaction, cells were covered with blocking solution [PBS (−) containing 3% bovine serum albumin] for 10 minutes to block non-specific antibody binding. After incubating the cells with an antibody against human CDCA5 (made against recombinant CDCA5; see above), Alexa Fluor 488 goat anti-rabbit secondary antibody (Molecular Probes) was used to detect endogenous CDCA5. added. Nuclei were stained with 4 ′, 6-diamidino-2-phenylindole (DAPI). Cells stained with the antibody were observed with a laser confocal microscope (TSC SP2 AOBS: Leica Microsystems).

Immunohistochemistry and tissue microarray analysis To examine the significance of CDCA5 expression in clinical NSCLC, tissue sections were stained with ENVISION + kit / horseradish peroxidase (HRP; DakoCytomation). After blocking endogenous peroxidase and protein, affinity purified anti-CDCA5 antibody was added and each section was incubated with HRP-labeled anti-rabbit IgG as a secondary antibody. A chromogenic substrate was added and the specimens were counterstained with hematoxylin. As previously published, tumor tissue microarrays were constructed using formalin-fixed NSCLC (Callagy G, Cattaneo E, Daigo Y, et al. Diagn Mol Pathol 2003; 12: 27-34, Callagy G, Pharoah P Chin SF, et al. J Pathol 2005; 205: 388-96, Chin SF, Daigo Y, Huang HE, et al. Molecular Pathology 2003; 56: 275-9). The tissue area for sampling was selected by visual alignment with the corresponding H & E stained section on the slide. Three, four or five tissue cores (0.6 mm diameter, 3-4 mm height) taken from the donor tumor block were placed in the recipient paraffin block using a tissue microarrayer (Beecher Instruments). A hole was drilled in the core of normal tissue from each case. 5 μm sections of the resulting microarray block were used for immunohistochemical analysis. CDCA5 positivity was evaluated semi-quantitatively by three independent investigators without prior knowledge of clinicopathological data. Since the intensity of staining in each tumor tissue core was nearly uniform, the intensity of CDCA5 staining in the nucleus and cytoplasm was negative (no staining was seen in the tumor cells) or positive (in the nucleus and cytoplasm of the tumor cells) Evaluation was made by recording that brown staining was observed). A case was accepted as positive only if all three evaluators independently defined the case as positive.

Statistical analysis Statistical analysis was performed using the Stat View statistical program (SAS). A contingency table was used to correlate clinicopathological variables such as age, gender, and pathological tumor-lymph node-metastasis (TNM) classification with CDCA5 protein positivity determined by tissue microarray analysis. Tumor-specific survival curves were calculated from the date of surgery to the time of NSCLC-related death or the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and CDCA5 expression, and survival time differences between patient subgroups were analyzed using the log rank test. To determine the association between clinicopathological variables and cancer-related mortality, univariate analysis was performed with a Cox proportional hazard regression model.

RNA interference assay Two CDCA5 siRNA oligonucleotides were designed using the CDCA5 sequence. SiRNA (600 pM) was transfected into NSCLC cell lines LC319 and A549 using 30 μl Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) According to the manufacturer's protocol. Transfected cells were cultured for 7 days. Cell number and viability were determined by Giemsa staining and three 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assays (cell-counting kit-8 solution; Dojindo Laboratories). ). The siRNA sequences used were as follows: control-1 (si-LUC: luciferase gene from Photinus pyralis), 5′-NNCGUACCGGGAAUCUCUCGA-3 ′ (SEQ ID NO: 29); 2 (CNT: ON-TARGETplus siCONTROL Non-targeting siRNAs pool of 5'-UGGUUUACAUGUCGACUUA-3 '(SEQ ID NO: 30-); '(SEQ ID NO: 32); 5'-UGGUUUACAUGUGUGUGA-3' (SEQ ID NO: 32) 33)); siRNA-CDCA5- # 1 (si-CDCA5- # 1: 5'-GCAGUUGAUCUCCUGUGUUU-3 '(SEQ ID NO: 34)); siRNA-CDCA5- # 2 (si-CDCA5- # 2: 5' -GCCAGAGACUGUGGAAAUGUU-3 '(SEQ ID NO: 35)). The cell line used for this assay confirmed down-regulation of CDCA5 expression by functional siRNA, but not the control.

Cell Proliferation Assay c-Myc / His tagged CDCA5 expression vector (pcDNA3.1-c-Myc / His-CDCA5) or mock vector (pcDNA3.1-c-Myc / His) using FuGENE6 transfection reagent (Roche). ) Were transfected into COS-7 cells or NIH3T3 cells. Transfected cells were incubated in medium containing 0.4 mg / ml neomycin (Geneticin, Invitrogen). After 7 days, cell viability was examined by MTT assay. In order to establish COS-7 cells that stably express CDCA5, FuGENE6 transfection reagent (Roche) was used to convert untagged CDCA5 expression vector (pCAGGSn-CDCA5) or mock vector (pCAGGSn) to endogenous CDCA5. Weakly expressed COS-7 cells were transfected. Transfected cells were incubated for 14 days in medium containing 0.4 mg / mL neomycin (Geneticin, Invitrogen). Fifty colonies were then trypsinized and screened for stable transfectants by limiting dilution assay. CDCA5 expression was determined in each clone by RT-PCR, Western blotting, and immunocytochemical staining. Cell viability was examined by MTT assay on days 1, 3, 5, and 7.

