JP2009505631A - Cancer-related gene RASEGEF1A - Google Patents

Abstract

The present invention provides a method for detecting and diagnosing cancer. According to certain embodiments, the diagnostic method comprises determining the expression level of the RASGEF1A gene that has been found to distinguish between cancer cells and normal cells. Furthermore, the present invention also provides a method of screening for therapeutic agents useful in the treatment of cancer, a method for treating cancer, and a method for vaccinating a subject against cancer.

Description

TECHNICAL FIELD The present invention relates to methods for detecting and diagnosing cancer, and methods for treating and preventing cancer.

  This application claims the benefit of US Provisional Application No. 60 / 704,054, filed Jul. 28, 2005, the entire contents of which are incorporated herein by reference.

BACKGROUND ART Intrahepatic cholangiocarcinoma (ICC) is the second most common type of primary hepatobiliary cancer. Its incidence is increasing in Japan and Western countries, especially the UK (Shaib YH et al., J Hepatol 2004, 40: 472-7; Taylor-Robinson SD et al., Lancet 1997, 350: 1142-3; Kato I et al., Jpn J Clin Oncol 1990, 20: 232-7). The most common liver malignancy, hepatocellular carcinoma, has been thoroughly researched and clinical progress has been realized through the development of various treatment strategies. However, the carcinogenic processes involved in ICC are not well understood and the prognosis for ICC patients remains poor. In terms of treatment, surgical resection of the tumor is almost the only way to overcome this disease (Okuda K et al., J Gastroenterol Hepatol 2002, 17: 1056-63), but it has advanced invasive ICC. It is almost impossible to completely remove cancer cells from a patient. Furthermore, chemotherapy for ICC is also unsatisfactory. For example, partial response rates for treatment plans based on 5-fluorouracil and gemcitabine are 20-30%, but no survival benefit has been shown (Gores GJ, Hepatology 2003, 37: 961-9). Therefore, the development of new treatments for ICC is an urgent issue.

  The Ras family of GTPases plays an important role in integrating signals from cell surface receptors and transmitting them to downstream effector pathways. Ras protein exists mainly in an inactive GDP-bound state in resting cells. These are convertible to each other and switch between active and inactive GDP-bound forms (Hall A, Annu. Rev Cell Biol 1994, 10: 31-54). These activities are regulated by interaction with regulatory proteins that stimulate GDP / GTP exchange (GEF protein) and regulatory proteins that stimulate GTP hydrolysis (GAP protein). When activated by extracellular stimuli, guanine nucleotide exchange factor (GEF) promotes GDP release from Ras, allowing GTP to bind to Ras. This GTP-bound Ras forms an active conformation that relays signals to downstream effector proteins such as Raf, phosphoinositide 3-kinase, and RalGDS (Campbell SL et al., Oncogene 1998, 17: 1395-413; et al., Bioessays 1995, 17: 395-404). Several Ras family GTPases have been identified, but the number of Ras GEFs that have been shown to be active is limited, such as Sos, GRF, and GRP for Ras, C3G, CalDAG1, and Epac for Rap1, and RalGDS family members against Ral (Quilliam LA et al., Bioessays 1995, 17: 395-404; Bos JL, EMBO J 1998, 17: 6776-82; de Rooij J et al., Nature 1998, 396: 474- 7; Ebinu JO et al., Science 1998, 280: 1082-6; Gotoh T et al., J Biol Chem 1997, 272: 18602-7; Kawasaki H et al., Proc Natl Acad Sci USA 1998, 95: 13278 -83).

  Gene expression profiles generated by cDNA microarray analysis can provide much more detailed content regarding the nature of individual cancers than conventional histopathological methods. The promising point of such information is its potential for improving clinical strategies for treating neoplastic diseases and for developing new drugs (Petricoin EF et al., Nat Genet 2002, 32 Suppl: 474-9). For this purpose, we analyzed the expression profiles of tumors from various tissues by cDNA microarray (Okabe H et al., Cancer Res 2001, 61: 2129-37; Hasegawa S et al., Cancer Res 2002, 62: 7012-7; Kaneta Y et al., Jpn J Cancer Res 2002, 93: 849-56; Kaneta, Y. et al., Int J Oncol 2003, 23: 681-91; Kitahara O et al., Cancer Res 2001, 61: 3544-9; Lin Y et al. Oncogene 2002, 21: 4120-8; Nagayama S et al., Cancer Res 2002, 62: 5859-66; Okutsu J et al., Mol Cancer Ther 2002, 1: 1035-42; Kikuchi T et al., Oncogene 2003, 22: 2192-205).

  Studies designed to elucidate the mechanisms of carcinogenesis have already facilitated the identification of molecular labels for certain antitumor agents. For example, a farnesyltransferase inhibitor (FTI), originally developed to inhibit the Ras-related growth signaling pathways whose activation is dependent on post-translational farnesylation, is a Ras-dependent tumor in animal models. Has been shown to be effective in treating (Sun J et al., Oncogene 1998, 16 (11): 1467-73). Similarly, human clinical trials using a combination of anti-tumor agents and anti-HER2 monoclonal antibody trastuzumab aimed at antagonizing the proto-oncogene receptor HER2 / neu also improved the clinical response and overall survival of cancer patients. (Molina MA et al., Cancer Res 2001; 61 (12): 4744-9). Finally, constitutive activation of bcr-abl tyrosine kinase selectively inactivates the bcr-abl fusion protein to treat chronic myeloid leukemia where it plays a crucial role in leukocyte transformation The tyrosine kinase inhibitor STI-571 has been developed. These types of drugs are designed to suppress the carcinogenic activity of certain gene products (O'Dwyer ME and Druker BJ, Curr Opin Oncol 2000; 12 (6): 594-7). Thus, it is clear that gene products that are generally up-regulated in cancer cells may serve as potential targets for developing new anti-tumor agents.

  It was further demonstrated that CD8 + cytotoxic T lymphocytes (CTL) recognize tumor peptides associated with tumor associated antigens (TAA) presented on MHC class I molecules and lyse tumor cells. Since the discovery of the MAGE family as the first example of TAA, many other TAAs have been discovered using immunological approaches (Boon, Int J Cancer 1993, 54: 177-80; Boon & van der Bruggen, J Exp Med 1996, 183: 725-9; van der Bruggen et al., Science 1991, 254: 1643-7; Brichard et al., J Exp Med 1993, 178: 489-95; Kawakami et al., J Exp Med 1994, 180: 347-52). Some of the newly discovered TAAs are currently in clinical development as targets for immunotherapy. TAA discovered so far includes MAGE (van der Bruggen et al., Science 1991, 254: 1643-7), gp100 (Kawakami et al., J Exp Med 1994, 180: 347-52), SART ( Shichijo et al., J Exp Med 1998, 187: 277-88), and NY-ESO-1 (Chen et al., Proc Natl Acad Sci USA 1997, 94: 1914-8). On the other hand, gene products that have been demonstrated to be specifically over-expressed in tumor cells have been shown to be recognized as targets that induce cellular immune responses. Such gene products include p53 (Umano et al., Brit J Cancer 2001, 84: 1052-7), HER2 / neu (Tanaka et al., Brit J Cancer 2001, 84: 94-9), CEA ( Nukaya et al., Int J Cancer 1999, 80: 92-7).

  Despite significant progress in basic and clinical research on TAA (Rosenberg et al., Nature Med 1998, 4: 321-7; Mukherji et al., Proc Natl Acad Sci USA 1995, 92: 8078 -82; Hu et al., Cancer Res 1996, 56: 2479-83), only a limited number of candidate TAAs are currently available for the treatment of adenocarcinoma including colorectal cancer. TAA, which is expressed in large amounts in cancer cells and is also restricted to cancer cells, would be a promising candidate for immunotherapy targets. Furthermore, the identification of new TAAs that induce potent and specific anti-tumor immune responses is expected to boost the clinical use of peptide vaccination against various types of cancer (Boon et al., J Exp Med 1996, 183: 725-9; van der Bruggen et al., Science 1991, 254: 1643-7; Brichard et al., J Exp Med 1993, 178: 489-95; Kawakami et al., J Exp Med 1994, 180: 347-52; Shichijo et al., J Exp Med 1998, 187: 277-88; Chen et al., Proc Natl Acad Sci USA 1997, 94: 1914-8; Harris, J Natl Cancer Inst 1996, 88: 1442-55; Butterfield et al., Cancer Res 1999, 59: 3134-42; Vissers et al., Cancer Res 1999, 59: 5554-9; van der Burg et al., J Immunol 1996, 156: 3308 Tanaka et al., Cancer Res 1997, 57: 4465-8; Fujie et al., Int J Cancer 1999, 80: 169-72; Kikuchi et al., Int J Cancer 1999, 81: 459-66; Oiso et al., Int J Cancer 1999, 81: 387-94).

