TW201204393A - Diagnostic agent and therapeutic agent of cancer - Google Patents

Abstract

The present invention provides a novel medicament or method for treatment or diagnosis of cancer. Namely, the present invention provides a substance capable of inhibiting the expression of PUF60 gene, a substance capable of inhibiting the activity of PUF60 protein and a pharmaceutical composition containing the same as active ingredient; these substances and the pharmaceutical composition can be used in treatment or diagnosis of cancer.

Description

201204393 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a pharmaceutical composition for diagnosis and treatment of cancer. In particular, the present invention relates to the expression inhibitor of the PUF60 gene, the inhibitor of the activity of the PUF60 protein, and the use of PUF60 as a diagnostic marker for cancer. [Prior Art] The first cause of death in 曰 is cancer. The death of colorectal cancer is second only to lung cancer and stomach cancer. About 100,000 people suffer from colorectal cancer in a year, and about 40,000 people eventually die. And there is a tendency to increase year after year. In terms of the increase in colorectal cancer, although genetic factors and environmental factors are considered, the Westernization of dietary life, especially the excessive intake of animal fat, is hard to blame. In addition, breast cancer is the leading cause of cancer death among Japanese women aged 30 to 64. The number of breast cancer deaths is about 10,000 a year. Now, one in 20 women has breast cancer during their career, and the number of women with breast cancer in one year is about 40,000. In the case of early detection, if it is stage I, it will be 90% if it can survive for 10 years after surgery, but it will be 20% if it is stage IV. Although it is systemic drug therapy, it is still difficult to cure. The establishment of novel therapeutics is expected. Chemotherapy, which is currently the focus of cancer drug therapy, although there are many cytocidal agents that cause cell death by directly acting on DNA or RNA of cancer cells, these agents also act on, for example, bone marrow cells, reproduction. Cells, hair cells, gastrointestinal epithelial cells and other normal cells that divide vigorously, and bring strong side effects. 3 322688 201204393

The special mechanism has a specific role in the development of molecularly targeted drugs. As a representative example, the Jingchang Factor Receptor is the target ^' In the case of colorectal cancer, it is recognized that EGFR (Erbitux (General name: Cetuximab) is suitable for / 〇 EGFR positive and cannot be surgically removed. Recurrent colorectal cancer 'in terms of breast cancer, Herceptin (_like name: Trastuzumab), which recognizes HER2 (epithelial growth factor receptor), is suitable for the treatment of HER2 breast cancer or as a post-operative lion chemistry Therapy, and see the efficiency of high performance. In these successful cases, cancer depends on a specific gene, and by inhibiting the function of the gene, cancer shows the possibility of treatment. However, there are still few effective molecular markers. In the future, it is expected to develop more effective molecular markers. Poly-U binding factor 60kDa (PUF60) (also known as FIR (FBP interaction factor), SIAHBP1) is a splicing factor ( Splicing fact〇r), homologous to U2AF65, which is known as a splicing factor, and has a similar domain. Also, in terms of function, it is also reported to be related to U2AF65 (?886-McCawP) S, et al., (1999) RNA. Dec; 5(12): 1548-60) PUF60, like U2AF65, has two RNA recognition motifs (RRM) and has a C-terminal side. U2 auxiliary factor homology motif (UHM) domain structure, belonging to P〇ly-U RNA-binding protein (Hastings 4 322688 201204393 ML. et al., (2007) PloS ONE. Jun 20; 2(6):e538). It has been reported that PUF60 binds to U2AF65, SF1, and SF3B1 in the UHM domain region and is considered to be the mobilizing U2snRNP role in splicing (Corsini L. et al., ( 2009) J Biol Chem. Jan 2:284(1): 630-9). For the splicing of mRNA precursors (pre-mRNA), specific splicing factors recognize and bind to the splice sites in the sequence, and the stage This was carried out (Maniatis T, Tasic B. (2002) Nature. Jul 11; 418 (6894): 236-43) (Rymond B. (2007) Nat Chem Biol. Sep; 3(9): 533-5). Splicing factor 1 (SF1) binds to the branching point (A) in the intron (Berglund JA. et al., (1997) Cell. May 30; 89(5): 781-7) (Liu Z, et Al., (2001) Science. Nov 2; 294 (5544): 1098-102); heterodimer U2AF65-U2AF35 binds to AG, AG is a polypyrimidine region and a 3' side splice site (WuS. et al., (1999) Nature. Dec 16; 402 (6763): 832-5) (Zorio DA & Bluraenthal T. (1999) Nature. Dec 16; 402 (6763): 835-8). It has been reported that the combination of SF1 at the branching point is weak and can be stabilized by U2AF65 (Berglund JA. et al., (1998) Genes Dev. Mar 15; 12(6): 858-67). PUF60 has been identified as a nuclear extract, a novel poly-U RNA-binding splicing factor that binds to a poly U-Sepharose column, and is thought to enhance the function of U2AF65 (Page-McCaw PS. et al., ( 1999) RNA. Dec; 5(12): 1548-60). Regarding the interaction between PUF60 and U2AF65, it has been reported that it can mention the binding activity of 5 322688 201204393 to the p〇lypyrimidine tract RNA and the choice of exon for gene splicing. There are several effects (Hastings ML. et al., (2007) PLoS ONE. Jun 20; 2(6): e538). In the early stages of splicing, the next stage after SF1 binds to the branching point, U2 small ribonucleoprotein particles (snRNP) are mobilized to the 3' splice site. At this time, pl4 is localized at the branching point by the N-terminal (373-415aa) of SF3B1 (SF3bl55, SAP155), which is a constituent of U2snRNP, which is a constituent of U2snRNP, and Schlumenberg MJ. et al., (2006) Proc Natl Acad Sci USA Jan 31; 103(5): 1266-71). In the binding of U2snRNP to pre-RNA, the binding of SF3B1 to U2AF65 and the dissociation of SF1 from the branching point are necessary (Gozani 0, et al., (1998) Mol Cell Biol. Aug; 18(8):4752- 60). It has been reported that PUF60 has a C-terminal UHM that binds to the NHM-terminal UHM ligand motif (ULM) of SF3B1 (194-229aa), and the binding site of SF3B1 to U2AF65 and its binding site to PUF60 (Corsini L. Et al., (2009) J Biol Chem. Jan 2: 284(1): 630-9). Further, it is reported that the UHM domain of PUF60 is subjected to crystal structure analysis, which interacts with HLM of SF3B1 (Corsini L. et al., (2009) J Biol Chem. Jan 2:284(1): 630-9). Therefore, it is speculated that the interaction of PUF60 with SF3B1 may be important in terms of U2 snRNP mobilization to 3' splice sites. It has been reported that PUF60 (FIR) is specifically expressed in the cancer of colorectal cancer (International Publication No. WO2004/018518). Further, it has been reported that PUF60 6 322688 201204393 (SIAHBP1) exhibits an increase in non-small cell lung cancer, and it is described that the proliferation of non-small cell lung cancer cells can be inhibited by antisense oligonucleotides (International Publication No. WO2004/031413). Until now, the development of therapeutic drugs targeting nuclear factors has been necessary to inhibit not only the activity of the target molecule, but also the performance of the target molecule through the cell membrane and further through the nucleus. The development of invasive drugs is becoming more difficult, so the development of such drugs is less enthusiastic. Further, it has been previously thought that it is difficult to inhibit the interaction between protein molecules having no enzyme-like activity by low molecular compounds. This may be due to the difficulty in regulating the broad interaction interface between proteins with small low molecules. However, 'in recent years, there have been reports that a natural substance that is isolated from microorganisms and shows an antitumor effect, ie, piadienolide B (Kotake Y. et al., (2007) Nat Chem Biol. Sep; 3 (9): 570-5) and SpHceostatin A (Kaida D. et al., (2007) Nat Chem Biol. Sep: 3(9): 576-83) with the SF3b subunit of U2 snRNP As the target, the development of an anticancer agent based on the splicing body (Spiice〇s〇me) is receiving attention. Pradenide (E71 〇7) is undergoing Phase I clinical trials (Phase 1) in the United States. In addition, it was also reported that the results of splicing inhibitors were explored by in vivo screening analysis, and the natural product from plants (Ginkgo biloba) was found to be soginkugetin (0, Brien K. et al., (2008) J Biol Chem Nov 28;283(48):33147-54), attention is being paid to inhibitors of Spl iceosome as a target for cancer treatment (Disney MD. (2008) Nat Chem Biol. Dec: 4(12) : 7 322688 201204393 • 723-4). [Prior Technical Literature].

[Patent Document 1] [Patent Document 1] International Publication No. WO2004/018518 [Patent Document 2] International Publication No. WO2004/031413 [Non-Patent Document] [Non-Patent Document 1] Page-McCaw PS. et al., (1999) RNA. Dec; 5(12): 1548-60.

[Non-Patent Document 2] Hastings M. et al., (2007) PLoS0NE. Jun 20; 2(6): e538.

[Non-Patent Document 3] Corsini L. et al., (2009) J Biol Chem. Jan 2: 284(1): 630-9.

[Non-Patent Document 4] ManiatisT, Tasic B. (2002) Nature. Jul 11; 418(6894): 236-43.

[Non-Patent Document 5] Rymond B. (2007) Nat Chem Biol. Sep; 3(9): 533-5.