In vitro kinase assay In vitro kinase assay was performed using full-length recombinant GST-CDCA5 (pGEX-6p-1 / CDCA5 cleaved with Precision Protease). Briefly, 1.0 μg GST-CDCA5, histone H1 (Upstate), MBP, or GST, respectively, 1 μCi [γ- ³² P] -ATP (GE Healthcare) and 2 units CDC2 (BioLabs) or 50 ng Kinase buffer (50 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM EGTA, 2 mM DTT, 0.01% Briji 35, 1 mM ATP, pH 7.5, 25 ° C.) with 20 μl of ERK2 (Upstate) at 30 ° C. And incubated for 20 minutes. Laemmli SDS sample buffer was added to a final volume of 30 μl to stop the reaction, half of the sample was subjected to a 5-15% gradient gel (Bio-Rad Laboratories), and phosphorylation was visualized by autoradiography. MBP was used as the ERK substrate and H1 was used as the CDC2 substrate (positive control). GST served as a negative control substrate.

MALDI-TOF mass spectrometry CDCA5 recombinant protein was incubated with ERK kinase (rhERK2) or CDC2 at 37 ° C. for 3.5 hours. Samples were separated on an SDS-PAGE gel. After electrophoresis, the gel was stained with R-250 (Bio-Rad). As previously described (Kato T et al. Clin Cancer Res 2008; 14: 2363-70), a specific band corresponding to CDCA5 was digested with trypsin and subjected to matrix-assisted laser desorption / ionization mass spectrometry (MALDI-QIT). -TOF; used for analysis by Shimadzu Biotech, Kyoto, Japan). To identify proteins from the primary sequence database, mass spectral data was examined using the Mascot search engine (http://www.matrixscience.com). To determine the in vivo ERK-dependent phosphorylation site of CDCA5 in cultured cells, exogenously expressed CDCA5 protein in EGF-treated HeLa cells transfected with both CDCA5 expression vector and ERK2 expression vector was treated with anti-CDCA5 antibody. After immunoprecipitation, colloidal CBB staining was performed. As described above, the band corresponding to CDCA5 was cut out for MS analysis.

EGF stimulation assay Cultured cervical squamous cell carcinoma HeLa cells were cultured for 20 hours in medium without FCS. Cells were then stimulated with 50 μg / ml EGF for 20 minutes with or without 10 μM MEK inhibitor U0126 (Promega).

Flow cytometric analysis The cell cycle of A549, LC319, and HeLa cells were synchronized by aphidicolin (Sigma-Aldrich) treatment for 16 hours, washed 3 times with PBS (-), fresh to release cell cycle arrest Medium was added. After releasing G1 / S, cells were collected for 14 hours (every 1-2 hours), fixed with 70% ethanol, and then kept at 4 ° C. before use. Cells were incubated for 30 minutes at 37 ° C. with PBS (−) containing 100 μg / ml RNase (Sigma-Aldrich) and stained with 50 μg / ml propidium iodide (Sigma-Aldrich) for 30 minutes at 4 ° C. . The cell suspension at each time point was analyzed by FACScan (Becton Dickinson, Franklin Lakes, NJ).

Results Expression of CDCA5 in lung and esophageal cancers and normal tissues Detect transcripts whose expression in cancer cells is more than 3 times that of normal control cells in more than 40% of the analyzed clinical lung and clinical esophageal cancer samples In order to do this, 27,648 genes on the cDNA microarray were previously screened. Among the genes that were up-regulated, CDCA5 transcripts were identified and by semi-quantitative RT-PCR experiments, 9 out of 10 representative NSCLC cases and all 5 out of 5 SCLC cases (FIG. 1A). ), It was confirmed that the CDCA5 transcript was highly expressed in all 23 lung cancer cell lines. High levels of CDCA5 expression were observed in all lung cancer cell lines (FIG. 1B), whereas CDCA5 transcripts were rarely detected in cells derived from normal small airway epithelium (SAEC) and normal esophageal samples. . Furthermore, strong expression of endogenous CDCA5 protein was confirmed in lung and esophageal cancer cell lines by Western blot analysis using anti-CDCA5 antibody (FIG. 1C). Immunofluorescence analysis was performed to examine the intracellular localization of endogenous CDCA5 in lung cancer LC319 cells, and it was found that CDCA5 was localized in the nucleus of interphase cells and weak in the cytoplasm (FIG. 1D). By Northern blot analysis using the CDCA5 cDNA fragment as a probe, a 2.8 kb transcript was highly expressed in the testis, but was hardly detectable in any other normal tissue (FIG. 2A). Furthermore, CDCA5 protein expression in five normal tissues (heart, lung, liver, kidney, and testis) was compared with CDCA5 protein expression in lung cancer by immunohistochemistry using anti-CDCA5 polyclonal antibody. CDCA5 expression was abundantly detected in the nuclei of testicular cells and lung cancer cells, weakly detected in the cytoplasm, but hardly detectable in the remaining four normal tissues (FIG. 2B).

Relationship between CDCA5 expression and poor prognosis in NSCLC patients Immunohistochemical analysis using anti-CDCA5 polyclonal antibody was performed using a tissue microarray prepared from conserved NSCLC. CDCA5 expression patterns were classified as negative or positive (FIG. 2C). Of 262 NSCLC cases examined, 192 (73.3%) cases may have positive staining, 70 (26.7%) cases or negative staining in any adjacent non-cancer cells. (Table 1A). It was found that CDCA5 expression in NSCLC patients treated with curative treatment was associated with various clinicopathological parameters, and that there was a significant relationship between NSCLC CDCA5 positivity and tumor-specific poor survival ( P = 0.0143 by log rank test; FIG. 2D). Patient prognosis, age, gender, histological type (ADC vs. non-ADC), pT classification (tumor size; T1 vs. T2 + T3), pN classification (nodular status; N0 vs. N1 + N2) and CDCA5 status (negative vs. positive expression) The univariate analysis was also applied to investigate the relationship with several factors including All of these parameters were significantly associated with poor prognosis (Table 1B). In multivariate analysis, CDCA5 status is an independent prognostic factor for lung cancer patients undergoing surgery enrolled in this study (P = 0.0237), but pT and pN classification and age may not be prognostic factors Indicated.