Peripheral blood mononuclear cells (PBMC) from a healthy donor, when stimulated by the peptide, produce significant levels of IFN-α in response to the peptide, but the ⁵¹ Cr release assay uses HLA-A24 or HLA- It has been repeatedly reported that A0201 is rarely cytotoxic to tumor cells in a restrictive manner (Kawano et al., Cancer Res 2000, 60: 3550-8; Nishizaka et al., Cancer Res 2000, 60: 4830-7; Tamura et al., Jpn J Cancer Res 2001, 92: 762-7). However, both HLA-A24 and HLA-A0201 are HLA alleles that are common in the Japanese and white populations (Date et al., Tissue Antigens 1996, 47: 93-101; Kondo et al., J Immunol 1995, 155 : 4307-12; Kubo et al., J Immunol 1994, 152: 3913-24; Imanishi et al., Proceeding of the eleventh International Histocompatibility Workshop and Conference Oxford University Press, Oxford, 1992, 1065; Williams et al., Tissue Antigen 1997, 49: 129). Therefore, these cancer antigen peptides presented by HLA may be particularly useful in the treatment of Japanese and Caucasian cancer. In addition, in vitro induction of low-affinity CTLs usually results in high levels of specific peptide / MHC complexes that effectively activate these CTLs on antigen-presenting cells (APC) using high concentrations of peptides. (Alexander-Miller et al., Proc Natl Acad Sci USA 1996, 93: 4102-7).

  The present invention is based on the discovery of a specific expression pattern of the RASGEF1A gene in cancer cells.

  Previous analysis of the genome-wide expression profiles of genes in 25 ICCs identified a set of genes whose expression is normally upregulated (Obama K et al., Hepatology 2005 Jun, 41 (6): 1339-48). In the present specification, among these genes, the focus is on the gene RASGEF1A (RasGEF domain family, member 1A). The present inventors have detected that the expression of the RASGEF1A gene is enhanced in bladder cancer (PCT / JP2006 / 302684) and suppressed in breast cancer (WO 2005/28678). Throughout the present invention, the RASGEF1A gene has a broad spectrum of intrahepatic cholangiocarcinoma (ICC), lung cancer, stomach cancer, colorectal cancer, prostate cancer, chronic myelogenous leukemia, kidney cancer, soft tissue sarcoma, pancreatic cancer, and seminoma. It was further revealed that human tumors are frequently up-regulated. Furthermore, the protein encoded by this gene was also found to enhance RAS activity. Furthermore, suppression of the RASGEF1A gene by small interfering RNA (siRNA) leads to suppression of ICC cell proliferation and / or cell death, and this gene may serve as a novel therapeutic target for various types of human neoplasms There is.

  The RASGEF1A gene and its transcripts and translation products identified herein are useful in diagnosis as markers for cancer and as oncogene targets, altering their expression and / or activity to alter cancer symptoms Can be treated or alleviated. Similarly, various compounds can be identified as agents for treating or preventing cancer by detecting changes in RASGEF1A gene expression caused by a compound.

  Accordingly, the present invention provides a method for diagnosing or determining a predisposition to cancer in a subject by determining the expression level of the RASGEF1A gene in a biological sample from the subject, such as a tissue sample. If the expression level of this gene is elevated compared to the normal control level, it indicates that the subject is suffering from or at risk of developing cancer. Normal control levels can be determined using normal cells obtained from non-cancerous tissue.

  In the present invention, the preferred cancer is ICC.

  In the context of the present invention, the phrase “control level” refers to the expression level of the RASGEF1A gene detected in a control sample and includes both normal and cancer control levels. The control level can be a single expression pattern or multiple expression patterns from a single reference population. For example, the control level may be a database of expression patterns from previously tested cells. “Normal control level” means the expression level of the RASGEF1A gene detected in normal healthy individuals or in a population of individuals known not to have cancer. A normal individual is an individual who has no clinical symptoms of cancer. In contrast, “cancer control level” means the expression level of the RASGEF1A gene found in individuals or populations suffering from cancer.

  If the expression level of the RASGEF1A gene detected in the sample is elevated compared to the normal control level, the subject (from which the sample was obtained) may have cancer or be at risk for cancer Is done.

  Alternatively, the expression level of a gene panel containing the RASGEF1A gene in a sample can be compared to the cancer control level of the same gene panel. If the expression level of a sample is similar to the cancer control level, it is suggested that the subject (who obtained the sample) is suffering from or at risk for cancer.

  As used herein, for example, 10%, 25%, or 50%, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold, or compared to a control level, or Further gene expression levels are considered “altered” if gene expression is increased or decreased. The expression level of the RASGEF1A gene can be determined, for example, by detecting the hybridization intensity of the nucleic acid probe to the gene transcript in the sample.

  In the context of the present invention, a tissue sample from a subject may be any tissue obtained from a test subject, eg, a patient known to have or suspected of having cancer. For example, the tissue may include epithelial cells. More specifically, the tissue may be cancer epithelial cells.

  The present invention further identifies a compound that inhibits or enhances the expression or activity of RASGEF1A by contacting a test cell expressing RASGEF1A with the test compound and determining the expression level of the RASGEF1A gene or the activity of its gene product Providing a method for The test cell may be an epithelial cell such as a cancer epithelial cell. In the absence of a test compound, if the expression level of the gene or the activity of its gene product is reduced compared to the control level, this suggests that the test compound can be used to reduce the symptoms of cancer. The

  The present invention also provides a kit comprising at least one detection reagent that binds to the transcription product or translation product of the RASGEF1A gene.

  The therapeutic methods of the present invention include a method for treating or preventing cancer in a subject comprising administering to the subject an antisense composition. In the context of the present invention, the antisense composition reduces the expression of a particular target gene (ie, the RASGEF1A gene). For example, the antisense composition can comprise nucleotides that are complementary to the RASGEF1A gene sequence. Alternatively, the methods of the invention can include administering an siRNA composition to the subject. In the context of the present invention, the siRNA composition decreases the expression of the RASGEF1A gene. In yet another method, treating or preventing cancer in a subject can be performed by administering a ribozyme composition to the subject. In the context of the present invention, a ribozyme composition specific for nucleic acids reduces the expression of the RASGEF1A gene. In fact, the present inventors confirmed the inhibitory effect of siRNA on the RASGEF1A gene. For example, inhibition of cell proliferation of cancer cells by siRNA is demonstrated in the Examples section, which supports the fact that the RASGEF1A gene serves as a preferred therapeutic target for cancer.

  The invention also includes vaccines and vaccination methods. For example, a method for treating or preventing cancer in a subject can include administering to the subject a vaccine comprising a polypeptide encoded by the RASGEF1A gene or an immunologically active fragment of the polypeptide. In the context of the present invention, an immunologically active fragment is a polypeptide that induces an immune response that is shorter in length than the full-length native protein but is similar to the immune response induced by the full-length protein. For example, the immunologically active fragment should be at least 8 amino acid residues long and be able to stimulate immune cells such as T cells or B cells. Immune cell stimulation can be measured by detecting cell proliferation, production of cytokines (eg, IL-2), or antibody production.

  One advantage of the methods described herein is that the disease is identified prior to detecting overt clinical symptoms of cancer. Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

DETAILED DESCRIPTION OF THE INVENTION The words “a”, “an”, and “the” as used herein mean “at least one” unless otherwise specified.