[Non-Patent Document 6] Berglund JA. et al., (1997) Cell. May 30; 89(5): 781-7.

[Non-Patent Document 7] LiuZ, et al., (2001) Science. Nov 2; 294 (5544): 1098-102.

[Non-Patent Document 8] Wu S. et al., (1999) Nature. Dec 16; 402 (6763): 832-5.

[Non-Patent Document 9] Zorio DA & Blumenthal T. (1999) Nature. Dec 16; 402 (6763): 835-8. 8 322688 201204393 [Non-Patent Document l〇] Berglund JA. et al., (1998) Genes

Dev. Mar 15:12(6):858-67.

[Non-Patent Document ll] Page-McCawPS· et al., (1999) RNA· Dec; 5(12): 1548-60.

[Non-Patent Document 12] Schellenberg MJ et al., (2006) Proc Natl Acad Sci USA Jan 31; 103(5): 1266-71. [Non-Patent Document 13] Gozani 0, et al., (1998) Mol Cell

Biol. Aug; 18(8): 4752-60.

[Non-Patent Document 14] Kotake Y. et al., (2007) Nat Chem Biol. Sep; 3(9): 570-5.

[Non-Patent Document 15] Kaida D. et al., (2007) Nat Chem Biol. Sep: 3(9): 576-83.

[Non-Patent Document 16] 0'Brien K. et al., (2008) J Biol Chem. Nov 28; 283(48): 33147-54.

[Non-Patent Document 17] Disney MD. (2008) Nat Chem Biol.

Dec: 4(12): 723-4. [Disclosure] [Problems to be Solved by the Invention] Under the conditions as described above, novel agents or methods for treating or diagnosing cancer are sought. [Means for Solving the Problem] The present inventors identified gene amplification, which is one of the characteristics of oncogene addict ions, as an index, and found out from among the human genomes of about 30 genes. A gene group that is an innovative drug. Furthermore, by 322688 201204393, genome-wide analysis by array-based comparative genomic hybridization (array CGH) method narrows the selection to a high frequency of gene amplification in cancer patients' samples. The gene was found to be a gene that causes a proliferation inhibition effect by gene knock down, that is, PUF60. Therefore, the present invention provides a pharmaceutical composition for cancer treatment or prevention of cancer progression or metastasis containing an inhibitory substance of the PUF60 gene as an active ingredient, and an activity-inhibiting substance containing PUF60 protein as an active ingredient for cancer The pharmaceutical composition for the treatment or prevention, the screening method for the activity inhibitory substance of the pUF6〇 gene or the activity inhibitory substance of the PUF60 protein, the screening kit, the diagnosis method for the cancer using the PUF60 as a diagnostic marker, the diagnostic composition, and the diagnosis Sets, etc. The present invention relates in particular to the use of small interfering RNA (siRNA) or antisense nucleic acids against the PUF60 gene as expression-inhibiting substances of the PUF60 gene. Accordingly, the present invention provides the following. (1) A step for inhibiting proliferation of cancer cells, which comprises an expression-inhibiting substance of a cyclin gene or an activity-inhibiting substance of a pUF6Q protein. (2) The composition according to the above (1), wherein the expression inhibitory substance of the 4 PUF6G gene is selected from any one of the following groups (4) to (6): (a) inhibiting by the RNAi effect puF6〇* a nucleic acid which acts as a function, (b) an antisense nucleic acid which is a transcription product of the PUF gene or a part thereof, and 322688 10 201204393 (c) a nucleic acid having ribzyme activity, the ribonucleic acid The enzyme specifically cleaves the transcription product of the PUF gene; the activity inhibitory substance of the PUF60 protein is selected from the following ((1) to any of the groups consisting of: (d) can specifically bind to PUF60 An antibody to a protein, (e) a low molecular compound that specifically binds to the PUF60 protein, and (f) a low molecular compound that inhibits the interaction of a molecule that interacts with the PUF60 protein. (3) As described above (1) or (2) The composition of the puF6〇 gene, wherein the expression inhibitor of the puF6〇 gene comprises a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. (4) as described in (1) to (3) above. a composition of any of the records, The cancer according to any one of the above (1) to (4), which is a medicine for preventing or treating cancer. (6) A nucleic acid molecule Is selected from the group consisting of (i) and (ii): (I) antisense nucleic acid molecule and siRNA molecule of (a) or (b) below (a) nucleic acid sequence of PUF60 gene, (b) coding a nucleic acid sequence of a PUF60 protein; and (II) a vector (c) and (d) (c) a vector comprising the above antisense nucleic acid molecule, and (d) a vector containing the siRNA molecule. (6) The nucleic acid molecule according to the above aspect, wherein the siRNA molecule comprises a nucleic acid sequence of 322688 11 201204393 SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. (8) as described in the above (6) or (7) A nucleic acid molecule for use in the prevention or treatment of colorectal cancer or breast cancer. (9) A method of using PUF60 as a diagnostic marker for cancer, comprising detecting a PUF60 gene, a transcription product thereof or a translation thereof from a subject's sample a product, or a step of such a fragment. (10) A method as described in the above (9), which comprises : (i) a step of detecting a PUF60 gene or a transcript thereof or a fragment thereof from a subject, wherein a nucleic acid sequence encoding the gene or a transcript thereof or a nucleic acid sequence encoding the PUF60 protein is used. Or a portion of the specifically hybridized nucleic acid molecule is subjected to the detection, or (11) a step of detecting a PUF60 protein from the subject, wherein the detection is carried out using an antibody that specifically binds to the protein or a fragment thereof; The presence of the PUF60 gene, its transcription product, the PUF60 protein or the fragment in the sample indicates that the subject may have cancer. (11) The method according to (9) or (10) above, wherein the cancer is colorectal cancer or breast cancer. (12) A diagnostic kit for cancer comprising: (i) a nucleic acid molecule which specifically hybridizes to a nucleic acid sequence of a PUF60 gene or a transcript thereof or a nucleic acid sequence encoding a PUF60 protein; or 12 322688 201204393 (ii) Antibodies that specifically bind to PUF60 protein or a fragment thereof; and (iii) instructions for use. (13) The kit according to the above (12), wherein the cancer is colorectal cancer or breast cancer. (14) A method for screening a performance inhibitor of a PUF60 gene, comprising: a step of culturing a cell expressing a PUF60 gene in the presence or absence of a test compound; and a quantity of a transcription product of the PUF60 gene or an amount of a PUF60 protein The step of determining the amount of PUF60 expression of the cultured cells for the indicator; and the step of comparing the amount of expression of the PUF60 in the presence or absence of the test compound. (15) A method for screening an activity inhibitor of PUF60 protein, comprising: a step of contacting a test compound with a multi-peptide or PUF60 protein encoded by a PUF60 gene; and measuring the biological activity of the above-mentioned multi-peptide or protein And a step of selecting a compound that inhibits the biological activity of the multi-peptide or protein as compared to the biological activity of the multi-peptide or protein in the absence of the test compound. (16) A screening method according to the above (14) or (15), which is used for screening a cancer preventive or therapeutic agent. (17) The screening method according to the above (16), wherein the cancer is colorectal cancer or breast cancer. [Effects of the Invention] As shown in the following examples, it can be seen that PUF60 is specifically expressed in cancer tissues, and since the gene repression (RANi) analysis in cancer cells, 13 322688 201204393 can be induced to cause significant cell death induction. The resulting proliferation inhibition effect PUF60^^^^fi]#J^ pup6〇H may be useful. The PUF6G $ splicing factor, salty, is believed to be caused by the inhibition of PUF60, which can cause AI to accept different books, which makes the abnormal _ and: white matter increase, and finally induces death through intracellular stress. And the regulation of the PUF6G gene in colorectal cancer and breast cancer, and it is observed that the function of inhibiting the function of RANi has cancer cells, so it is possible to use PUF60 as a diagnosis of colorectal cancer and breast cancer. In addition, the performance-inhibiting substance of PUF60 or the activity-inhibiting substance of puf60 may be used as an anticancer agent of the next-generation. [Embodiment] [Embodiment of the Invention] L has an agent for inhibiting cancer. The embodiment is a pharmaceutical composition for the treatment or prevention of cancer, such as PUF60 囡+ + expression inhibitor, PUF60 protein activity inhibitor ′, and such a substance as an active ingredient. In the present specification, the term “PUF60 gene” Refers to PUF60 (Poly-binding splicing) from humans encoded by Gene ID: 22827 (http://www.ncbl. nim. nih.gov/gene /22827) in the NCBI Gene Database Gene 60kDa) and its functional equivalents. The protein encoded by the PuF6〇 gene (PUF60 protein) is a Ro ribonucleoprotein (RNp) binding protein, and its interaction with R〇 RNP is recognized. 14 322688 201204393 It is possible to (evolutionally) function the Ro RNP. The PUF60 protein is also a FUSE binding protein that can form a = meta complex with FUSE (far upstream element). The PUF60 protein can also inhibit the c-myc reporter via fuse. The PUF60 protein is known to be transcribed as a transcription factor and inhibits activated transcription. The PUF60 gene is associated with pigmented dry skin disease. It is known that in the case of the PUF60 gene, there are two alternative transcriptional variants which are alternatively spliced, which encode two distinct isoforms (http://www.ncbi.nlm.nih. Gov/gene/22827). For the transcripts (ie, mRM) and translation products (ie, proteins) of the PUF60 gene, as described above, including two transcriptional variants resulting from alternative splicing, that is, three species are known, respectively, in the NCBI database. The registration number is _078480 and NP-51095 (equivalent type a), NM-014821 and NPJ) 55096 (equivalent type b), N1001136033 and NPj) 〇ii29505 (equivalent type c) are specified. In the present specification, the "transcription product of the PUF60 gene" refers to the mRNA produced by the transcription of the PUF60 gene, that is, the so-called "PUF60 mRNAj °" in the present specification, the "translated product of PUF60" refers to the PUF60. The "PUF60 protein" synthesized by the nucleic acid sequence of mRNA. In the present specification, the term "PUF60" means "PUF60 gene, PUF60 mRNA, PUF60 protein, any of these, or all of these, when "PUF60" is used alone. As to what meaning it is, those skilled in the art will naturally understand from the literature using the term. 15 322688 201204393 The term "inhibition of gene expression" as used in this specification means a series of events (including, for example, transcription (production of mRNA), translation (production of protein)), from inhibition of genes to production of proteins. One or two items' inhibits the production of a protein encoded by the gene. In the present specification, a gene encoding human-derived PUF60 (hereinafter referred to as "original gene") is specifically designated by gene numbering (NCBI): 22827. "Functional equivalent" means that although the encoded protein retains the same biological activity as the human PUF60 protein, it has a number of nucleotide changes compared to the human PUF6(R) protein gene (eg, by degeneracy of the genetic code) Producer, or by coding different variants, etc.) or, more generally, 'meaning that by having a substitution of more than one nucleotide 'deletion, addition, insertion, or the like a variation of two or more combinations, the nucleic acid sequence is changed from the nucleic acid sequence of the original gene, but the function or health of the protein encoded thereby In the present specification, the term "PUF60 protein" means any of the above three kinds of PUF60 proteins (hereinafter, referred to as "PUF60 protein"), which is the same as the protein encoded by the original gene. "original protein"); and maintain the same function or biological activity as these proteins (binding to the pyrimidine-rich region (polypyridamole region) downstream of the branching point, 3, upstream of the splicing site; The constitutive factors of U2AF65 and U2 snRNP, ie, the splicing-related factors such as SF3B1 and SF1 of the SF3b subunit, have splicing regulatory activity; and the R〇RNP binding ability, the ability to form a ternary complex with FUSE, and the inhibition of c-myc The ability to transcribe, the ability to inhibit transcriptional activation by TFIIH, etc., and the deletion, substitution, insertion, and addition of 1 to a plurality of amino acid residues from 16 322 688 201204393 compared with the amino acid sequence of the proteins a natural or artificial variant protein consisting of a variant amino acid sequence of a combination of any two or more thereof. The variation site and number of the base acid are not particularly limited as long as the variant protein maintains the same function or biological activity as the original protein, and the number of mutations is, for example, 1 to 50, 1 to 4, 1 to 30, 1 Up to 25, 1炱20, 1 to 15, 1 to 1〇, 1 to 9, 1 to 8, 1 to 7, 1 to 6 (1 to several), 1 to 5 1 to 4, 1 to 3, 1 to 2, and 1. The number of mutations is generally preferably less. Moreover, the proteins of such variations comprise: about 70 amino acid sequences with the original protein. % or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, and 93°/. Above, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the identity of the amino acid sequence' and having the same function or biological activity as the original protein . The above homologous (homo 1 ogy ) value is generally larger. The "partial peptide" (or "fragment") of the PUF60 protein is also included in the above PUF60 protein. A part of the peptide of the PUF60 protein is a partial peptide consisting of a sequence of a partially amino acid of one of the amino acid sequences of the above PUF60 protein, preferably, as long as it has the same activity as the activity of the above pUFgo protein, One can be. For example, at least 2, preferably at least 50, more preferably at least 7, more preferably at least 1 , and most preferably 2, of the amino acid sequences of the above three modifications may be mentioned. A multipeptide of an amino acid sequence consisting of an amino acid residue. Preferably, the multi-peptide of 322688 17 201204393 contains an amino acid sequence corresponding to a portion of the PUF protein activity > v king. Further, a part of the peptide used in the present invention may be one or a plurality of amino acid sequences (for example, about 1 i to 20, more preferably about i to H) in the above multiple wins. Preferably, the steps are from about i to 5) the kine acid residue is deleted, added, replaced, inserted, or a variation of the gradation of any two or more thereof. The 60 protein used in the present invention can be prepared from cells or tissues expressing the protein. Further, these proteins can be synthesized by a known peptide synthesizer or by a recombinant method using a suitable host cell selected from a prokaryotic or eukaryotic organism. The PUF60 protein used in the present invention may be derived from any species, preferably from humans. By "same function or biological activity" it is meant that such functions or activities are of the same nature. Therefore, 'for example, as long as it has an activity such as 1 binding ability, U2AF65 binding ability, SF3B1 binding ability, and r〇rnp binding ability, which is homogenous to the activity of the original protein, the degree of such activity, the molecular weight of the protein, and the like It may be different (for example, about 1 to 100 times, preferably about 0.5 to 20 times, more preferably about 〇. 5 to 2 times). The activity can be determined according to the poly-U binding ability (Page-MaCaw PS, et al., (1999) RNA. Dec; 5(12): 1548-60), U2AF65 binding ability (Hastings ML, et al., ( 2007) PLoS ONE. Jun 20; 2(6): e538), SF3B1 binding capacity (Corsini L, et al., (2009) J Biol Chem. Jan 2; 284(1): 630-9) and Ro RNP binding The ability is carried out by a known method described in the literature (Bouifard P. et al., (2000) RNA. Jan; 6(1): 66-78). 18 322688 201204393 Furthermore, for the identity of amino acid sequences or base sequences, the algorithm of Karlin and Altschul can be used for BLAST (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc. Natl. Acad. Sci USA 90: 5873, 1993) to decide. A program called BLASTN and BLASH was developed according to the BLAST algorithm (Altschul SF. et al., J Mol Biol 215; 403, 1990). In the case of analyzing the base sequence using BLASTN, the parameter is set to, for example, score = 100, word length = 12. Also, in the case of analyzing the amino acid sequence using BLASTX, the parameter is set to, for example,