ＡＤＣ、腺癌
非ＡＤＣ、扁平上皮癌＋大細胞癌および腺扁平上皮癌
^＊Ｐ＜０．０５（χ^２検定）

（表１Ｂ）ＮＳＣＬＣ患者における予後因子のＣｏｘ比例ハザードモデル解析

ＡＤＣ、腺癌
非ＡＤＣ、扁平上皮癌＋大細胞癌および腺扁平上皮癌
＊Ｐ＜０．０５ Table 1A: Relationship between CDCA5 positivity in NSCLC tissue and patient characteristics (n = 322)

ADC, adenocarcinoma non-ADC, squamous cell carcinoma + large cell carcinoma and adenosquamous cell carcinoma
^* P <0.05 ^{(χ 2} test)

Table 1B: Cox proportional hazards model analysis of prognostic factors in NSCLC patients

ADC, adenocarcinoma non-ADC, squamous cell carcinoma + large cell carcinoma and adenosquamous cell carcinoma * P <0.05

Growth promoting activity of CDCA5 In lung cancer cell lines A549 and LC319 showing high levels of CDCA5 expression, endogenous CDCA5 expression was knocked down by siRNA. CDCA5 mRNA and protein expression levels were examined. Two CDCA5-specific siRNAs (si-CDCA5- # 1 and si-CDCA5- # 2) are expressed in CDCA5 mRNA and protein compared to control siRNA constructs (si-LUC and si-CNT; FIG. 3A). Was found to be significantly suppressed. Colony formation assay and MTT assay revealed that when CDCA5 expression was reduced by two types of si-CDCA5, the knockdown effect on CDCA5 expression significantly suppressed the proliferation of both A549 and LC319 cells (FIGS. 3B and 3C). Next, the possible role of CDCA5 in the cell growth promoting effect was examined. Two types of plasmids designed to express untagged full-length CDCA5 (pcDNA3.1-CDCA5) or mock plasmids, transfected into COS-7 cells or NIH3T3 cells and overexpressing exogenous CDCA5 COS-7 cell lines (COS-7-CDCA5- # A and-# B) and two control cells (COS-7-mock- # A and-# B) were established. These COS-7-derived transfectants were then subjected to MTT assays to compare the growth of COS-7-CDCA5 cells with that of control COS-7-mock cells. Transfection of CDCA5 cDNA into COS-7 or NIH3T3 cells significantly enhanced cell proliferation compared to mock vector transfectants. According to the CDCA5 expression level detected by Western blot analysis, the proliferation of two types of COS-7-CDCA5 cells was promoted to a significant degree (FIG. 3D).

In vitro CDCA5 phosphorylation by ERK and CDC2 protein kinases An in silico approach, one of the key downstream components of the oncogenic MAPK pathway, several consensus phosphorylation sites of CDCA5 protein by ERK kinase [xx-S / TP] has been suggested, in order to analyze the function of CDCA5 in carcinogenesis, we focused on the possible phosphorylation of CDCA5 protein. According to previous reports using proteome phosphorylated peptide screening, CDCA5 was estimated to be phosphorylated on serine-75, serine-79, and threonine-115 (Olsen Jv et al. Cell 2006; 127 ( 3): 635-648). To identify cognate kinases for CDCA5 phosphorylation, the peptide sequences of CDCA5, including serine-75, serine-79, and threonine-115, with potential phosphorylation sites were compared. Serine-75 of CDCA5 is a perfect match with the consensus CDC2 protein kinase phosphorylation site [S / TPPxR / K], whereas serine-79 and threonine-115 are ERK phosphorylation sites [x -X-S / TP] was found to be in good agreement. These consensus sequences are highly conserved in many species. Subsequently, an in vitro kinase assay was performed by incubating recombinant rhERK2 and rhCDCA5. CDCA5 was found to be directly phosphorylated by both ERK kinases (FIG. 4A). This result suggested that CDCA5 may be involved in the ERK pathway.

  To determine the direct phosphorylation site of CDCA5 by ERK, an in vitro kinase assay followed by mass spectrometry was performed. After protein separation by kinase assay and subsequent SDS-PAGE, three bands corresponding to rhCDCA5 protein incubated with rhERK2 or rhCDCA5 protein not incubated with rhERK2 were digested with trypsin and subjected to MALDI-QIT-TOF MS analysis (FIG. 4B).