  The terms `` isolated '' and `` purified '' as used with respect to a substance (e.g., a polypeptide, antibody, polynucleotide, etc.) are at least those substances that may otherwise be included in natural sources. Indicates that one substance is not substantially contained. Thus, an isolated or purified antibody is substantially free of cellular material such as carbohydrates, lipids, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or chemically. When specifically synthesized means an antibody substantially free of chemical precursors or other chemicals. The term “substantially free of cellular material” includes preparations of a polypeptide from which the polypeptide is isolated or separated from cellular components of cells that are produced recombinantly. Thus, a polypeptide substantially free of cellular material includes (by dry weight) less than about 30%, 20%, 10%, or 5% heterologous protein (also referred to herein as “contaminating protein”) Polypeptide preparations having the Similarly, where the polypeptide is produced recombinantly, preferably a polypeptide preparation that is substantially free of medium and that is less than about 20%, 10%, or 5% of the volume of the protein preparation. included. Where the polypeptide is made by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, and the chemical precursors or other chemicals involved in protein synthesis are present in the protein preparation. Polypeptide preparations that are less than about 30%, 20%, 10%, or 5% by volume (by dry weight) are included. A specific protein preparation contains an isolated or purified polypeptide, such as a single band following the protein preparation, such as sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and coomassie brilliant blue staining of the gel. Can be indicated by the appearance of. In preferred embodiments, the antibodies of the invention are isolated or purified.

  An “isolated” or “purified” nucleic acid molecule, such as a cDNA molecule, is substantially free of other cellular material or media or chemically synthesized when made by recombinant techniques. If present, it may be substantially free of chemical precursors or other chemicals. In a preferred embodiment, the nucleic acid molecule encoding the antibody of the present invention is isolated or purified.

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

  The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Natural amino acids are those encoded by the genetic code, as well as those post-translationally modified in cells (eg, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). The phrase “amino acid analog” has the same basic chemical structure as a natural amino acid (the α-carbon is attached to a hydrogen, carboxy group, amino group, and R group), but a modified R group or modification A compound having a main chain formed (for example, homoserine, norleucine, methionine, sulfoxide, methionine methylsulfonium). The phrase “amino acid mimetic” means a chemical compound that has a different structure but has the same function as a normal amino acid.

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

  The terms “polynucleotide”, “nucleotide”, “nucleic acid”, and “nucleic acid molecule” are used interchangeably unless otherwise specified and are generally accepted single letters as well as amino acids. Indicated by notation. Like amino acids, these include both natural and non-natural nucleic acid polymers.

  The present invention is based in part on the discovery of increased RASGEF1A gene expression in cells from patients with various cancers. The nucleotide sequence of the human RASGEF1A gene is shown in SEQ ID NO: 1 and is also available as GenBank accession number AK095136. As used herein, the RASGEF1A gene encompasses the human RASGEF1A gene, as well as the RASGEF1A gene of other animals including, but not limited to, non-human primates, mice, rats, dogs, cats, horses, and cows, and alleles Included are genes present in other animals, such as those corresponding to the mutant and RASGEF1A genes.

  The amino acid sequence encoded by the human RASGEF1A gene is shown in SEQ ID NO: 2 and is also available as GenBank accession number BAC04491. In the present invention, the polypeptide encoded by the RASGEF1A gene is referred to as “RASGEF1A” and may also be referred to as “RASGEF1A polypeptide” or “RASGEF1A protein”.

  According to one aspect of the invention, functional equivalents are also included in the RASGEF1A polypeptide. 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 the biological ability of the RASGEF1A protein may be used as such a functional equivalent in the present invention. Such functional equivalents include those in which one or more amino acids have been substituted, deleted, added, or inserted into the native amino acid sequence of the RASGEF1A protein. Alternatively, the polypeptide may comprise an amino acid sequence having at least about 80% homology (also referred to as sequence identity) to the sequence of the respective protein. In other embodiments, the polypeptide may be encoded by a polynucleotide that hybridizes under stringent conditions to the native nucleotide sequence of the RASGEF1A gene.

The phrase “stringent conditions” means that a nucleic acid molecule hybridizes to its target sequence, typically in a complex mixture of nucleic acids, but is not detectable to other sequences. It means the condition that does not hybridize. 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 5-10 ° C. lower than the thermal melting point (T _m ) for the specific sequence at a defined ionic strength pH. T _m is the temperature at which 50% of the probe complementary to the target (under a defined ionic strength, pH, and nucleic acid concentration) hybridizes to the target sequence at equilibrium (because the target sequence is present in excess). In _Tm , 50% of the probe is occupied in 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 are possible, such as: 50% formamide, 5 × SSC, and 1% SDS, 42 ° C. incubation, or 5 × SSC, 1% SDS, 65 ° C. Wash at 50 ° C. in 0.2 × 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. Those skilled in the art will recognize that individual additions, deletions, insertions, or substitutions to an amino acid sequence that alter a single amino acid or a low ratio of amino acids result in a protein in which the alteration of the protein results in a similar function. It is thought that it is. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (d), glutamic acid (E);
3) 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 RASGEF1A protein of the present invention. However, the present invention is not limited thereto, and as long as they retain at least one biological activity of the RASGEF1A protein, the RASGEF1A protein contains non-conservative modifications. Furthermore, modified proteins do not exclude polymorphic variants, interspecies homologs, and those encoded by alleles of these proteins.

  Furthermore, the RASGEF1A gene of the present invention encompasses a polynucleotide encoding such a functional equivalent of the RASGEF1A protein.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

I. Cancer diagnosis
I-1. Methods for diagnosing cancer or a predisposition to develop cancer
The expression of RASGEF1A gene was found to be specifically increased in cancer patients. Thus, the genes identified herein, as well as their transcripts and translation products, are useful in diagnosis as cancer markers and diagnose cancer by measuring the expression of the RASGEF1A gene in cell samples Can do. Specifically, the present invention provides a method for diagnosing cancer or a predisposition to develop cancer in a subject by determining the expression level of the RASGEF1A gene in the subject.

  Cancers that can be diagnosed by the methods of the present invention include ICC, bladder cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, chronic myelogenous leukemia, kidney cancer, soft tissue sarcoma, pancreatic cancer, and seminoma. Including, but not limited to. The method of the present invention is particularly suitable for diagnosing ICC.

  According to another aspect of the present invention, it is a predisposition to develop at least one of such cancers.

  In the context of the present invention, the term “diagnose” is intended to encompass prediction and likelihood analysis. The methods of the present invention are intended to be used clinically in making decisions regarding therapeutic interventions, diagnostic criteria such as stage, and treatment modalities including disease monitoring and cancer surveillance.

  The subject diagnosed by the present invention is preferably a mammal. Exemplary mammals include, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, and cows.

  It is preferable to carry out the diagnosis by collecting a biological sample from the subject to be diagnosed. Any biological material can be used as a biological sample for determination as long as it contains the desired transcript or translation product of the RASGEF1A gene. Biological samples include, but are not limited to, body tissues and body fluids such as blood, sputum, and urine. Preferably, the biological sample comprises a cell population comprising epithelial cells, more preferably cancer epithelial cells, or epithelial cells from a tissue suspected of being cancerous. Furthermore, if necessary, the cells may be purified from the obtained body tissues and fluids and then used as a biological sample.

  According to the present invention, the expression level of the RASGEF1A gene is determined in a subject-derived biological sample. Expression levels can be determined at the transcription (nucleic acid) product level using methods known in the art. For example, the mRNA of the RASGEF1A gene may be quantified using a probe by a hybridization method (eg, Northern hybridization). Detection may be performed on a chip or array. Use of the array is preferred for detecting the expression level of a plurality of genes (eg, various cancer-specific genes) including the RASGEF1A gene of the present invention. One skilled in the art can prepare such probes using the sequence information of the RASGEF1A gene (SEQ ID NO: 1; GenBank Accession No. AK095136). For example, the cDNA of the RASGEF1A gene may be used as a probe. If necessary, the probe may be labeled with a suitable label, such as a dye and isotope, and the expression level of the gene can be detected as the intensity of the hybridized label.

  Furthermore, the transcription product of the RASGEF1A gene can be quantified using primers by an amplification-based detection method (eg, RT-PCR). Such primers can also be prepared based on the available sequence information of the gene. For example, the primers used in the Examples (SEQ ID NO: 5 and 6; or SEQ ID NO: 7 and 8) can be used for detection by RT-PCR, but the invention is not limited thereto.