For example, score = 50, word length = 3. In the case of using the BLAST and Gapped BLAST programs, the default parameters of each program are used. ° In this specification, the term "cancer" is used in a generally recognized sense in the art, and includes Malignant diseases of epithelial tissues or endocrine tissues such as respiratory cancer, gastrointestinal system cancer, genitourinary system cancer, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. For example, cancer includes those formed from tissues of the cervix, lungs, prostate, breast, head and neck, colon (or large intestine), and ovaries. This term also includes cancerous sarcoma, which contains malignant tumors composed of cancerous tissues and tumors. "Adenocarcinoma" refers to a cancer derived from glandular grades, or a cancer that can form a glandular structure that recognizes tumor cells. Furthermore, in the present specification, the terms "cancer" and "tumor" have the same meaning. Examples of cancer which can be treated or prevented by the method of the present invention include colon cancer, breast cancer, gastric cancer, lung cancer, prostate cancer, esophageal cancer, liver cancer, bladder cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, 322688 19 201204393 Pancreatic cancer, ovarian cancer, brain tumors, blood tumors, etc. In the present specification, the "pharmaceutical composition for the treatment or prevention of cancer" or the "prophylactic or therapeutic agent for cancer" is an anti-cancer agent, a cancer metastasis inhibitor, a cancer regulating agent, a cancer inducer, and a cancer. It is used in the sense of a cell proliferation inhibitor, a cancer cell infiltration inhibitor, a cancer preventive agent, and the like. Further, in the present specification, "prevention of cancer" means inhibiting the progression or metastasis of cancer. Thus, 'in one embodiment of the present invention', an expression-inhibiting substance of the PUF60 gene is provided, which comprises the following (a) to (h). Examples of substances that inhibit transcription from the PUF60 gene into PUF60 mRNA are: == the gene or a portion thereof, an antisense nucleic acid, (b) for the PUF gene or a portion thereof (〇 for the PUF gene or a nucleic acid thereof, negatives acting) _ = birth reduction (―, (4) other compounds that inhibit transcription. Gene variants, and, in turn, inhibition of translation from PUF60 gene into p. Examples are: 叩6〇 protein substances (e) for PUF6〇mRM or its — A thief (eg, si_, . . . has an effect on (f) against PUF60 mRNA or a portion thereof (g) cake on PUF60 mRNA or an antisense polynucleotide thereof, a poly(tetra) acid, and a D knife with ribose Other compounds that inhibit the translation of nucleic acid enzymes. In this specification, the term "nucleic acid 4 (poly) nucleotide" means 322688 20 201204393 awake or hidden. The so-called "nucleic acid" is not only for those who have a discussion or a base. And may include modified persons, such as having other heterocyclic type test groups. These U substances may be those containing methylation, ticketing and β-bite, thiolated hydrazine and shouting, or other heterocyclic ring. Modified fresh and modified (tetra) acid can also be sugar For example, in the cancer therapeutic agent of the present invention, the effect of the RNAi can be used instead of the above-mentioned secrets. A nucleic acid having an effect of inhibiting the expression of the PUF6G gene is effective as a cleavage. The RNAi is a gene that introduces a foreign gene and a target intrinsic gene when a double-stranded RNA having the same or similar sequence as the target gene sequence is introduced into the cell. The performance is suppressed. For the use of the term, for example, a double-stranded RNA that causes 19 to 30 bases of RNA interference, such as dsRNA (double stranded rna), siRNA (small interfering RNA) Or shRNA (short scorpion RNA). These RNAs can be locally delivered to a desired site by a delivery system such as a ribosome. The double-stranded RNA can also be partially expressed by a vector capable of producing the above-described double-stranded RNA. A method for preparing a double-stranded RNA (dsRNA, siRNA, or shRNA), a method of using the same, and the like are known in the literature (Japanese Patent Publication No. 2002-516062; US Patent Publication No. 2002/086356A; Nature Genetics, 24) (2), Feb. 180-183; Genesis, 26(4), April, 240-244; Nature Spe. 21, 407: 6802, 319-20; Genes & Dev., Vol. 16(8). Apr.16, 948-958 ; Proc.Natl. Acad. Sci. USA., 99(8), 16 Apr., 5515-5520; Science. 296(5567), 19 Apr., 550-553; Proc Natl. Acad. Sci. USA, Apr 30, 99:9, 6047-6052; Nature 21 322688 201204393 • Biotechnology, V〇1.20(5), May, 497-500; Nature-Biotechnology, Vol. 20(5), etc.). The length of the double-stranded RNA which exerts the RNAi effect used in the present invention is usually 19 to 30 bases, preferably 20 to 27 bases, more preferably 21 to 25 bases, most preferably 21 to 23 Bases. In the present invention, specifically, the following siRNA (used in Example 3) can be used. siRNA a : UGUACGACCAGGAGCGUUUUU (SEQ ID NO: 1) siRNA b : CAGCCUACAGUGCGGAUAAUU (SEQ ID NO: 2) siRNA c : GCUUCAUUGAGUACGAGAAUU (SEQ ID NO: 3) siRNA d : CCAUCAAGAGCAUCGACAUUU (SEQ ID NO: 4) 'Antisense nucleic acid' or 'antisense' in this specification "Polynucleotide" refers to a nucleic acid having a polynucleotide that is complementary to at least a portion of a DNA region of a subject, and which can hybridize to at least a portion of the region. The present invention = antisense nucleic acid is RNA, DNA or modified nucleic acid (rna, DNA). These can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and hybridized with RNA. a specific example of the modified nucleic acid + a sulfur derivative or a phosphorothioate derivative of the nucleic acid, and further, the decomposition of the guanamine or the oligonucleotide of the oligodeoxynucleotide is resistant to Wait. Unlimited