Identification of ERK-dependent CDCA5 phosphorylation in vivo To demonstrate that endogenous CDCA5 is phosphorylated by ERK in mammalian cells, serum was depleted in the presence or absence of the MEK inhibitor U0126 HeLa cells were stimulated with EGF. Western blotting with anti-ERK antibody detected upward shifted bands and found that ERK was significantly activated 15 and 30 minutes after EGF stimulation, but decreased at 60 minutes. (FIG. 5A, left panel). According to the high level of ERK phosphorylation, the CDCA5 band detected by the anti-CDCA5 antibody shifted to the high molecular weight side. In contrast, treatment of cells with EGF and MEK inhibitor U0126 decreased ERK phosphorylation levels, decreased ERK phosphorylation levels, and completely inhibited the upward CDCA5 band shift (FIG. 5A, right panel). ). These results demonstrated the possibility of endogenous CDCA5 protein phosphorylation by the ERK pathway. In order to confirm MAP kinase pathway-dependent CDCA5 phosphorylation in cultured cells and to identify the phosphorylation site, HeLa cells transfected with a plasmid designed to express myc-tagged CDCA5 were treated with MEK inhibitor U0126. Stimulated with EGF in the presence or absence. These cell extracts were used for 2D-Western blotting using anti-myc antibody. Two spots were detected in HeLa cells that were not treated with EGF and U0126 (spot numbers 1 and 2). However, EGF treatment significantly increased the signal of one of the spots (spot number 2), whereas two new spot signals (spot numbers 3 and 4) with more acidic pI values. Induced. These shifted spots with an acidic pI were greatly reduced by preincubation of cells with the MEK inhibitor U0126. Furthermore, the signal of spot number 2 increased by EGF stimulation was also decreased by U0126 treatment. These results suggest that CDCA5 was specifically phosphorylated by the MAPK cascade in response to EGF ligand stimulation.

Identification of ERK-dependent CDCA5 phosphorylation in vivo Phosphorylated CDCA5 was detected as a band shift according to an in vitro kinase assay using ERK kinase and CDCA5 as a substrate. Furthermore, endogenous CDCA5 was detected as a band shift in HeLa cells under EGF-stimulated conditions (FIG. 5A). To determine the phosphorylation status of untagged CDCA5 in cultured cells, HeLa cells transfected with exogenous CDCA5 expression vector were stimulated with EGF. It was found that endogenous ERK was activated in the cell by the band shift of ERK2. However, phosphorylated CDCA5 protein was not detected as a band shift (FIG. 5B). To identify ERK-dependent phosphorylation sites in cultured cells, an immunoprecipitation assay using anti-CDCA5 antibody was performed to immunoprecipitate untagged CDCA5 protein from EGF-untreated cell lysates. Only Ser21 was confirmed in all samples. These results indicated that endogenous ERK in cancer cells may not be sufficient to stimulate overexpression of exogenous CDCA5 protein. Subsequently, exogenous ERK1 or 2 was transfected into HeLa cells. After stimulation of cells with EGF, activation of exogenous ERK1 or 2 as well as endogenous ERK was detected in EGF stimulated cells, but not with U0126 MEK inhibitor treatment. By activation of ERK, phosphorylated exogenous CDCA5 could be detected as a band shift (FIG. 6A). From these results, it was found that CDCA5 is phosphorylated by overexpressed ERK in HeLa cells. In order to determine the ERK-dependent phosphorylation site of CDCA5, this untagged CDCA5 protein was immunoprecipitated with anti-CDCA5 antibody, followed by colloid CBB staining. Cells were prepared under conditions of EGF untreated, EGF stimulation, or stimulation with EGF and MEK inhibitor U0126. For MS analysis using a 4800 plus MALDI-TOF-TOF analyzer, a band corresponding to CDCA5 was cut out (FIG. 6B). MS analysis showed that 70%, 77%, and 66% of the CDCA5 sequence were covered in untreated, EGF stimulation, and EGF and MEK inhibitor stimulation, respectively. Two ERK-dependent phosphorylation sites Ser79 and Ser209 were identified. Phospho-Ser21 was found in all samples, indicating that it was not an ERK-dependent phosphorylation site (FIGS. 6C-6E). To confirm the results of MS analysis, Western blot analysis was performed using cells transfected with an untagged CDCA5 vector in which Ser79 and / or Ser209 residues were replaced with alanine. ERK2 expression vectors were cotransfected with wild type and mutant CDCA5 expressing vectors under untreated or EGF stimulated conditions. As shown in Western blotting, ERK2 band shift revealed that ERK was activated by EGF stimulation. Wild type CDCA5 was detected as a band shift after EGF stimulation. The level of the upward shifted bands of CDCA5-S79A and CDCA5-S209A was weak compared to wild type CDCA5, but no shift band of double mutant CDCA5 was detected (FIG. 6F). From these results, it was found that Ser79 and Ser209 can be phosphorylated by ERK in cells.

  To date, there is a lot of evidence to report that the MARK pathway promotes carcinogenesis. Furthermore, these data suggested that CDCA5 is characterized as a cancer testis antigen that is one of the causes of carcinogenesis in the lung or esophagus. In order to investigate whether the function of ERK-dependent phosphorylation sites of CDCA5 could play an important role in cancer progression, the dominant negative effect of these phosphorylation sites on cancer cell proliferation was examined. Proliferation assays were performed using alanine substitutes of CDCA5-Ser79 or Ser209. MTT assay showed that transfection of Ser209 alanine substitute inhibited the growth of lung cancer cell lines A549 and LC319 lung cancer cell lines. This may suggest that the Ser209 alanine substitute showed a dominant negative effect on these cell lines (FIG. 7A). To confirm the effect of these phosphorylation sites on cell proliferation, Ser79 or Ser209 phosphorylation mimetic constructs in which Ser residues were replaced with aspartate or glutamate were expressed in LC319 lung cancer cell lines. MTT assay showed that only cells transfected with Ser209 phosphorylated mimetic vector could significantly promote the growth of LC319 and A549 cells (FIG. 7B). This result may strongly suggest that Ser209 phosphorylation of CDCA5 plays an important role in cancer cell proliferation.