Specifically, the probe or primer used for the method of the invention hybridizes to the mRNA of the RASGEF1A gene under stringent, moderately stringent, or low stringent conditions. . As used herein, the phrase “stringent (hybridization) conditions” means conditions under which a probe or primer hybridizes to its target sequence but not to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of stringent conditions is selected to be about 5 ° C. lower than the thermal melting point (T _m ) for the specific sequence at a defined ionic strength and pH. Tm is the temperature at which 50% of a probe complementary to a target sequence (under a defined ionic strength, pH, and nucleic acid concentration) hybridizes to the target sequence in equilibrium. Since the target sequence is generally present in excess, at Tm 50% of the probes are occupied in equilibrium. Typically, stringent conditions are between pH 7.0 and pH 8.3, with salt concentrations of less than about 1.0M sodium ions, typically about 0.01M to 1.0M sodium ions (or other salts). And it is believed that the temperature is at least about 30 ° C. for short probes or primers (eg, 10-50 nucleotides) and at least about 60 ° C. for longer probes or primers. Stringent conditions may be achieved by the addition of destabilizing agents such as formamide.

Alternatively, translation products can be detected for the diagnosis of the present invention. For example, the amount of RASGEF1A protein may be determined. Methods for determining the amount of a protein as a translation product include immunoassay methods that use an antibody that specifically recognizes the protein. The antibody may be monoclonal or polyclonal. In addition, any fragment or variant of an antibody (e.g., chimeric antibody, scFv, Fab, F (ab ') ₂ , Fv, etc.) can be detected as long as the fragment retains the ability to bind to the RASGEF1A protein. May be used for Methods for preparing these types of antibodies for protein detection are well known in the art, and any method can be used in the present invention to prepare such antibodies and their equivalents. .

  As another method for detecting the expression level of the RASGEF1A gene based on its translation product, the intensity of staining may be observed through immunohistochemical analysis using an antibody against the RASGEF1A protein. That is, when strong staining is observed, it indicates an increased presence of protein and at the same time a high expression level of the RASGEF1A gene.

  Furthermore, translation products can also be detected based on their biological activity. Specifically, it has been demonstrated herein that the RASGEF1A protein has Ras nucleotide dissociation activity, RAS enhancing activity, and is involved in cancer cell migration. Therefore, such activity of RASGEF1A protein can be used as an indicator of RASGEF1A protein present in a biological sample. For example, such activity may be detected according to the methods described in the examples.

  Furthermore, in addition to the expression level of the RASGEF1A gene, the expression level of other cancer-related genes, such as genes that are known to be differentially expressed in ICC, can also be determined to improve the accuracy of diagnosis. .

  The expression level of a cancer marker gene comprising the RASGEF1A gene in a biological sample is increased or decreased, eg, 10%, 25%, or 50%, from the control level of the corresponding cancer marker gene; or more than 1.1 times, more than 1.5 times, Rise to more than 2.0 times, more than 5.0 times, more than 10.0 times or more; or at least 0.5 times, at least 0.2 times, at least 0.1 times, or less it can.

  The control level can be determined at the same time as the test biological sample by using a sample previously collected and stored from a subject whose disease state (cancerous or non-cancerous) is known. Alternatively, the control level may be determined by statistical methods based on the results obtained by analyzing the predetermined expression level of the RASGEF1A gene in a sample from a subject whose disease state is known. Furthermore, the control level may be a database of expression patterns from previously tested cells. Furthermore, according to one aspect of the invention, the expression level of the RASGEF1A gene in the biological sample may 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 from a tissue type similar to that of the patient-derived biological sample. Furthermore, it is preferable to use a standard value for the expression level of the RASGEF1A gene in a population whose disease state is known. The standard value may be obtained by any method known in the art. For example, an average value ± 2S.D. Or a range of average values ± 3S.D. May be used as the standard value.

  In the context of the present invention, a control level determined from a biological sample that is known not to be cancerous is referred to as a “normal control level”. On the other hand, if the control level is determined from a cancerous biological sample, this is called the “cancerous control level”.

  If the expression level of the RASGEF1A gene is elevated compared to the normal control level or similar to the cancerous control level, the subject is diagnosed with or at risk of developing cancer Can be done. In addition, when the expression levels of multiple cancer-related genes are compared, the similarity in gene expression pattern between the sample and the cancerous reference suggests that the subject is suffering from or at risk of developing cancer To do.

  The difference between the expression level of the test biological sample and the control level can be normalized to the expression level of a control nucleic acid, such as a housekeeping gene. It is a gene whose expression level is known not to vary depending on the cancerous state or non-cancerous state of cells. Exemplary control genes include, but are not limited to, β-actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1.

I-2. Evaluating the effectiveness of cancer treatment The RASGEF1A gene differentially expressed between normal and cancerous cells makes it possible to monitor the progress of cancer treatment and diagnose cancer. The method described above can be applied to the evaluation of the effectiveness of cancer treatment. Specifically, the effectiveness of cancer treatment can be assessed by determining the expression level of the RASGEF1A gene in cells from the subject undergoing treatment. If desired, the test cell population is taken from the subject at various time points, before treatment, during treatment, and / or after treatment. The expression level of the RASGEF1A gene can be determined, for example, according to the method described above in the section “I-1. Method for diagnosing cancer or a predisposition to develop cancer”. In the context of the present invention, the control level compared to the detected expression level is preferably determined from the RASGEF1A gene expression in cells not exposed to the treatment of interest.

  When comparing the expression level of the RASGEF1A gene to a control level determined from a cell population that does not include normal cells or cancer cells, the similarity in expression level suggests that the treatment of interest is effective and The difference suggests that the clinical outcome or prognosis of the treatment is less favorable. On the other hand, when the comparison is performed against a control level determined from a cancer cell or a cell population containing cancer cells, a difference in expression level suggests an effective treatment, whereas similarity in expression level is clinical Suggests that outcome or prognosis is less favorable.

  Furthermore, the expression level of the RASGEF1A gene before and after treatment can be compared according to the method of the present invention to evaluate the effectiveness of the treatment. Specifically, the level of expression detected in a biological sample from a subject after treatment (i.e., the post-treatment level) is the level of expression detected in a biological sample taken from the same subject prior to treatment (i.e., treatment Compared with previous level). If the post-treatment level is reduced compared to the pre-treatment level, the treatment of interest is suggested to be effective, whereas the post-treatment level is increased compared to the pre-treatment level, Or, if similar, suggests that clinical outcome or prognosis is less favorable.

  As used herein, the term “effective” refers to a decrease in the expression of a gene whose treatment is pathologically up-regulated, an increase in the expression of a gene whose pathology is down-regulated, or a subject. Show a reduction in the size, morbidity, or metastatic potential of carcinomas. When the treatment of interest is applied prophylactically, "effective" means that the treatment delays or prevents the formation of a tumor or delays, prevents or alleviates at least one clinical symptom of cancer. Means. Assessment of tumor status in a subject can be performed using standard clinical protocols.

  Furthermore, the effectiveness of the treatment can be determined in connection with any known method for diagnosing cancer. Cancer can be diagnosed, for example, by identifying symptomatic abnormalities such as weight loss, abdominal pain, back pain, loss of appetite, nausea, vomiting, and general malaise, weakness, and jaundice.

I-3. Evaluation of Prognosis of Subjects Having Cancer The method for diagnosing cancer described above can also be used to evaluate the prognosis of a subject's cancer. Accordingly, the present invention also provides a method for assessing the prognosis of a subject having cancer. The expression level of the RASGEF1A gene can be determined, for example, according to the method described above in the section “I-1. Method for diagnosing cancer or a predisposition to develop cancer”. For example, the expression level of the RASGEF1A gene in biological samples from patients at various stages can be used as a control level to compare with the expression level of this gene detected in a subject. By comparing the expression level of the RASGEF1A gene in a subject with a control level, the prognosis of the subject can be assessed. Alternatively, the prognosis of a subject can be assessed by comparing the pattern of expression levels in the subject over time.

  For example, if the expression level of the RASGEF1A gene in the subject is increased compared to the normal control level, it is suggested that the prognosis is less favorable. Conversely, if the expression level is similar compared to the normal control level, it indicates that the subject's prognosis is more favorable.