Dankao, the antisense nucleic acid used by DNA, is linked downstream of the appropriate promoter to the sequence containing the transcription termination signal at the 3' side. The ruthenium acid is thus prepared, and the desired animal can be transformed by using a known method. The sequence of the nuclear anger is preferably a sequence complementary to the intrinsic gene or the deficiencies of the morphing animal, but it is not necessary to be completely complementary as long as the gene can be effectively inhibited 322688 22 201204393. For example, the right sigh & ten and PUF6 〇 gene 瀬 A 5, the end of the non-translated region of the opposite antisense phase, the financial effective inhibition of the translation of the gene. A sequence complementary to the coding region or the non-translated region on the 3' side can also be used. The antisense nucleic acid of the gene can be effectively inhibited from the transcription of the target gene by about 7 G% or more, more preferably about 犠 or more, and most preferably about 95% or more. In terms of using an antisense nucleic acid and effectively inhibiting the expression of the target gene, the length of the antisense nucleoside is at least about 1 碱基 base or more (for example, about one), preferably about 15 bases or more, more preferably about More than 100 test bases, better step-by-step test. Antisense nucleic acids can be designed with reference to well-known literature (see, for example, 'Pingdao and Inoue, Lectures on Fresh Chemistry Experiment 2, Replication and Expression of Nucleic Acid IV Genes, edited by the Japanese Biochemical Society, Tokyo Chemicals, 1993, p. 319 347; J. Kawakami et al., Pharm Tech Japan. V〇l. 8, ρ·247, 1992; ST Crooke et al., ed·, Antisense Research and Applications, CRC Press, 1993, etc.). Further, in the cancer therapeutic agent of the present invention, a nucleic acid can be used as an active ingredient. The nucleic acid has a ribonuclease activity capable of specifically cleaving a transcription product of the PUF60 gene. The term "ribonuclease activity" refers to a nucleic acid which can specifically cleave a gene which is a target gene transcription product, i.e., mRNA. Regarding ribonuclease, although there are macromolecules of 400 nucleotides or more like the M1RNA contained in the first group intron group or RNaseP, there are also about 40 cores called hammerhead or scorpion type. Active structure of glucosinolates 23 322688 201204393 Domain (Protein Nucleic Acid Enzyme, 1990, 35, p. 2191). For hammerhead ribonuclease, for example, FEBS Lett, 1988, 228, ρ. 228; FEBS Lett, 1988, 239, p. 285; Protein Nucleic Acid, 1990, 35, P. 2191; Nucl Acids Res, 1989 , 17, ρ·7059, etc. Further, regarding an indole-type ribonucleic acid enzyme, for example, Nature, 1986, 323, P. 349: Nucl Acids Res, 1991, 19, p. 6751: Kikuyo, Chemistry and Biology, 1992, 30, p. 112 Wait. By specifically cleaving the transcription product of the PUF60 gene of the present invention by using these ribonuclease, the expression of the gene can be suppressed. Further, in the present invention, a compound other than the nucleic acid which inhibits the transcriptional activity of the PUF60 gene can be used as an active ingredient. Such compounds are, for example, compounds which bind to factors involved in the expression and transcription of the PUF60 gene. These compounds "T are natural or synthetic compounds. Such compounds can be obtained by the screening methods described hereinafter. In another embodiment of the present invention, 'a living inhibitory substance providing a puF6 〇 protein' comprises the following (a) to (d). (a) an antibody that specifically binds to a PUF60 protein, (b) a puF60 protein variant having a property of dominantly deactivating the PUF60 protein, and (c) a (low molecular) compound that specifically binds to the PUF60 protein (described above) (except for antibodies and variants), and (e) a (low molecular) compound that inhibits the interaction of a molecule that interacts with the PUF60 protein, and the term "antibody" as used herein means the full length of the protein 322688 24 201204393 or Fragment reaction antibody. The aspect of the antibody of the present invention is not particularly limited as long as it can specifically bind to the PUF6G protein of the present invention, and may include a human antibody in addition to a plurality of antibodies and monoclonal antibodies, and is formed by recombination. Humanized antibodies' and such antibody fragments or anti-defective modifications. An antibody that specifically binds to prase protein f (anti-PUF6() anti-PUF6() anti-PUF6() anti-PUF6() anti-PUF6() anti-PUF6() anti-PUF6() anti-PUF6() anti-PUF6() anti-PUF6() anti-PUF6() anti-PUF6() anti-PUF6() anti-PUF6 (4) A protein exhibiting the gene expression of the P诵 protein variant, and the activity of the intrinsic wild-type protein is lost or reduced. Further, in the present invention, the activity of the protein can be suppressed. An antibody or a variant which binds to the above variant of the Ρ_protein; or an antibody or a variant other than the interaction of the sputum; the compound may be, for example, a combination A compound which inhibits P-deletion activity by a compound which deletes a protein and inhibits its activity or a molecule which inhibits the interaction of a suspension molecule of I (which can exert the biological activity of (10).) It may be a natural product' or a synthetic compound. These compounds are obtained by the selection method. The above-mentioned invention can inhibit the activity of deleting the protein. As a prophylactic or therapeutic agent for cancer. 2. Method for using PUF60 as a cancer marker The present inventors have found that the PUF60 gene is genetically amplified in a specimen derived from cancer, particularly colorectal cancer 322688 25 201204393 . The frequency is high (Examples i and .2). Therefore, in one embodiment of the present invention, there is provided a method of using puF6〇 as a diagnostic marker for cancer. More specifically, the present invention provides a method of using PUF6〇 as a large intestine. A method for diagnosing a marker for cancer or breast cancer, comprising the step of detecting a PUF60 gene, or a transcript thereof or a translation product thereof, in a sample from a subject. In the present specification, the term "subject" refers to a human Patients and healthy people, especially those who are at risk of cancer, those who are suspected of having cancer, those who are suffering from cancer, etc. In this specification, the "sample" contains organ tissues, cells or body fluids (such as blood) from the subject. (including whole blood, plasma, serum, etc.) urine, lymph, saliva, sweat, semen, etc.) In the PUF60 aspect, the invention is used as a diagnostic marker. Specific implementation of the method (1) detecting the pUF6〇 gene or a fragment thereof or a fragment thereof in a sample taken from a subject, wherein the hybridization specifically hybridizes with the gene or its transcript: acid sequence The subtraction molecule (probe) into the stone 雉 ^ ^ in the sample of the presence of PUF60 gene, complex ' can be diagnosed as the subject: = colorectal cancer or (four) high energy-cutting ability. Or "(poly)nucleotide" means DNA戋RNa, which may be a double-stranded ridge, or may be a ship: RM hybrid _ 322688 26 201204393 'In this specification, a nucleic acid molecule "specifically hybridizes" It is meant that the nucleic acid* molecule hybridizes to a particular nucleic acid sequence under stringent hybridization conditions, i.e., does not bind to any nucleic acid sequence, but only binds to a particular nucleic acid sequence. Hybridization can be carried out according to known methods or methods based thereon, for example in