Discussion Due to the mechanism of action that has been clarified in detail, molecular targeting drugs are expected to have high specificity for malignant cells and few side effects. Despite improvements in model surgery and adjunct chemoradiotherapy, lung cancer and ESCC are known to show the worst prognosis of any malignant tumor. Thus, today, new diagnostic biomarkers for the early detection of these cancers and better selection of ancillary treatment modalities for individual patients, as well as new types of anticancer drugs and / or cancer vaccines There is an urgent need to develop. In order to identify appropriate diagnostic and therapeutic target molecules, whole genome expression analysis was combined (Kikuchi T et al. Oncogene 2003; 22: 2192-2205, Kakiuchi S et al. Mol Cancer Res 2003; 1: 485 -499, Kakiuchi S et al. Hum Mol Genet 2004; 13: 3029-3043, Kikuchi T et al. Int J Oncol 2006; 28: 799-805, Taniwaki M et al. Int J Oncol 2006; 29: 567-75 , Yamabuki T et al. Int J Oncol 2006; 28: 1375-84) for selecting genes that were overexpressed in lung and esophageal cancer cells by high-throughput screening of loss-of-function effects by means of the RNAi technique (Suzuki C et al. Cancer Res 2003; 63: 7038-7041, Ishikawa N et al. Clin Cancer Res 2004; 10: 8363-8370, Kato T et al. Cancer Res. 2005; 65: 5638-46, Furukawa C et al. Cancer Res. 2005; 65 (16): 7102-10, Ishikawa N et al. Cancer Res. 2005; 65 (20): 9176-84, Suzuki C et al. Cancer Res. 2005; 65: 11314-25, Ishikawa N et al. Cancer Sci 2006; 97: 737-45, Takahashi K et al. Cancer Res 2006; 66: 9408-19, Hayama S et al. Cancer Res 2 006; 66: 10339-48, Kato T et al. Clin Cancer Res 2007; 13: 434-42, Suzuki C et al. Mol Cancer Ther 2007; 6: 542-551, Yamabuki T et al. Cancer Res 2007; 67 : 2517-25, Hayama S et al. Cancer Res 2007; 67: 4113-22, Kato T et al. Cancer Res 2007; 67: 8544-53, Taniwaki M et al. Clin Cancer Res 2007; 13: 6624-31 , Ishikawa N, et al. Cancer Res 2007; 67: 11601-11, Mano Y et al. Cancer Sci 2007; 98: 1902-13, Suda T et al. Cancer Sci 2007; 98: 1803-8, Mizukami Y et al. Cancer Sci 2008; 99: 1448-54). Using this systematic approach, CDCA5 is found to be frequently overexpressed in clinical lung cancer and clinical ESCC samples, and overexpression of this gene product plays an essential role in the growth of lung cancer cells. It was shown that.

  Previous studies have demonstrated that CDCA5 interacts with cohesion on chromatin and functions to support sister chromatid adhesion during interphase. Sister chromatids usually segregate further in most G2 cells, suggesting the possibility that CDCA5 is already required for establishment of adhesion already in S phase (Schmitz J et al. Curr Biol 2007; 17: 630-636). To date, there is only one other protein known that is particularly necessary for the establishment of adhesion: Saccharomyces cerevisiae acetyltransferase Eco1 / Ctf7 (Skibbens RV et al. Genes Dev 1999; 13: 307-319, Toth A et al. Gene Dev 1999; 13: 320-333, Ivanov D et al. Curr. Biol 2002; 12: 323-328). In addition, homologs of this enzyme are also required for adhesion in Drosophila and human cells (Williams BC et al. Curr. Biol 2003; 13: 2025-2036, Hou F and Zou H. Mol Biol Cell 2005; 16 : 3908-3918), it is still unknown whether these proteins function even in S phase. It would therefore be interesting to address whether CDCA5 and Eco1 / Ctf7 homologs work together to establish cancer cell adhesion.

  In order to effectively perform accurate chromosome segregation, sister chromatid adhesions must be established and disassembled at the appropriate time in the cell cycle. It has been shown previously that activation of APCCdc20 controls adhesion disruption with respect to degradation by targeting the late inhibitor securin. This allows for separase-dependent cleavage of Scc1 / Rad21 and induces the late phase. The degradation of most cell cycle substrates of APC is logical in terms of these functions. Degradation prevents the activity from appearing out of time and promotes cell cycle progression in stages. The function of CDCA5 may also overlap with the function of other factors that regulate adhesion, and their activity is combined to ensure that the chromosome replicates and segregates (Rankin S et al. Mol. Cell 2005 ; 18: 185-200). According to our microarray data, APC and CDC20 are highly expressed in lung and esophageal cancers, but their expression in normal tissues is low. CDC20 was also confirmed to be highly expressed in clinical small cell lung cancer using semi-quantitative RT-PCR and immunohistochemical analysis (Taniwaki M et al. Int J Oncol 2006; 29: 567-75). These data imply that CDCA5, along with CDC20, can enhance cancer cell growth by promoting cell cycle progression, but these molecules can interact directly with CDCA5. There is no evidence.

  CDCA5 has previously been reported to be located in the nucleus during interphase and in the cytoplasm during mitosis (Rankin S et al. Mol. Cell 2005; 18: 185-200). However, its physiological function remains unclear. It was confirmed that CDCA5 was localized in the nucleus. The nucleus contains genetic material, whose main function is to maintain gene integrity and regulate gene expression. The nucleus is a dynamic structure that changes according to the needs of the cell. In order to control the function of the nucleus, the process of entering and exiting the nucleus is regulated. Localization of CDCA5 in the nucleus indicates that this molecule may play a role as an essential factor in controlling the cell cycle (Kho CJ, et al. Cell Growth Differ 1996; 7: 1157- 1166, Bader N, et al. Exp Gerontol 2007 [electronic version]). CDCA5 is known to play an important role in cell cycle control by interacting with cohesion on chromatin (Schmitz J, Watrin E, Lenart P, et al. Curr Biol 2007; 17: 630- 636), there is no study demonstrating how CDCA5 is related to the carcinogenic process. CDCA5 was confirmed to be a putative oncogene that is abnormally expressed in lung cancer cells. Tissue microarray analysis found that NSCLC patients showing high expression of CDCA5 protein had a short tumor-specific survival. This strongly indicates that CDCA5 plays an important role in lung cancer progression.