II. Kit The present invention also provides a reagent for detecting cancer, that is, a reagent capable of detecting the transcription product or translation product of the RASGEF1A gene. Specifically, it is a nucleic acid that specifically binds to or identifies the transcript of the RASGEF1A gene. For example, nucleic acids that specifically bind to or identify a transcript of the RASGEF1A gene include oligonucleotides (eg, probes and primers) that have a sequence complementary to a portion of the RASGEF1A gene transcript. . Alternatively, an antibody can be exemplified as a reagent for detecting a translation product of a gene. Probes, primers, and antibodies described above in the section “I-1. Method for diagnosing cancer or a predisposition to develop cancer” can be mentioned as suitable examples of such reagents.

  Furthermore, the translation product may be detected based on its biological activity. As shown in the Examples, it was demonstrated that the RASGEF1A protein has Ras nucleotide dissociation activity, RAS enhancing activity, and is involved in cancer cell migration. Thus, materials necessary for detection of Ras nucleotide dissociation assay, Ras activity or cancer cell migration may also be used as reagents for detecting the expression level of the RASGEF1A gene according to the present invention.

  These reagents may be used for the aforementioned cancer diagnosis. Assay formats for using these reagents may be Northern hybridizations or sandwich ELISAs, both of which are well known in the art.

  The detection reagent may be packaged together in the form of a kit. For example, the detection reagent may be packaged in a separate container. Further, the detection reagent may be packaged together with other reagents necessary for detection. For example, the kit may be a nucleic acid or antibody as a detection reagent (either bound to a solid matrix or packaged separately with a reagent for binding them to the matrix), a control reagent (positive and / or Or negative), and / or a detectable label. Instructions (eg, documentation, tape, VCR, CD-ROM, etc.) for performing the assay may also be included in the kit.

  As one aspect of the present invention, a reagent for detecting cancer can be immobilized on a solid matrix such as a porous strip to form at least one site for detecting cancer. The measurement region or detection region of the porous strip may include a plurality of sites each containing a detection reagent (eg, nucleic acid). The test strip may also include sites for negative and / or positive controls. Alternatively, the control site may be located on a separate strip from the test strip. Optionally, different detection sites may contain different amounts of immobilized detection reagent (eg, nucleic acid). That is, a larger amount may be included in the first detection site and a smaller amount may be included in the next site. When a test biological sample is added, the number of sites presenting a detectable signal provides a quantitative indicator of the expression level of the RASGEF1A gene in the sample. The detection site may be formed in any shape that can be properly detected and is typically in the shape of a bar or dot that spans the width of the test strip.

III. Screening method
The RASGEF1A gene, a polypeptide encoded by this gene or a fragment thereof, or a transcriptional regulatory region of this gene is used to screen for agents that alter the expression of this gene or the biological activity of the polypeptide encoded by this gene It is possible. Such agents can be used as agents for treating or preventing cancer. Accordingly, the present invention provides a method of screening for an agent for treating or preventing cancer using the RASGEF1A gene, a polypeptide encoded by this gene or a fragment thereof, or the transcriptional regulatory region of this gene.

  The substance isolated by the screening method of the present invention is a substance that is expected to inhibit the expression of the RASGEF1A gene or the activity of the translation product of this gene, and therefore, a disease caused by a cell proliferative disease such as cancer is detected. Candidate for treatment or prevention. Such agents may be cancers selected from the group of ICC, bladder cancer, lung cancer, stomach cancer, colorectal cancer, prostate cancer, chronic myelogenous leukemia, kidney cancer, soft tissue sarcoma, pancreatic cancer, and seminoma. Expected to be treated or prevented. That is, substances screened by the methods of the present invention are considered to have clinical benefit and can be further tested for the ability to prevent cancer cell growth in animal models or test subjects.

  In the context of the present invention, the substance identified by the screening method of the present invention may be any compound or a composition comprising several compounds. Furthermore, the test substance exposed to the cell or protein by the screening method of the present invention may be a single compound or a combination of compounds. If combinations of compounds are used in these methods, these compounds may be contacted sequentially or simultaneously.

  Any test substance, e.g. cell extract, cell culture supernatant, fermentation microorganism product, marine organism extract, plant extract, purified or crude protein, peptide, non-peptide compound, synthetic micromolecular compound ( Antisense RNA, siRNA, including nucleic acid constructs such as ribozymes), and natural compounds can be used in the screening methods of the present invention. The test substance of the present invention also comprises (1) a biological library, (2) a spatially addressable parallel solid phase library or liquid phase library, (3) a synthetic library method that requires 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) synthetic library method using affinity chromatography selection Can also be obtained. Biological library methods using affinity chromatography selection are limited to peptide libraries, but the other four approaches apply to peptide libraries, non-peptide oligomer libraries, or small molecule libraries of compounds It is possible (Lam, Anticancer Drug Des 1997, 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al., Proc Natl Acad Sci USA 1993, 90: 6909-13; Erb et al., Proc Natl Acad Sci USA 1994, 91 : 11422-6; Zuckermann et al., J Med Chem 37: 2678-85, 1994; Cho et al., Science 1993, 261: 1303-5; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2059 Carell et al., Angew Chem Int Ed Engl 1994, 33: 2061; Gallop et al., J Med Chem 1994, 37: 1233-51). Compound libraries can be in solution (see Houghten, Bio / Techniques 1992, 13: 412-21) or on beads (Lam, Nature 1991, 354: 82-4), on chip (Fodor, Nature 1993). , 364: 555-6), on bacteria (US Pat. No. 5,223,409), on spores (US Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), on plasmids (Cull et al., Proc Natl Acad Sci USA) 1992, 89: 1865-9) or on phage (Scott and Smith, Science 1990, 249: 386-90; Devlin, Science 1990, 249: 404-6; Cwirla et al., Proc Natl Acad Sci USA 1990, 87 : 6378-82; Felici, J Mol Biol 1991, 222: 301-10; US Patent Application No. 2002103360).

  A compound in which a part of the structure of a compound screened by any of the screening methods of the present invention is converted by addition, deletion, and / or substitution is included in the agent obtained by the screening method of the present invention. .

  Further, when the test substance to be screened is a protein, in order to obtain DNA encoding the protein, the entire amino acid sequence of the protein may be determined, and the nucleic acid sequence encoding the protein may be estimated or obtained. A part of the amino acid sequence of the obtained protein may be analyzed, an oligo DNA may be prepared based on the sequence as a probe, and a cDNA library may be screened using the probe to obtain DNA encoding the protein. The resulting DNA is used in preparing test substances that are candidates for treating or preventing cancer.

III-1. Protein-based screening method According to the present invention, it was suggested that the expression of RASGEF1A gene is very important for the growth and / or survival of cancer cells. Therefore, a substance that suppresses the function of the polypeptide encoded by this gene was considered to be used in inhibiting cancer cell growth and / or survival and treating or preventing cancer. Accordingly, the present invention provides a method of screening for agents for treating or preventing cancer using the RASGEF1A polypeptide.

  In addition to the RASGEF1A polypeptide, fragments of this polypeptide can also be used for the screening of the present invention so long as they retain at least one biological activity of the native RASGEF1A polypeptide.

  The polypeptide or fragment thereof may be further linked to other substances as long as the polypeptide and fragment retain at least one of its biological activities. Usable materials include peptides, lipids, sugars and sugar chains, acetyl groups, natural and synthetic polymers, and the like. These types of modifications may be performed to provide additional functions or to stabilize polypeptides and fragments.

The polypeptide or fragment used in the method of the present invention can be obtained from nature as a natural protein through conventional purification methods or through chemical synthesis based on a selected amino acid sequence. For example, conventional peptide synthesis methods that can be employed for synthesis include the following:
1) Peptide Synthesis, Interscience, New York, 1966;
2) The Proteins, Vol. 2, Academic Press, New York, 1976;
3) Peptide Synthesis (Japanese), Maruzen Co., 1975;
4) Basics and Experiment of Peptide Synthesis (Japanese), Maruzen Co., 1985;
5) Development of Pharmaceuticals (Vol.2) (Japanese), Vol.14 (peptide synthesis), Hirokawa, 1991;
6) WO99 / 67288; and
7) Barany G. and Merrifield RB, Peptides Vol. 2, “Solid Phase Peptide Synthesis”, Academic Press, New York, 1980, 100-118.