The method described in Molecular Cloning Third Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press. 2001 was carried out. Further, in the case of using a commercially available library, it can be carried out in accordance with the method described in the attached instruction manual. Here, regarding the "stringent hybridization conditions", the specific nucleic acid sequence can be distinguished from any other nucleic acid by the probe nucleic acid used in the present invention, which can be low-duty conditions, moderately harsh conditions, and south. Any of the harsh conditions. r Low severity conditions" For example: 5xSSC, 5x Denhalt solution, 〇·5% SDS, 50% guanamine, 32 °C conditions. The "moderately harsh conditions" are, for example, 5xSSC, 5x denhard solution, 0.5% SDS, 50% guanamine, and 42 °C. The "highly harsh conditions" are, for example, 5xSSC, 5x denhard solution, 〇. 5% SDS, 50% guanamine, 5 〇. (The conditions of: Under these conditions, it is expected that the higher the temperature is, the more efficiently the DNA with high homology can be obtained. However, in terms of the factors affecting the severity of hybridization, salt is considered to have temperature and exploration. A plurality of elements such as the concentration of the needle, the length of the probe, the ionic strength, the time, and the salt concentration. Those skilled in the art can achieve the same severity by appropriately selecting these elements. For example, when the software is searched for by homology such as fasta or blast, and the calculation is performed using a default parameter, the nucleic acid sequence with PUF60 has, for example, 70% or more, 27 322688 201204393 75% or more, 80%. A nucleic acid molecule of μ or above, above, above, above, above 91%, above 92%, above 99%, above coffee, above coffee, above 97%, 98% < In the diagnostic method of the sequence, 'the nucleus based on the PUF6° gene can be used, such as the 'spoon: two probes or primers. Specifically, these diagnostic methods, the squat/the living specimens from the subject and the poly Nuclear acid (probe) The polynucleic acid (probe) is a step of detecting and/or quantifying a hybridization of a deletion gene or a fragment thereof in the above-described sample by a nucleic acid sequence which hybridizes to a nucleic acid sequence of a severe gene or a fragment thereof. ^, The above method of the present invention uses the above probe to detect and/or quantify the difficulty or _ or basin fragment of the PUF60 gene in the living specimen of the examiner. The length of the nucleic acid sequence used as the probe may be, for example, 12 or more, 15 or more, 18 or more, 21, 1, 24, or more, and 27 or more. 30 test bases above, or: more: length of polynuclear acid fragments. In terms of hybridization, the above-mentioned low- and medium-duty "severe stricter can be used. In addition, in this (4) book towel, "the nucleic acid sequence which can hybridize under the stringent hybridization conditions to the minus sequence of the _0 gene or the W segment" Of course, it includes a subtraction (4) complementary to the nucleic acid sequence of the fragment of the P-cookie. The method of hybridization of a probe and a nucleic acid is known to those skilled in the art and is described in, for example, International Publication No. __, EP-A 0 020 362, U.S. Patent No. 2,915, No. 82, EP-A0063879 'EP -A0173251 > EP-A0128018 In the diagnostic method of the present invention, the sequence of the target can be detected or quantified by a known method using a heteropolynucleotide probe or primer for the PUF60 gene of 322688 28 201204393. For such known methods, for example, Southern Crossing, Northern Crossing, RT-PCR, PCR-SSCP (Genomics, Vol. 5, pp. 874-879 (1989)), Proceedings of the National

Academy of Sciences of the United States of America, Vol. 86, pp. 2766~2770 (1989)), FISH method, DNA wafer or array CGH (comparative genomic hybridization) method, and the like. Quantitative detection can be performed by quantitative RT-PCR. Array CGH is a method of applying the chromosome CGH method (Kallionicmi, A. et al. (1992) Science 258, 818-821), that is, by using a DNA fragment (BAC, which covers a chromosomal region at a high density). PAC, YAC, etc.) DNA wafers were spotted on a glass slide, and the DM and normal DNA from the cancer, which are labeled with the pigment, were simultaneously hybridized with the genomic DNA fragments on the slide, and then the binding state was detected. A method for detecting abnormal DNA sets in cancer with high resolution (Pinkel, D. et. al. (1998) Nat. Genet. 20, 207-211). Furthermore, in the present invention, in order to detect whether the expression of the PUF60 gene is regulated upstream, the amount of PUF60 mRNA of the cell is compared with a standard gene (for housekeeping genes (for example, Shaper, NL, J. Mammary Gland Biol. Neoplasia 3 (1998) 315 -324; Wu, YY and Rees. JL, Acta Derm. Venereol. 80 (2000) 2-3)) Comparison of mRNA quantities, preferably by RT-PCR. In the case of detecting and/or quantifying the target sequence (DNA, mRNA, etc.) by the above method and confirming the performance (or excessive expression) of the PUF60 gene, it is possible to diagnose 29 322688 201204393 as: for example, 'because of PUF60 performance (or excess performance) The disease caused by the disease (such as cancer (such as colorectal cancer, breast cancer)) is high, or the possibility of future disease is high. Alternatively, in an alternative aspect of the specific embodiment of the method of the invention described above, (ii) detecting the PUF60 protein from the subject, wherein the antibody is specifically bound to the protein or fragment thereof. The test. As a result, if it is confirmed that the PUF60 protein or a fragment thereof is present in the sample, the subject can be diagnosed as having a high possibility of suffering or having a high possibility of suffering in the future. In the present specification, the phrase "an antibody that specifically binds to a PUF60 protein or a fragment thereof" is used interchangeably with an "anti-PUF60 antibody", and means an antibody that specifically binds to a PUF60 protein, a fragment thereof (partial peptide) or a salt thereof. . The anti-PUF60 antibody used in the present invention may be a plurality of antibodies or a monoclonal antibody. The type of the antibody is not particularly limited, and includes an antibody having any isotype of IgG, IgM, IgA, igj) or IgE. IgG or IgM is preferred, and IgG is more preferable in view of ease of purification and the like. Further, the term "r antibody" is used in the sense of including any antibody fragment or derivative. For example, it includes Fab, Fab, 2, CDR, humanized steroid, multifunctional antibody, single chain antibody (ScFv) and the like. The antibody of the present invention can be produced by a known method. Methods for the manufacture of such antibodies are well known in the art (for example, see Harlow E. & Lane D., Antibody, Göld Sprin S Harbor Laboratory Press (1988)). As described above for detecting PUF60 30 322688 201204393 performance in a living specimen from a subject, the immunoassay includes: a living body sample taken from a subject suspected of having cancer or having a risk of developing cancer 2 The anti-PUF60 antibody is contacted under conditions in which specific antigen-antibody binding is generated, and then the immunospecific 陧 and 纟 σ conjugates of the antibody are determined. The presence and/or increased performance of the PUF60 protein is detected using the binding of such antibodies. In this case, the increase in PUF60 protein expression was detected as an indicator of disease status. The amount of PUF60 protein in a living body can be compared with the amount in a healthy person who does not have cancer, as needed. In one aspect of the above immunoassay, in order to fix all the proteins present in the sample, it is possible to use, for example, a solid sample such as a serum sample and a solid phase support such as a cellulose or a carrier. )contact. Next, the support is washed with a buffer and then treated with an anti-PUF60 antibody that is detectably labeled. Next, the solid phase support was washed twice with a buffer to remove unbound antibody. The amount of antibody bound to the solid support is determined by well known methods. The detection conditions suitable for each measurement can be appropriately determined by those skilled in the art using conventional test methods. In one of the methods for detecting an anti-PUF60 antibody in a detectable manner, the antibody is bound to an enzyme, for example, an enzyme used in an enzyme immunoassay (EIA) [Voiler, A., The enzyme Linked Immunosorbent Assay ELISA, 1978, Diagnostic Horizons, 2:1-7, Microbiological Associates Quarterly Publication, Walkersvi1le. MD; Voiler, A., J. Clin. Pathol., 31:507-520, 1978; Butier, JE, Meth. Enzymol., 73:482- 523, 1981]. The enzyme bound to the antibody is reacted with a suitable matrix by, for example, a method of producing a chemical molecule 31 322688 201204393 which can be detected by spectrophotometry or by fluorescence measurement (fluorescence by visible means). It can be used to test the 撵 "two color developing enzymes and secret phosphatase, but the enzyme contains a peroxygen colorimetric method, and the colorimetric method uses leaven; the == can also be used in the present invention. Other methods used may be exemplified by radioactive immunoassay (Guan, San_Qi_Technology, New Analysis, Immunoturbidimetry (_helQmetry), Glory Immunoassay (fia), and Time Fluorescence Immunoassay ( «, enzyme decantation (eia), luminescent immunoassay (LIA), electrochemiluminescence immunoassay (ECUa), latex agglutination, immunoprecipitation, sedimentation, gel diffusion, sedimentation, immunoassay An immunoassay or the like in a group consisting of a diffusion assay, a lectin assay, a complement-binding assay, an immunoradiometric assay, and a protein A immunoassay (for example, WOOO/14227, ep1111〇47A2). As described above, by using the PUF60 protein quantification method using the antibody of the present invention, various diseases associated with the dysfunction of the PUF60 protein can be diagnosed. For example, if the concentration of the PUF60 protein is increased, Then, it can be diagnosed as 'for example, the possibility of suffering from diseases caused by excessive expression of PUF60 protein (for example, colorectal cancer, breast cancer) is high or the possibility of future disease is high. Furthermore, the anti-PUF60 antibody of the present invention can be used for diagnosis in vivo. Methods of preparing and using antibody preparations useful in this diagnosis are known in the art. For example, regarding antibody-chelating agents, described in Nucl. Med. Bi〇i. 1990 17:247-254. An antibody having a paramagnetic ion as a marker for magnetic resonance imaging is described, for example, in Magnetic Resonance in 32 322688 201204393