  Our data also show that CDCA5 is phosphorylated by ERK at two phosphorylation sites, serine-79 and serine-209, which have sequences that are highly conserved in many species as consensus ERK phosphorylation sites. It was also likely to be oxidized (data not shown), suggesting that phosphorylation of serine-209 by ERK appears to be important for cell growth. ERK functions as an integration point for multiple biochemical signals and is a member of the MAP kinase family proteins involved in various cellular processes such as proliferation, differentiation, transcriptional regulation, and development (Roux PP and Blenis J. Microbiol. Mol Biol Rev 2004; 68: 320-44, Chang L and Karin M. Nature 2001; 410: 37-40, Ferrell JE Jr. Trends Biochem Sci 1996; 21: 460-6). It was well known that abnormal regulation of the MAPK cascade contributes to carcinogenesis (Cowley S, Paterson H, Kemp P, Marshall CJ. Cell 1994; 77: 841-52, Mansour SJ, Matten WT, Hermann AS, et al. Science 1994; 265: 966-70). The Raf-MEK-ERK pathway is an important downstream effector of Ras small GTPase, and Ras small GTPase is an important downstream effector of EGFR (Roux PP and Blenis J. Microbiol. Mol Biol Rev 2004; 68: 320-44, Chang L and Karin M. Nature 2001; 410: 37-40, Ferrell JE Jr. Trends Biochem Sci 1996; 21: 460-6). Thus, the EGFR-Ras-Raf-MEK-ERK signaling network was the subject of intense research that led to the discovery of new anticancer drugs. Currently, Raf-MEK-ERK cascade inhibitors such as the geldanamycin analog 17-allylamino-17-demethoxygeldanamycin (17-AAG) are being evaluated in clinical trials (Smith RA, Dumas J, Adnane L, Wilhelm SM. Curr Top Med Chem 2006; 6: 1071-89). Furthermore, Ras inhibitors, which are small molecule MEK inhibitors, have been developed (English JM and Cobb MH. Trends Pharmacol Sci 2002; 23: 40-5). Although these drugs have been found to be effective in preclinical studies and / or in a certain proportion of cancer patients, the clinical response was unlikely to correlate accurately with the activation level of the target protein. In addition, in some cases, serious adverse reactions due to nonspecific cytotoxicity were reported. Therefore, specifically targeting this pathway based on the elucidation of unknown downstream canceration signals may be one promising approach.

  It was proved that the proliferation of lung cancer cells overexpressing CDCA5 can be effectively suppressed by the dominant negative effect by the mutant CDCA5 protein in which serine-209 which is an ERK-dependent phosphorylated residue is substituted with alanine. Since phosphorylation of CDCA5 at this site is likely essential for lung cancer cell growth / survival and CDCA5 may belong to cancer testis antigen, selective targeting of CDCA5-ERK enzyme activity, and Cancer immunotherapy, such as cancer vaccines, can be a promising treatment strategy that has strong biological activity against cancer and is expected to have little risk of adverse events.

  In summary, CDCA5 is likely to play a critical role in carcinogenesis in the lung by phosphorylation of serine-209 through the MAPK pathway. Inhibition of CDCA5 expression and functional interaction between CDCA5 and ERK kinase may be a promising therapeutic strategy for developing new types of anticancer drugs.

Industrial Applicability As proved herein, ERK has a kinase activity against CDCA5, and suppressing this activity inhibits cell growth of cancer cells. Therefore, a drug that inhibits ERK kinase activity against CDCA5 is useful for treatment as an anticancer agent for treating lung cancer or esophageal cancer. The phosphorylation site of CDCA5 by ERK is, for example, Ser79 or Ser209.

  Furthermore, the present invention provides a method for screening an anticancer agent that inhibits ERK kinase activity against CDCA5. Thus, it is expected that candidate compounds that inhibit key steps of cell growth can be isolated by the present invention.

  Furthermore, the present invention demonstrates that treatment of cancer cells with a CDCA5 variant, such as CDCA5 (S209A), suppresses ERK kinase activity of CDCA5 against Ser209 and thus suppresses the growth of cancer cells. This data means that upregulation of ERK function and enhancement of ERK kinase activity against CDCA5 are common features of carcinogenesis in the lung. Thus, selective inhibition of ERK kinase activity may be a promising therapeutic strategy for treating lung cancer patients and esophageal cancer patients.

  All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. However, nothing in this specification should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

  Although the invention has been described in detail with respect to particular embodiments thereof, the above description is illustrative and explanatory in nature and is intended to describe the invention and preferred embodiments thereof. Should be understood. Those skilled in the art will readily appreciate through routine experimentation that various changes and modifications can be made without departing from the spirit and scope of the invention. Further advantages and features will be apparent from the appended claims, such claims being determined by their reasonable equivalents as will be understood by those skilled in the art. Thus, the invention is intended to be defined not by the above description, but by the following claims and their equivalents.