  Alternatively, proteins can be obtained by employing any known genetic engineering method for producing polypeptides (eg, Morrison J., J Bacteriology 1977, 132: 349-51; Clark-Curtiss and Curtiss, Methods in Enzymology (Edited by Wu et al.) 1983, 101: 347-62). For example, first an appropriate vector containing a polynucleotide encoding the protein of interest in an expressible form (e.g., downstream of a regulatory sequence containing a promoter) is prepared, transformed into an appropriate host cell, and then the host Cells are cultured to produce the protein. More specifically, by inserting the gene encoding the RASGEF1A polypeptide into a vector for expressing a foreign gene such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8, the gene (for example, Animal) Expressed in cells. A promoter may be used for expression. Any commonly used promoter may be used, such as the SV40 early promoter (Rigby, Williamson (ed.), Genetic Engineering, vol. 3.Academic Press, London, 1982, 83-141), EF-α promoter ( Kim et al., Gene 1990, 91: 217-23), CAG promoter (Niwa et al., Gene 1991, 108: 193), RSV LTR promoter (Cullen, Methods in Enzymology 1987, 152: 684-704), SRα Promoter (Takebe et al., Mol Cell Biol 1988, 8: 466), CMV early promoter (Seed et al., Proc Natl Acad Sci USA 1987, 84: 3365-9), SV40 late promoter (Gheysen et al., J Mol Appl Genet 1982, 1: 385-94), adenovirus late promoter (Kaufman et al., Mol Cell Biol 1989, 9: 946), and the HSV TK promoter. The vector can be introduced into a host cell for expressing the RASGEF1A gene by any method such as electroporation (Chu et al., Nucleic Acids Res 1987, 15: 1311-26), calcium phosphate method (Chen et al , Mol Cell Biol 1987, 7: 2745-52), DEAE dextran method (Lopata et al., Nucleic Acids Res 1984, 12: 5707-17; Sussman et al., Mol Cell Biol 1985, 4: 1642-3) And the lipofectin method (Derijard B, Cell 1994, 7: 1025-37); Lamb et al., Nature Genetics 1993, 5: 22-30; Rabindran et al., Science 1993, 259: 230-4) can do.

  The RASGEF1A protein may also be produced in vitro employing an in vitro translation system.

  The RASGEF1A polypeptide that is contacted with the test substance may be, for example, a purified polypeptide, a soluble protein, or a fusion protein fused with other polypeptides.

III-1-1. Identification of an agent that binds to the RASGEF1A polypeptide An agent that binds to a protein is thought to alter the expression of the gene encoding the protein or the biological activity of the protein. Accordingly, as one aspect, the present invention provides a method of screening for an agent for treating or preventing cancer comprising the following steps:
(a) contacting a test substance with a RASGEF1A polypeptide or fragment thereof;
(b) detecting the binding between the polypeptide and the test substance; and
(c) selecting a test substance that binds to the polypeptide.

  Binding of the test substance to the RASGEF1A polypeptide can be detected, for example, by immunoprecipitation using an antibody against the polypeptide. Therefore, for the purpose of such detection, the RASGEF1A polypeptide or fragment thereof used for screening preferably contains an antibody recognition site. The antibody used for the screening may be an antibody that recognizes an antigenic region (for example, an epitope) of the RASGEF1A polypeptide of the present invention, the preparation method of which is well known in the art, and any method is used in the present invention. It may be used to prepare such antibodies and their equivalents.

  Alternatively, the RASGEF1A polypeptide or fragment thereof is expressed as a fusion protein containing a monoclonal antibody recognition site (epitope) at the N-terminus or C-terminus that has been shown to be specific for the N-terminus or C-terminus of the polypeptide. obtain. A commercially available epitope-antibody system can be used (Experimental Medicine 1995, 13: 85-90). For example, vectors that can express a fusion protein with β-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescent protein (GFP), etc. by using the multiple cloning site are commercially available. Can be used. In addition, fusion proteins containing very small epitopes that can be detected by immunoprecipitation with antibodies to the epitope are also known in the art (Experimental Medicine 1995, 13: 85-90). Such epitopes consisting of several to several tens of amino acids that do not alter the properties of the RASGEF1A polypeptide or fragments thereof can also be used in the present invention. Examples include polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human herpes simplex virus Glycoprotein (HSV-tag), E-tag (epitope on monoclonal phage), etc. are included.

  Glutathione S-transferase (GST) is also well known as the counterpart of fusion proteins detected by immunoprecipitation. When GST is used as a protein to be fused with a RASGEF1A polypeptide or fragment thereof to form a fusion protein, the fusion protein is an antibody against GST or a substance that specifically binds to GST, i.e. glutathione (e.g. 4B) can be used for detection.

  In immunoprecipitation, immunization is performed by adding an antibody (recognizing the RASGEF1A polypeptide or fragment thereof itself or an epitope tagged to the polypeptide or fragment) to the reaction mixture of the RASGEF1A polypeptide and the test substance. A complex is formed. If the test substance has the ability to bind to the polypeptide, the immune complex formed will consist of the RASGEF1A polypeptide, the test substance, and the antibody. In contrast, if the test substance lacks such ability, then the immune complex that is formed consists only of the RASGEF1A polypeptide and antibody. Therefore, the ability of the test substance to bind to the RASGEF1A polypeptide can be examined, for example, by measuring the size of the formed immune complex. Any method for detecting the size of a substance can be used, including chromatography, electrophoresis and the like. For example, when mouse IgG antibodies are used for detection, protein A or protein G sepharose can be used to quantify the formed immune complexes.

  For more details on immunoprecipitation, see, for example, Harlow et al., Antibodies, Cold Spring Harbor Laboratory publications, New York, 1988, 511-52.

  Furthermore, the RASGEF1A polypeptide or fragment thereof used to screen for agents that bind to the RASGEF1A polypeptide or fragment thereof may be bound to a carrier. Examples of carriers that can be used to bind to the polypeptide include insoluble polysaccharides such as agarose, cellulose, and dextran, and synthetic resins such as polyacrylamide, polystyrene, and silicone, preferably from the materials described above. Prepared, commercially available beads and plates (eg, multi-well plates, biosensor chips, etc.) can be used. If beads are used, they may be packed in a column. Alternatively, the use of magnetic beads is also known in the art, which makes it possible to easily isolate polypeptides and agents bound to beads using magnetism.

  The polypeptide may be bound to the carrier according to a conventional method such as chemical binding and physical adsorption. Alternatively, the polypeptide can be bound to the carrier via an antibody that specifically recognizes the protein. Furthermore, the binding of the polypeptide to the carrier can also be performed using interacting molecules such as a combination of avidin and biotin.

  Screening using such a RASGEF1A polypeptide or a fragment thereof bound to a carrier includes, for example, contacting a test substance with a polypeptide bound to the carrier, incubating the mixture, washing the carrier, and the carrier. Detecting and / or measuring the bound agent. As long as the buffer does not inhibit binding, binding may be performed in a buffer, such as, but not limited to, a phosphate buffer and a Tris buffer buffer.

  In screening methods in which a RASGEF1A polypeptide or fragment and composition (eg, cell extract, cell lysate, etc.) bound to such a carrier is used as a test substance, such a method is commonly referred to as affinity chromatography. . For example, the RASGEF1A polypeptide may be immobilized on an affinity column carrier, and a test substance containing a substance capable of binding to the polypeptide is added to the column. After adding the test substance, the column is washed, and then the substance bound to the polypeptide is eluted with an appropriate buffer.

  A biosensor using the surface plasmon resonance phenomenon can be used as a means for detecting or quantifying the bound substance in the present invention. When such a biosensor is used, the interaction between the RASGEF1A polypeptide and the test substance is observed in real time as a surface plasmon resonance signal with a negligible amount of polypeptide and without labeling. (Eg BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide and the test substance using a biosensor such as BIAcore.