Medicine 1991 22:339-342. 3. Diagnostic Kits The present invention also provides kits for detecting and/or quantifying a PUF60 gene or a fragment thereof as a cancer marker in a sample from a subject, which is capable of interacting with the PUF60 gene under stringent hybridization conditions. A nucleic acid sequence in which a nucleic acid sequence thereof or a portion thereof is hybridized. Furthermore, the present invention provides a kit for detecting and/or quantifying a PUF6〇 protein or a fragment thereof as a cancer marker in a sample from a subject, which comprises an anti-PUF60 antibody. These kits are used to detect cancer markers by the above methods of hybridization or immunology. For such cancers, for example, large cancer, breast cancer, stomach cancer, lung cancer, prostate cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, Pancreatic cancer, ovarian cancer, brain tumors, blood tumors, etc. The diagnostic kit of the present invention is particularly useful for the diagnosis of colorectal cancer and breast cancer. The kit of the first aspect described above comprises a nucleic acid sequence which is hybridized under stringent hybridization conditions to the P U F 60 gene or a partial nucleic acid sequence thereof. For example, the kit of the present invention may contain the above-described polynuclear acid immobilized on a DNA wafer. The kit of the first aspect described above contains a PUF60 antigen (a component of a PUF60 protein and a partial peptide thereof (or a fragment thereof) that can be detected and/or quantified from a subject's body fluid sample. For example, by ELISA and/or For the quantification of PUF60 protein, these components can be used for detection and/or quantification, for example, tissue sections, or amounts of pUF6〇 in liquid samples such as blood and urine. These antibodies can be used for radioactivity, fluorescence, and ratio. The kit of the present invention may contain a labeled secondary antibody. 33 322688 201204393 The kit of the present invention comprises, in addition to a nucleic acid sequence which hybridizes to the PUF60 gene or a portion thereof of the nucleic acid sequence under stringent hybridization conditions. In addition to anti-PUF60 antibodies, the container and the label are also included. The label on the container or attached to the container may indicate that the drug is used to detect a colorectal cancer marker or a breast cancer marker. In addition, other items may be included, for example, Instructions, etc. 4. Screening method for substances which inhibit the activity or expression of PUF60 protein The present invention provides a screening method for a candidate compound having a cancer suppressing effect. A preferred embodiment is a method in which the binding of the PUF60 protein to the test compound is used as an index. In general, a compound which binds to the PUF60 protein can be expected to have an effect of inhibiting the activity of the PUF60 protein, wherein the compound is activated by binding to the PUF60 protein. Preferably, in the method, the test compound is first contacted with the PUF60 protein. The form of the PUF60 protein depends on the index for detecting the binding to the test compound, for example, it can be a refined form of the PUF60 protein. The form of the intracellular or extracellular form, or the form of the affinity column. The test compound used in the method can be appropriately identified as needed. Examples of the label include a radioactive label, a fluorescent label, and the like. In the method, the binding of the PUF60 protein to the test compound is detected. The test compound used in the method is not particularly limited, and examples thereof include a single compound such as a natural compound, an organic compound, an inorganic compound, a protein, and a peptide. , as well as compound libraries, gene library performance products, cell extracts Cell culture supernatant, fermented microbial product, marine organism 34 322688 201204393 Extract, plant extract, etc., but not limited thereto. The combination of PUF60 protein and test compound can be, for example, put into combination It is detected by the labeling of the test compound on the PUF60 protein, and the activity of the PUF60 protein caused by binding of the test compound to the PUF60 protein expressed in or outside the cell can be used as an index. The binding activity of the protein to the test compound It can be determined by a known method (for example, see Sullivan, FX, et al. (1998) J. Biol. Chem. 273, 8193-8202; Ohyama, C. et al. (1998) J. Biol. Chem. 273 , 14582-14587: Noda, K., et al. (2003) Cancer Res. 63, 6282-6289). In the method, a test compound which binds to the PUF60 protein and inhibits its activity is next selected. The compound which is isolated by the present method is expected to have a cancer suppressing action and is useful as a prophylactic or therapeutic agent for cancer. Another aspect of the screening method of the present invention is a method using the expression of the PUF60 gene as an index. In the method, the test compound is first contacted with a cell expressing the UF 60 gene. Examples of the source of the "cell" used include cells derived from humans, mice, cats, dogs, cows, sheep, birds, pets, and livestock, but are not limited thereto. For the "cell expressing the PUF60 gene", a cell expressing the endogenous PUF60 gene or a cell expressing the exogenous PUF60 gene and expressing the gene can be used. Cells expressing the exogenous PUF60 gene can usually be produced by introducing a expression vector into which the PUF60 gene is inserted, into a host cell. The expression vector can be produced by general genetic engineering techniques 35 322688 201204393 . As the test compound used in the method, 'no special restrictions' can be used, for example, a single compound such as a natural compound, an organic compound, an inorganic compound, a protein, a peptide, and a compound library, a performance product of a gene bank, Cell extracts, cell culture supernatants, fermented microbial products, marine organism extracts, plant extracts, and the like. The "contact" between the test compound and the cell expressing the PUF60 gene is usually carried out by separately adding the test compound to the culture solution of the cell expressing the PUF60 gene, but is not limited thereto. In the case where the test compound is a protein or the like, "contact" can be carried out by introducing a DNA vector expressing the protein into the cell. In the present method, the amount of expression of the puF6〇 gene is then determined. Among them, "gene performance" includes both transcription and translation. The measurement of the amount of expression of the gene can be carried out by methods known to those skilled in the art. For example, mRNA is extracted from cells expressing the PUF60 gene according to a conventional method. The transcription amount of the gene was measured by performing a Northern hybridization method using the mRNA as a template or an RT_pCR method. Or 'you can also separate the promoter region of the gene according to the F-Finance method, and connect the marker gene (fluorescent, color-developing, etc., which can be detected as a gene such as lacquerase, etc.). Limited to this), "GFR half-marker gene miscellaneous, the transcription amount of the gene ^ ^ by observing the recovery of the protein from the cells expressing the P-contact gene, ^, can be detected by SDS-PAGE electrophoresis Do not use pUF6〇 protein knife and then use the translation of the gene. In addition, the antibody against PUF60 322688 36 201204393 protein can be used to detect the protein expression by Western Blotting. The antibody for detecting the PUF60 protein is not particularly limited as long as it is a detectable antibody, and for example, a single antibody or a plurality of antibodies can be used. Then, a compound which lowers the amount of expression is selected in comparison with the case where the test compound is not contacted (control group). The compound thus selected becomes a candidate for a cancer therapeutic agent. 5. The preparation method and the preparation method of the preparation of the present invention, the prophylactic or therapeutic agent for cancer containing the expression-inhibiting substance of the PUF60 gene, the prophylactic or therapeutic agent for cancer containing the activity-inhibiting substance of PUF60 protein, the present invention A prophylactic or therapeutic agent for a cancer containing an anti-PUF60 antibody, or an anti-PUF60 antibody and a radioisotope, a therapeutic protein, a low molecular agent, and a viral vector or a non-viral vector carrying the therapeutic gene used in the present invention A prophylactic or therapeutic agent for cancer which is combined by chemical or genetic engineering methods, or any combination thereof, may be formulated according to a known method. When the prophylactic or therapeutic agent of the present invention is formulated, It is necessary to add a pharmaceutically acceptable carrier, and examples thereof include a surfactant, an excipient, a coloring agent, a flavoring agent, a preservative, a stabilizer, a buffer, a suspending agent, an isotonic agent, a binder, a disintegrating agent, The lubricant, the fluidity promoter, the flavoring agent, and the like are not limited thereto, and other conventional carriers may be appropriately used. Specifically, light anhydrous citric acid may be mentioned. Sugar, crystalline cellulose, mannitol, starch, calcium carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetic acid Ester, polyethylene ° ratio 37 322688 201204393 rancidone, gelatin, medium chain fatty acid type triglyceride, polyoxyethylene ethyl hardened castor oil 60, white sugar, carboxymethyl cellulose, corn starch, inorganic salts, etc. The type of the prophylactic or therapeutic agent of the present invention may, for example, be a key as an oral agent: a powder, a powder, a pill, a powder, a granule, a fine granule, a soft capsule, and a hard capsule. a film coating agent, a pel let, a sublingual agent, a paste, etc.; an injection, a suppository, a transdermal agent, an ointment, a plaster, a topical solution, etc. as a parenteral preparation; The most appropriate dosage form is selected according to the route of administration and the subject to be administered. The activity of the PUF60 protein (or the expression of the PUF60 gene) as an active ingredient is contained in the preparation in an amount of from 0.1 to 99.9 wt. The administration amount of the active ingredient of the agent of the present invention varies depending on the subject, the subject organ, the symptom, the administration method, and the like, but in the case of oral administration, generally, for example, for a patient (60 kg) per至优选为1. 0至50毫克。 Preferably, about 0.1 to 100 mg, preferably about 1. 0 to 100 mg, more preferably about 1. 0 to 50 mg. In the case of non-oral administration, the amount of administration varies depending on the subject, subject organ, symptoms, administration method, etc., but for example, in the form of an injection, for example, for a patient (60 kg), usually 1至约10毫克。 By daily injection of about 0.01 to about 30 mg, preferably from about 0.1 to about 20 mg, more preferably from about 0.1 to about 10 mg. However, it is ultimately up to the physician or veterinarian to make an appropriate decision based on the type of the dosage form, the method of administration, the age and weight of the patient, and the symptoms of the patient. The preparation thus obtained can be administered, for example, to humans and other mammals (e.g., rats, rabbits, sheep, pigs, cows, cats, dogs, marmosets, etc.). 38 322688 201204393 The condition of the animal outside, the dose of the animal; the amount of the 6 〇 kg of the cancer, :: Ming Γ 剂 或 or therapeutic agent can be used for cancer (such as colorectal cancer, stomach cancer, prostate cancer, esophageal cancer, Liver cancer, biliary tract cancer, spleen cancer, kidney cancer, makeup ^ ^ dirty cancer, broken: cystic cancer, uterine cancer, thyroid cancer 1 for colorectal cancer 4 = tumor, blood _, etc.) and treatment, preferably the present invention Lion and secret. ^ or PUF6G gene = ' As an active ingredient, the anti-cancer agent and the cancer metastasis inhibitor are active ingredients of PUF6G 0 f. Therefore, it can be used as a fine preparation for use, an apoptosis-inducing agent for cancer cells, and the like. The type of the agent = woven, organ or cancer of the present invention is not particularly limited. Further, Table I of the deleted gene: both the activity inhibitory substance of the PUF6G protein and the inhibitory substance. In the present invention, the pre-JJ Dajie 4 can be used according to the known means = 丨; the use of antisense nucleic acid in the therapeutic agent '\-nucleic acid alone, or inserted into the anti-virus r, the body, adenovirus vector, gland After the virus-associated virus and the physiologically acceptable carrier are formulated, they are administered by a catheter such as a gene grab or hydrogel catheter. The combination of the viral vector and the anti-ρ_antibody