Claims (25)

A method for measuring the kinase activity of an ERK polypeptide against a CDCA5 polypeptide, comprising the following steps:
a. Incubating an ERK polypeptide or functional equivalent thereof with a CDCA5 polypeptide or functional equivalent thereof under conditions suitable for phosphorylation of the CDCA5 polypeptide by the ERK polypeptide comprising:
The functional equivalents of ERK polypeptides are:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, and ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2 wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is SEQ ID NO: 1 or 2 Selected from the group consisting of polypeptides having a kinase activity equivalent to a polypeptide consisting of the amino acid sequence of
The functional equivalents of CDCA5 polypeptide are:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5,
ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5, wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is derived from the amino acid sequence of SEQ ID NO: 5 A polypeptide having a kinase activity equivalent to that of the polypeptide, and iii. A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, provided that the resulting polypeptide is SEQ ID NO: 5 A process selected from the group consisting of polypeptides having a biological activity equivalent to a polypeptide consisting of an amino acid sequence;
b. Detecting phosphorylation of CDCA5 polypeptide at amino acid residue serine 79 or 209 of SEQ ID NO: 5 to determine the phosphorylation level of CDCA5 polypeptide; and c. Determining the kinase activity of the measured ERK polypeptide by correlating with the phosphorylation level of the CDCA5 polypeptide determined in step (b). A method for identifying an agent that modulates the kinase activity of an ERK polypeptide against a CDCA5 polypeptide, or a method for screening for an agent for treating or preventing lung cancer or esophageal cancer, comprising the following steps:
a. Incubating the ERK polypeptide or functional equivalent thereof with the CDCA5 polypeptide or functional equivalent in the presence of a test compound under conditions suitable for phosphorylation of the CDCA5 polypeptide by the ERK polypeptide. And
The functional equivalents of ERK polypeptides are:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, and ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2 wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is SEQ ID NO: 1 or 2 Selected from the group consisting of polypeptides having a kinase activity equivalent to a polypeptide consisting of the amino acid sequence of
The functional equivalents of CDCA5 polypeptide are:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5,
ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5, wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is derived from the amino acid sequence of SEQ ID NO: 5 A polypeptide having a kinase activity equivalent to that of the polypeptide, and iii. A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, provided that the resulting polypeptide is SEQ ID NO: 5 A process selected from the group consisting of polypeptides having a biological activity equivalent to a polypeptide consisting of an amino acid sequence;
b. Detecting phosphorylation of CDCA5 polypeptide at amino acid residue serine 79 or 209 of SEQ ID NO: 5 to determine the phosphorylation level of CDCA5 polypeptide;
c. Comparing the level determined in step (b) with the level determined in the absence of the test agent; and d. Selecting a test agent that has reduced the level of phosphorylation of the CDCA5 polypeptide relative to the level determined in the absence of the test agent in step (c). A method for identifying an agent that modulates the kinase activity of an ERK polypeptide against a CDCA5 polypeptide, or a method for screening for an agent for treating or preventing lung cancer or esophageal cancer, comprising the following steps:
a. A cell expressing, in the presence of EGF, a test agent, a polynucleotide encoding a CDCA5 polypeptide or functional equivalent thereof, and a polynucleotide encoding an ERK polypeptide or functional equivalent thereof,
The functional equivalents of CDCA5 polypeptide are:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5,
ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5, wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is derived from the amino acid sequence of SEQ ID NO: 5 A polypeptide having a kinase activity equivalent to that of the polypeptide, and iii. A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, provided that the resulting polypeptide is SEQ ID NO: 5 Selected from the group consisting of polypeptides having a biological activity equivalent to a polypeptide consisting of an amino acid sequence,
The functional equivalents of ERK polypeptides are:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, and ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2 wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is SEQ ID NO: 1 or 2 Contacting with a cell selected from the group consisting of polypeptides having a kinase activity equivalent to a polypeptide consisting of the amino acid sequence of
b. Detecting phosphorylation of CDCA5 polypeptide at amino acid residue serine 79 or 209 of SEQ ID NO: 5 and determining the phosphorylation level of CDCA5 polypeptide;
c. Comparing the level detected in step (b) with the level determined in the absence of the test agent; and d. Selecting a test agent that has reduced the level of phosphorylation of the CDCA5 polypeptide relative to the level determined in the absence of the test agent in step (c). A kit for identifying an agent that modulates ERK kinase activity against CDCA5, comprising the following components:
a. A CDCA5 polypeptide or functional equivalent thereof, comprising:
The functional equivalents of CDCA5 polypeptide are:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5,
ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5, wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is derived from the amino acid sequence of SEQ ID NO: 5 A polypeptide having a kinase activity equivalent to that of the polypeptide, and iii. A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, provided that the resulting polypeptide is SEQ ID NO: 5 A CDCA5 polypeptide or a functional equivalent thereof selected from the group consisting of polypeptides having a biological activity equivalent to a polypeptide consisting of an amino acid sequence;
b. An ERK polypeptide or functional equivalent thereof, wherein the functional equivalent of the ERK polypeptide is:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, and ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2 wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is SEQ ID NO: 1 or 2 A polypeptide having a kinase activity equivalent to a polypeptide consisting of the amino acid sequence of