  From synthetic chemical compounds, or molecules in natural substance banks or random peptide phage display libraries, molecules that bind to specific proteins by exposing them to specific molecules immobilized on a carrier. Methods of screening and high-throughput screening methods based on combinatorial chemistry techniques that isolate not only proteins but also chemical compounds (Wrighton et al., Science 1996, 273: 458-64; Verdine, Nature 1996, 384: 11-3 ) Is also well known to those skilled in the art. These methods can also be used to screen for substances (including agonists and antagonists) that bind to the RASGEF1A protein or fragments thereof.

  When the test substance is a protein, for example, West Western blot analysis (Skolnik et al., Cell 1991, 65: 83-90) can be used in the method of the present invention. Specifically, the protein that binds to the RASGEF1A polypeptide is a cell, tissue, organ, or a protein that is expected to express at least one protein that binds to the RASGEF1A polypeptide using a phage vector (e.g., ZAP). A cDNA library prepared from cultured cells (e.g. SSP25) is first prepared, the protein encoded by the cDNA library vector is expressed on LB agarose, and the expressed protein is immobilized on a filter and purified. And by reacting the labeled RASGEF1A polypeptide with the aforementioned filter and detecting plaques expressing the protein to which the RASGEF1A polypeptide is bound according to the label of the RASGEF1A polypeptide.

Radioisotopes (e.g. ³ H, ¹⁴ C, ³² P, ³³ P, ³⁵ S, ¹²⁵ I, ¹³¹ I), enzymes (e.g. alkaline phosphatase, horseradish peroxidase, β-galactosidase, β-glucosidase), fluorescent Substances (eg, fluorescein isothiocyanate (FITC), rhodamine) and labeling substances such as biotin / avidin may be used for labeling the RASGEF1A polypeptide in the methods of the invention. If the protein is labeled with a radioisotope, detection or measurement can be performed by liquid scintillation. Alternatively, if the protein is labeled with an enzyme, the protein can be detected or measured by adding a substrate for the enzyme and detecting an enzymatic change in the substrate, such as color production, with an absorptiometer. Furthermore, when a fluorescent substance is used as a label, the bound protein may be detected or measured using a fluorometer.

  In addition, the RASGEF1A polypeptide bound to the protein can be detected or measured by using an antibody that specifically binds to the RASGEF1A polypeptide, or a peptide or polypeptide fused to the RASGEF1A polypeptide (e.g., GST). Can do. When an antibody is used in the screening of the present invention, the antibody is preferably labeled with one of the aforementioned labeling substances, and detected or measured based on the labeling substance. Alternatively, an antibody against the RASGEF1A polypeptide may be used as a primary antibody detected by a secondary antibody labeled with a labeling substance. Furthermore, the antibody that binds to the RASGEF1A polypeptide in the screening of the present invention may be detected or measured using a protein G column or protein A column.

  Alternatively, in another embodiment of the screening method of the present invention, a two-hybrid system using cells can be used (`` MATCHMAKER Two-Hybrid system '', `` Mammalian MATCHMAKER Two-Hybrid Assay Kit '', `` MATCHMAKER one-Hybrid system '' (Clontech); "HybriZAP Two-Hybrid Vector System" (Stratagene); references "Dalton et al., Cell 1992, 68: 597-612" and "Fields et al., Trends Genet 1994, 10: 286-92" ). In the two-hybrid system, the RASGEF1A polypeptide or fragment thereof is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells. From a cell that is expected to express at least one protein that binds to the RASGEF1A polypeptide, the cDNA library can be fused to the transcriptional activation region of VP16 or GAL4 when expressed. Prepare. The cDNA library is then introduced into the aforementioned yeast cell and the cDNA derived from the library is isolated from the detected positive clone (if the protein that binds to the RASGEF1A polypeptide is expressed in the yeast cell, These two types of binding activate the reporter gene, allowing positive clones to be detected). A protein encoded by cDNA can be prepared by introducing the cDNA isolated above into E. coli and expressing the protein.

  As the reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and the like can be used in addition to the HIS3 gene.

  The agent isolated by this screening is a candidate agonist or antagonist of the RASGEF1A polypeptide. The term “agonist” means a molecule that activates the function of a polypeptide by binding to the polypeptide. In contrast, the term “antagonist” means a molecule that inhibits the function of a polypeptide by binding to the polypeptide. In addition, agents isolated as antagonists by this screening are candidates for inhibiting the in vivo interaction of RASGEF1A polypeptides with molecules (including nucleic acids (RNA and DNA) and proteins).

III-1-2. Identification of Agents by Detecting Biological Activity of RASGEF1A Polypeptide According to the present invention, the expression of RASGEF1A gene was shown to be very important for the growth and / or survival of cancer cells. . Therefore, an agent that suppresses or inhibits the biological function of the translation product of the RASGEF1A gene is considered useful as a candidate for treating or preventing cancer. Accordingly, the present invention also provides a method for screening a compound for treating or preventing cancer using the RASGEF1A polypeptide or a fragment thereof, comprising the following steps:
(a) contacting a test substance with a RASGEF1A polypeptide or fragment thereof; and
(b) detecting the biological activity of the polypeptide of step (a).

  Any polypeptide can be used for screening as long as it has one biological activity of the RASGEF1A polypeptide that can be used as an indicator in the screening method of the present invention. Since RASGEF1A polypeptide has activity to promote cell growth of cancer cells, the biological activity of RASGEF1A polypeptide that can be used as an indicator for screening includes such cells of human RASGEF1A polypeptide Proliferative activity is included. For example, a human RASGEF1A polypeptide can be used, and a polypeptide that includes a fragment thereof and is functionally equivalent thereto can also be used. Such polypeptides can be expressed endogenously or exogenously by appropriate cells.

  When the biological activity detected in the method of the present invention is cell proliferation, this can be accomplished, for example, by preparing cells expressing a RASGEF1A polypeptide or fragment thereof, culturing these cells in the presence of a test substance, and cell cycle Detection of wound healing activity, performance of a Matrigel invasion assay, and colony forming activity, such as by determining cell growth rate, such as by measuring and as described in the Examples (see Materials and Methods) It can be detected by measuring.

According to one aspect of the present invention, the screening further comprises the following steps after the aforementioned step (b):
c) selecting a test substance that inhibits the biological activity of the polypeptide as compared to the biological activity detected in the absence of the test substance.

  Agents isolated by this screen are candidates for antagonists of the RASGEF1A polypeptide, and thus candidates that inhibit in vivo interactions of this polypeptide with molecules (including nucleic acids (RNA and DNA) and proteins). is there.

As another aspect of the present invention, an agent that is a candidate for an agonist of RASGEF1A polypeptide can be screened using the biological activity of the polypeptide as an indicator. According to such a method, an agent that enhances the biological activity of the RASGEF1A polypeptide is selected. Specifically, the screening method further comprises the following steps after the aforementioned step (b):
c) selecting a test substance that enhances the biological activity of the polypeptide relative to the biological activity detected in the absence of the test substance.

Herein, it was revealed that the RASGEF1A polypeptide enhances the activity of RAS. Thus, agents that reduce RAS enhancing activity, ie, one of the biological activities of a RASGEF1A polypeptide, can be used to treat or prevent cancer. Accordingly, the present invention further provides a method of screening for an agent for treating or preventing cancer. One embodiment of this screening method includes the following steps:
(a) contacting the RASGEF1A polypeptide or fragment thereof and RAS in the presence of a test substance;
(b) detecting RAS activity; and
(c) selecting a test substance that reduces RAS activity compared to the activity detected in the absence of the test substance.

  RAS activity can be measured by methods known in the art, such as GDP detection (see, eg, Hall BE et al., J Biol Chem 2001, 276: 27629-37) or RAS activation assay. it can. In the present invention, in order to estimate the RAS activation activity of the RASGEF1A polypeptide in the presence or absence of a test substance, for example, a cell lysate of a cell expressing the RASGEF1A gene is used in the presence or absence of the test compound. Incubate with GST-Ras fusion protein, GDP, and GTPγS in the presence. After incubation, active Ras or GTP-Ras is detected by Western blot analysis using an anti-Ras antibody that detects 21 kDa active Ras or GTP-Ras. Active Ras or GTP-Ras pull-down assays can be performed using glutathione disks. Commercially available RAS activation assay kits can also be used for the screening methods of the present invention. For example, `` StressXpress® RAS Activation Kit '' (Stressgen Bioreagents Corp.) or `` EZ-DetectTM Ras Activation Kit '' (PIERCE Biotechnology) contains all the elements for this assay, such as anti-RAS antibodies, glutathione disks, Includes GST fusion protein, GDP, and GTPγS containing Ras binding domain (RBD) of Raf1. Furthermore, Ras activity can be detected as described in the Examples (see “8. Ras Nucleotide Dissociation Assay” and “9. Analysis of Ras Activity” in “Materials and Methods” below). Wanna).