Recombinant adenosis f (4) silk therapy H but general and pharmaceutical loading _ Ichio used this as a carrier as described above; and water: from use. For these carriers, the water such as protein, etc., is easy to ride, such as water; 8 sugar, human white, and thousands of good. In addition, it can be added to pharmaceuticals 322688 39 201204393 deposits, preservatives, measuring agents, etc. Such a preparation = music composition can be based on the treatment, (4) the form of investment, == path cast. In terms of administration form, sugar, capsules, troches, granules, injections, ointments and the like. In the case of administering the anti-P_) antibody of the present invention, a diseased body particle or a pharmaceutical composition containing a heterodule for treatment, usually 1 to 3 to 10 sick persons per administration are administered for each adult. Health, except for the state of the disease, the nature of the target cells, tissue changes. The number of applications can be from one to several times a day, and the investment period can be from the next day to several months. The investment can be made into one set (set) and long-term cast. Further, the viral vector or virus thief molecule used in the present invention can be used for the detection of specific cells and/or tissues, or for the diagnosis of disease states. For example, a viral vector particle obtained by loading a detectable marker genome into a viral vector and transfecting it into an appropriate host cell can be used in combination with an anti-PUF60 antibody for detecting and diagnosing tumor cells. Alternatively, a detectable tag can be combined with an anti-PUF60 antibody and then used to detect and diagnose tumor cells. Hereinafter, the present invention will be more specifically described by way of examples, but the scope of the invention is not limited to the examples. [Examples] Example 1: Identification of a cancer-specific amplified gene by array CGH In the present example, in order to identify a colorectal cancer-specific gene amplification, a sample of 100 colorectal cancer cases was performed by an array type. CGH method (this is a well-known technique in the art (Snijders AM, et al,, (;2〇〇3;) Brief Funct Genomic Proteomic. Apr; 2(1): 37.45. de Leeuw RJ, 322688 40 201204393 et al. , (2004) Hum Mol Genet, Sep 1; 13(17): 1827-37; Veltman JA, et al., (2002) Am J Hum Genet. May; 70(5):1269-76.)) . As a result, it was found that the PUF60 (poly-U-binding factor 60 kDa) (NCBI accession number: NM_078480) gene was amplified at a high frequency in a colorectal cancer sample (Table 1). Furthermore, gene amplification was also observed for the evaluation of 46 breast cancer cases (Table 1). [Table 1] _ _Amplification frequency 64 / 100 (64%) Colorectal cancer breast cancer ___10 / 46 (22%) __ Table 1 PUF60 gene amplification frequency in cancer samples 100 specimens of colorectal cancer cases and Array CGH analysis results of 46 breast cancer cases. The brightness ratio (G/R) of the cancer (green mark) to the normal (red mark) sample is 1.2 or more and is determined to be amplification. Example 2: Cancer-specific hypersensitivity as measured by immunohistochemical staining The cancer-specific expression of the pUF6 purine gene in cancer specimen tissues was evaluated by immunohistochemical staining methods well known in the art. Specifically, Lu's colorectal cancer tissue array slice (Super Bio wafer, model. CDA) or breast cancer tissue array slice (Bi〇chain, model: z7〇2〇〇〇5) was incubated in a thermostat at 60 ° C for 丨 hours. Thereafter, it was immersed in dimethyl strepone for 3 minutes, and then washed twice with fresh dimethyl sting to remove paraffin. After that, after treatment with a series of stepwise concentrations (100% to 75%) of ethanol, the tissue will be 41 322688 201204393

The slices were hydrated with pure water. The sections were immersed in 10 mM citrate buffer (PH6. 〇) and subjected to antigen-activation treatment in an autoclave at 121 ° C for 15 minutes, and then left to cool at room temperature for 30 minutes. Slice with TBST (25 mM)

Tris-HCl pH 7. 4, 130 mM NaCl, 2.5 mM KC1, 0·1% Tween 20) After 3 minutes of washing in 5 minutes, immersed in an aqueous solution of hydrogen peroxide diluted to 3% with decyl alcohol for 15 minutes. . After washing with TBST for 3 times for 3 minutes, blocking reaction was carried out for 30 minutes using Block Ace (DS Pharma), and a 100-fold diluted PUF60 antibody (abeam, model: ab22819) was reacted at room temperature for 1 hour. After 5 minutes of washing with TBST for 5 minutes, VECTASTAIN Elite ABC Goat igG kit (vect〇r Laboratories) and metal-reinforced DAB matrix kit (Thermo)

Scient i f ic) 'Detects the antibody response once in accordance with the specified procedure of use. After immersing in TBST to stop the detection reaction, it was washed three times with TBST for 5 minutes, and subjected to contrast staining by Sakura Finetek. Dehydrated by a series of stepwise concentrations of ethanol (75% to 1%), and cleared by diphenylbenzene, sealed with Mount-Quick (Avenue), using BX51-34-FL-l (01ympus) for microscopic examination of the slice. RESULTS: Tissue sections of 10 cases of colorectal cancer and breast cancer were used to perform tissue-free staining. As a result, one-to-one history of a total of one specimen was observed to exhibit cancer-specific staining. 〇; PUF60 drug standard, showing cancer filaments, its residue can be used as a cancer treatment and is also useful as a diagnostic marker. 322688 42 201204393 • [Table 2] Immunohistochemical positive negative 10/10 (100%) 0/10 (0%) 10/10 (100%) _0/10 (0%) Colorectal cancer breast cancer Table 2 In colorectal cancer and The expression of PUF60 in breast cancer samples was analyzed by immunohistochemical staining in samples of 10 cases of colorectal cancer and breast cancer. The staining in the nucleus was evaluated in 4 segments (3+, 2+, Γ, 0), and 2+ or more was judged as positive. Example 3: Evaluation of antitumor effect by RNAi analysis of PUF60 using colorectal cancer and breast cancer cell lines. About the gene amplification and the PUF60 gene observed in colorectal cancer and breast cancer samples, by RANi The method evaluates the effect on cancer cells when their function is inhibited. The rate of gene repression caused by the RANi method was evaluated by quantitative RT-PCR analysis. The effect on cancer cells was determined by analyzing the number of viable cells and the survival rate of the cells was evaluated. <RANi analysis> The cell line was purchased from ATCC and cultured according to the designated protocol. For the siRNA', ON-TARGET plus SMART pool siRNA (Pharmacon) was used. The siRNA was modified to avoid leaving the target, and the following four siRNAs were further mixed. siRNA a : UGUACGACCAGGAGCGUUUUU (SEQ ID NO: 1) siRNA b : CAGCCUACAGUGCGGAUAAUU (SEQ ID NO: 2) 43 322688 201204393 siRNA c : GCUUCAUUGAGUACGAGAAUU (SEQ ID NO: 3) siRNA d : CCAUCAAGAGCAUCGACAUUU (SEQ ID NO: 4) Introduction of siRNA into cultured cells using Lipofectamine RNAiMAX (Invitrogen), 10 nM of siRNA was introduced into the cells according to the procedure used in the reagents. Use ON-TARGET plus Non-Targeting Pool (Pharmacon) as a control. <Quantitative RT-PCR analysis> In order to evaluate the gene knockdown rate, the effect of siRNA was verified by the amount of mRNA. All RNA was extracted from cells 24 hours after introduction of siRNA using the SV96 Total RNA I so 1 on i on System (Promega) and in accordance with the procedure attached to the reagents. Thereafter, cdnA was synthesized using a Super Script III First-Strand Synthesis System (Invitrogen) for RT-PCR and in accordance with the procedure used in the reagent. Quantitative RT-PCR was carried out using this cDNA as a template. For quantitative PCR, use Power SYBR Green Master Mix (Applied)