An ERK polypeptide or a functional equivalent thereof selected from the group consisting of:
c. A reagent for detecting phosphorylation of CDCA5 at amino acid residue serine 79 or 209 of SEQ ID NO: 5; and d. ATP and kinase buffer. A kit for identifying an agent that modulates ERK kinase activity against CDCA5, comprising the following components:
a. A polynucleotide encoding a CDCA5 polypeptide or functional equivalent thereof, and a cell expressing a polynucleotide encoding an ERK polypeptide or functional equivalent thereof, comprising:
The functional equivalents of CDCA5 polypeptide are:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5,
ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 5, wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is derived from the amino acid sequence of SEQ ID NO: 5 A polypeptide having a kinase activity equivalent to that of the polypeptide, and iii. A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, provided that the resulting polypeptide is SEQ ID NO: 5 A polypeptide having biological activity equivalent to a polypeptide consisting of an amino acid sequence,
Selected from the group consisting of
The functional equivalents of ERK polypeptides are:
i. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, and ii. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2 wherein one or more amino acids are substituted, deleted or inserted, provided that the resulting polypeptide is SEQ ID NO: 1 or 2 A cell selected from the group consisting of polypeptides having a kinase activity equivalent to a polypeptide consisting of the amino acid sequence of
b. EGF, and c. A reagent for detecting phosphorylation of CDCA5 polypeptide at amino acid residue serine 79 or 209 of SEQ ID NO: 5.   The kit according to claim 4 or 5, wherein the reagent is an antibody that recognizes phosphorylation of CDCA5 polypeptide at amino acid residue serine 79 or 209 of SEQ ID NO: 5. A substantially pure polypeptide selected from the group consisting of:
a. A polypeptide comprising the amino acid sequence of SEQ ID NO: 7;
b. Biological activity equivalent to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7, comprising the amino acid sequence of SEQ ID NO: 7, wherein one or more amino acids are substituted, deleted, inserted and / or added A polypeptide having, and c. A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6, and comprising the amino acid sequence of any one of SEQ ID NO: 7 A polypeptide having a biological activity equivalent to   An isolated polynucleotide encoding the polypeptide of claim 7.   A vector comprising the polynucleotide of claim 8.   A host cell comprising the polynucleotide of claim 8 or the vector of claim 9. A method for the treatment and / or prevention of lung cancer or esophageal cancer in a subject comprising the following steps:
Administering a CDCA5 polypeptide variant having a dominant negative effect, a polynucleotide encoding the variant, or a vector comprising the polynucleotide.   12. The method of claim 11, wherein the CDCA5 polypeptide variant comprises an amino acid sequence in which at least one ERK-dependent phosphorylation site in the CDCA5 polypeptide is replaced with an amino acid residue other than a wild-type amino acid residue.   13. The method of claim 12, wherein the ERK-dependent phosphorylation site is one or both of Ser-79 and Ser-209.   14. The method of claim 13, wherein the CDCA5 polypeptide variant comprises the amino acid sequence of SEQ ID NO: 7.   A CDCA5 polypeptide variant has the general formula: [R]-[D], wherein [R] is a membrane-introducing agent and [D] is a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 15. The method of claim 14, wherein: The membrane introduction agent
Poly-arginine;
Tat / RKKRRQRRR / SEQ ID NO: 8;
Penetratin / RQIKIWFQNRRMKWKK / SEQ ID NO: 9;
Buforin II / TRSSRAGLQFPVGRVHRRLRK / SEQ ID NO: 10;
Transportan / GWTLNSAGYLLLGKINLKALAALAKIL / SEQ ID NO: 11;
MAP (Model Amphiphilic Peptide) / KLALKLALKALKALKALA / SEQ ID NO: 12;
K-FGF / AAVALLPAVLLALLAP / SEQ ID NO: 13;
Ku70 / VPMLK / SEQ ID NO: 14;
Ku70 / PMLKE / SEQ ID NO: 15;
Prion / MANLGYWLLALFVTMWTDVGLCKKKRPKP / SEQ ID NO: 16;
pVEC / LLIILRRRIRKQAHAHSK / SEQ ID NO: 17;
Pep-1 / KETWWETWWTEWSQPKKRKV / SEQ ID NO: 18;
SynB1 / RGGRRLSYSRRRRSTSTGR / SEQ ID NO: 19;
Pep-7 / SDLWEMMVSLACQY / SEQ ID NO: 20; and HN-1 / TSPLNIHNGQKL / SEQ ID NO: 21
The method of claim 15, wherein the method is selected from the group consisting of:   A composition for the treatment and / or prevention of lung cancer or esophageal cancer, which is a pharmaceutically effective amount of a CDCA5 polypeptide variant having a dominant negative effect as an active ingredient, which encodes the variant A composition comprising a polynucleotide, or a vector comprising the polynucleotide, and a pharmaceutically acceptable carrier. A method for assessing or determining the prognosis of a patient with lung cancer comprising the following steps:
(A) detecting the expression level of the CDCA5 gene in a biological sample derived from a patient;
(B) comparing the detected expression level with a control level; and (c) determining the prognosis of the patient based on the comparison of (b).   19. The method of claim 18, wherein the control level is a good prognosis control level and an increase in expression level compared to the control level is determined to have a poor prognosis.   19. The method of claim 18, wherein the increase is at least 10% higher than the control level. Expression levels are as follows:
(A) a method for detecting mRNA of CDCA5,
19. The method of claim 18, wherein the method is determined by any one method selected from the group consisting of (b) a method for detecting CDCA5 protein and (c) a method for detecting biological activity of CDCA5 protein.   19. The method of claim 18, wherein the biological sample from the patient comprises biopsy material, sputum or blood, pleural effusion, or urine. (A) a reagent for detecting mRNA of the CDCA5 gene;
(B) a reagent for detecting a protein encoded by the gene; and (c) a reagent selected from the group consisting of a reagent for detecting the biological activity of the protein, for diagnosing lung cancer Kits for assessing or determining the prognosis of patients with lung cancer.   24. The kit according to claim 23, wherein the reagent is a probe for a gene transcript of the gene.   24. The kit according to claim 23, wherein the reagent is an antibody against a protein encoded by the gene.

Images