Biosystems) ‘According to the procedure used in the reagents, use 75〇〇 Real One

Time PCR System (Applied Biosystem) to implement. TATA binding protein (TBP) was used as an intrinsic control group, and the relative ratio to the negative control group (NC) was calculated by comparing the Ct method (a Δ Ct). <Analysis of the number of viable cells> Using Alamar Blue (Biosource) according to the procedure attached to the reagents by Wal lac 1420 Multi label / Luminescence

Counter ARVO (PerkinElmer) or Infinite M200 (TECAN) 322688 44 201204393 - The results of the measurement In Figure 2, the results of quantitative analysis using colorectal cancer and breast cancer cells, including the colorectal cancer and breast cancer fine p'uFrtt^ SlRM ^ 24 ^ ^ ^ ® ^ ^ The performance of i compared to the negative control group (NC) relative NC, is the siRNA that will (4) the sequence of any transcript of the gene. As shown in Fig. 2, by quantitative RT_pcR evaluation of the 跗 table: the inhibition effect (in terms of the amount of RNA), it was observed that the introduction of the second, compared with the NC, will fully inhibit the performance" in Figure 3 'Shows the results of RNAi analysis of genes for colorectal cancer and breast cancer cell lines. After siRM was transfected into each cell line, the number of viable cells was analyzed on the fourth day, and the relative value compared with the negative control group was calculated as the survival rate (ViabiHty). ...', as a result, significant proliferation inhibition was observed in colorectal cancer cell lines RK0, RK〇E6, Wi plus sputum, and about 67%, 39%, and 58%, respectively. ^ =, in the breast cancer cell strains Rainbow 1 () 86, Leg 231.2 () cell strain, a significant proliferation inhibition effect of about 56%, 69%, 58% was observed. From the results of detailed evaluation of the cell phenotype of _ 〇 gene pressure by microscopic observation, it is apparent that the proliferation inhibition effect is caused by the prolongation of the cell death cell cycle. 3 _?, ', Tian p_ base two fruit weight: in:: by cancer 2:: proliferation of the plant proliferation] so implied _. Functional suppression of genes is cut in:::::: 322688 45 201204393 May be valid. From the above examples, it was observed that pu just showed gene amplification and hyperactivity particularly in colorectal cancer, and it was observed that the proliferation inhibition effect of cancer cells was exerted by the inhibition of the function of the legs, and & PUF6G is useful as a colorectal cancer agent and a breast cancer diagnostic sputum expression inhibitor or a PU activity inhibitory substance as an anticancer agent. n [Simple diagram of the diagram] Fig. 1 shows the performance of immunohistochemical staining by removing antibodies in the case of colorectal cancer and breast cancer specimens (in terms of protein = analysis results. Strong signals are seen in the nucleus of cancer cells) This figure shows the observed images of intestinal cancer (a to C) and breast cancer (d to f) tissues. Fig. 2 is a graph showing the gene repression rate of the p gene measured by quantitative RT-PCR analysis. It shows the inhibitory effect by the performance of p just after (in terms of flooding). a to c: colorectal cancer fine d to f: breast cancer cell line. Nc means negative control group, NT means untreated: Fig. 3 (a To f) shows the results of analysis of the genes for colorectal cancer and breast cancer cells. The siRNA was transfected into each cell line, and the number of viable cells was counted on the 4th day to calculate the relative value relative to the negative pair (NC). As a survival rate (viabiu忭). ",,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Relative value of negative control group (10). a to C: colorectal cancer cell line; d to f: fine breast cancer Cell line. The figure shows differential diffraction around the 4th day after siRNA introduction in 322688 46 201204393 - cells (g), RK0E6 cells (h). Values indicate survival rate. NC indicates negative control group, NT indicates no [Major component symbol description] None 0 47 322688 201204393 Sequence Listing <110> First Sankyo Co., Ltd. <120> Cancer Diagnostic Agent & Therapeutic Agent <130> G10-0091TW <150><151><160> JP2010-095078 2010-04-16 4 <170> Patentln version 3.5 <210><211><212><213> 1 21 RNA artificial sequence <220><223><400> si RNA 1 uguacgacca ggagcguuuu u 21 <210><211><212><213> 2 21 RNA artificial sequence <220><223> siRNA <400> 2 cagccuacag ugcggauaau u 21 <210><211><212><213> 3 21 RNA artificial sequence <220><223> siRNA <400> 3 gcuucauuga guacgagaau u 21

<210><211><212><213> 4 21 RNA artificial sequence <220><223> siRNA 1 322688 201204393 <400> 4 ccaucaagag caucgacauu u 21 2 322688

Claims (1)

201204393 VII. Patent application scope: The expression-inhibiting substance of the gene is selected from the group consisting of an activity-inhibiting substance of a PUF60 gene protein. The composition according to any one of the items, wherein the pUF6 is selected from the group consisting of the following (a) to (c) (a) inhibiting the expression of the PUF60 gene by the RNAi effect (1) A nucleic acid having ribonucleotide (rib. (4)) activity, which specifically cleaves the transcription of the PUF gene Any one of the group consisting of: (d) an antibody that specifically binds to the ρυρ(9) protein, (e) a low molecular compound that specifically binds to the PUF60 protein, and (f) inhibits interaction with the PUF6 〇 protein A low molecular compound in which the phase of the molecule acts. 3. The composition of the PUF60 gene, wherein the expression inhibitor of the PUF60 gene comprises the nucleic acid sequence of the sequence numbering sequence 2, the sequence of the salt 3 or the sequence number 4. 4. The composition according to any one of claims 1 to 3, wherein the cancer is colorectal cancer or breast cancer in 322688 201204393. 5. The composition according to any one of claims 1 to 4, which is a medicament for the prevention or treatment of cancer. 6. A nucleic acid molecule selected from the group consisting of (i) and (ii): (i) an antisense nucleic acid molecule and siRNA molecule (a) PUF60 gene directed against (a) or (b) below a nucleic acid sequence, (b) a nucleic acid sequence encoding a PUF60 protein; and (ii) a vector (c) and (d) (c) a vector comprising the antisense nucleic acid molecule, and (d) a vector comprising the siRNA molecule. 7. The nucleic acid molecule of claim 6, wherein the siRNA molecule comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. 8. The nucleic acid molecule according to claim 6 or 7, which is for use in the prevention or treatment of colorectal cancer or breast cancer. 9. A method of using PUF60 as a diagnostic marker for cancer comprising the step of detecting a PUF60 gene, a transcript thereof or a translation product thereof, or a fragment thereof, from a subject's sample. 10. The method of claim 9, comprising: (i) the step of detecting a PUF60 gene or a transcript thereof or a fragment thereof from a subject, wherein the gene is compatible with the gene a nucleic acid sequence of a nucleic acid sequence thereof or a nucleic acid sequence encoding the PUF60 protein, or a nucleic acid molecule which specifically hybridizes with all or a portion of the nucleic acid sequence of the PUF60 protein, or 2 322688 201204393 (ii) a step of detecting a PUF60 protein from a subject's sample, Wherein the detection is carried out using an antibody which specifically binds to the protein or a fragment thereof; the presence of the PUF60 gene, a transcription product thereof, the PUF60 protein or the fragment in the sample indicates that the subject may have cancer. 11. The method of claim 9 or claim 10, wherein the cancer is colorectal cancer or breast cancer. 12. A diagnostic kit for cancer, comprising: (i) a nucleic acid molecule that specifically hybridizes to a nucleic acid sequence of a PUF60 gene or a transcript thereof, or a nucleic acid sequence encoding a PUF60 protein; or (ii) An antibody that specifically binds to a PUF60 protein or a fragment thereof; and (iii) instructions for use. 13. The kit of claim 12, wherein the cancer is colorectal cancer or breast cancer. A screening method for screening a performance inhibitor of a PUF60 gene, comprising: a step of culturing a cell expressing a PUF60 gene in the presence or absence of a test compound; and a quantity of a transcription product of the PUF60 gene or PUF60 The amount of protein is a step of determining the amount of PUF60 expression of the cultured cells; and the step of comparing the amount of PUF60 present in the presence or absence of the test compound. 3 322688 201204393 15. A screening method for screening an activity inhibitor of PUF60 protein, comprising: a step of contacting a test compound with a multi-peptide or PUF60 protein encoded by a PUF60 gene; determining the multi-peptide or a step of biological activity of the protein; and a step of selecting a compound that inhibits the biological activity of the multi-peptide or protein as compared to the biological activity of the multi-peptide or protein in the absence of the test compound. 16. The screening method according to claim 14 or 15, which is for use in the screening of a prophylactic or therapeutic agent for cancer. 17. The screening method according to claim 16, wherein the cancer is colorectal cancer or breast cancer. 4 322688