III-2. Nucleotide-based screening methods
III-2-1. Screening Method Using RASGEF1A Gene As described in detail above, the onset and progression of cancer can be controlled by controlling the expression level of the RASGEF1A gene. Therefore, an agent that can be used in the treatment or prevention of cancer can be identified by screening using the expression level of the RASGEF1A gene as an indicator. In the context of the present invention, such screening may include, for example, the following steps:
(a) contacting the test substance with a cell expressing the RASGEF1A gene;
(b) detecting the expression level of the RASGEF1A gene; and
(c) selecting a test substance that reduces the expression level of the RASGEF1A gene compared to the level detected in the absence of the test substance.

  An agent that inhibits the expression of the RASGEF1A gene or the activity of its gene product can be identified by contacting a cell that expresses the RASGEF1A gene with a test substance and then determining the expression level of the RASGEF1A gene. Of course, identification may be performed using a population of cells expressing this gene instead of a single cell. If the expression level detected in the presence of the agent is reduced compared to the expression level in the absence of the agent, the agent is shown to be an inhibitor of the RASGEF1A gene, The agent is useful for inhibiting cancer, thus suggesting the possibility of being a candidate substance that can be used for the treatment or prevention of cancer.

  The expression level of a gene can be estimated by methods well known to those skilled in the art. The expression level of the RASGEF1A gene can be determined, for example, according to the method described above in the section “I-1. Method for diagnosing cancer or a predisposition to develop cancer”.

  The cell or cell population used for such identification may be any cell or any cell population as long as it expresses the RASGEF1A gene. For example, the cell or population may be or include an epithelial cell derived from a tissue. Alternatively, the cell or population is a cancer cell, including those derived from ICC, bladder cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, chronic myelogenous leukemia, kidney cancer, soft tissue sarcoma, pancreatic cancer, and seminoma It may be an immortalized cell of origin or may contain them. The cell expressing the RASGEF1A gene includes, for example, a cell line established from cancer (for example, SSP25). Further, the cell or population may be or contain a cell transfected with the RASGEF1A gene.

  The methods of the present invention are particularly suitable for screening functional nucleic acid molecules that allow screening of the various agents described above and that include antisense RNA, siRNA, and the like.

III-2-2. Screening method using transcriptional regulatory region of RASGEF1A gene According to another aspect, the present invention provides a method comprising the following steps:
(a) contacting a test substance with a cell into which a vector containing a transcriptional regulatory region of the RASGEF1A gene and a reporter gene expressed under the control of the transcriptional regulatory region is introduced;
(b) detecting the expression or activity of the reporter gene; and
(c) selecting a test substance that reduces the expression or activity of the reporter gene compared to the level detected in the absence of the test substance.

  Suitable reporter genes and host cells are well known in the art. The reporter construct required for screening can be prepared using the transcriptional regulatory region of the RASGEF1A gene, which can be obtained as a nucleotide segment containing the transcriptional regulatory region from a genomic library based on the nucleotide sequence information of the gene.

  The transcription regulatory region may be, for example, the promoter sequence of the RASGEF1A gene.

  When using a cell transfected with a reporter gene that is operably linked to a regulatory sequence of the RASGEF1A gene (for example, a promoter sequence), the expression level of the reporter gene product is detected to inhibit or enhance the expression of the RASGEF1A gene The agent can be identified as

  As the reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene, HIS3 gene and the like well known in the art can be used. Methods for detecting the expression of these genes are well known in the art.

III-3. Selection of appropriate therapeutic agents for individual individuals Due to differences in individual genetic makeup, the relative ability to metabolize various drugs may vary. Substances that are metabolized in the subject and act as antitumor substances change the gene expression pattern in the target cell from the gene expression pattern characteristic of the cancerous state to the gene expression pattern characteristic of the non-cancerous state It can be revealed by inducing. Thus, a putative therapeutic inhibitor or prevention of cancer in a test cell population derived from a selected subject due to the RASGEF1A gene differentially expressed between cancer and non-cancer cells disclosed herein. Can be tested to determine if the agent is an appropriate inhibitor of cancer in the subject.

  To identify an inhibitor of cancer appropriate for an individual subject, a test cell population from the subject is exposed to a candidate therapeutic agent and the expression of the RASGEF1A gene is determined.

  In the context of the method of the invention, the test cell population comprises cancer cells that express the RASGEF1A gene. Preferably, the test cell is an epithelial cell.

  Specifically, the test cell population may be incubated in the presence of a candidate therapeutic agent, and the expression of the RASGEF1A gene in the test cell population is measured and one or more reference profiles, eg, cancerous reference expression A profile or a non-cancerous reference expression profile may be compared.

  A decrease in the expression of the RASGEF1A gene in the test cell population as compared to a reference cell population containing cancer indicates that the substance has therapeutic potential.

IV. Pharmaceutical Composition for Treating or Preventing Cancer A substance screened by any of the screening methods of the present invention, an antisense nucleic acid and siRNA of the RASGEF1A gene, and an antibody against the RASGEF1A polypeptide are expressed or expressed by the RASGEF1A gene. Inhibits or suppresses the biological activity of a RASGEF1A polypeptide and inhibits or disrupts cell cycle regulation and cell proliferation. Accordingly, the present invention provides a composition for treating or preventing cancer, comprising a substance screened by any of the screening methods of the present invention, an antisense nucleic acid and siRNA of RASGEF1A gene, or an antibody against RASGEF1A polypeptide. The composition of the present invention treats or prevents cancers such as ICC, bladder cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, chronic myelogenous leukemia, kidney cancer, soft tissue sarcoma, pancreatic cancer, and seminoma. Can be used to

  These compositions may be used as drugs for humans and other mammals such as mice, rats, guinea pigs, rabbits, cats, dogs, sheep, pigs, cows, monkeys, baboons, and chimpanzees.

  In the context of the present invention, pharmaceutical formulations suitable for the active ingredients of the present invention (including screened agents, antisense nucleic acids, siRNA, antibodies, etc.) detailed below include oral, rectal, Those suitable for nasal administration, topical administration (including buccal and sublingual), vaginal or parenteral administration (including intramuscular, subcutaneous, and intravenous), or administration by inhalation or insufflation are included. Preferably administration is intravenous. The formulation is optionally packaged in individual dosage units.

  Pharmaceutical formulations suitable for oral administration include capsules, microcapsules, cachets and tablets, each containing a predetermined amount of the active ingredient. Suitable formulations also include powders, elixirs, granules, solutions, suspensions, and emulsions. The active ingredient is optionally administered as a bolus electuary or paste. Alternatively, if desired, the pharmaceutical composition can be administered parenterally in the form of a sterile solution or suspension suspension in water or any other pharmaceutically acceptable liquid. For example, the active ingredients of the present invention are in a unit dosage form required for accepted drug manufacture, in a pharmaceutically acceptable carrier or vehicle, specifically sterile water, saline, vegetable oil, It can be mixed with emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, solvents, preservatives, binders and the like. Depending on the amount of active ingredient contained in such a formulation, an appropriate dosage within the specified range can be obtained.

  Examples of additives that can be mixed in tablets and capsules include binders such as gelatin, corn starch, tragacanth gum, and gum arabic; excipients such as crystalline cellulose; corn starch, gelatin, and alginic acid Swelling agents; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, or saccharin; and flavoring agents such as peppermint, Gaultheria adenothrix oil, and cherries, but are not limited to these . A tablet may be made by compression or molding, optionally with one or more pharmaceutical ingredients. The active ingredient in free-flowing form, such as powder or granules, optionally mixed with binders, lubricants, inert diluents, lubricants, surfactants or dispersants and compressed in a suitable machine By doing so, a compressed tablet may be prepared. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Tablets may be coated by methods well known in the art. Tablets may optionally be formulated to provide sustained or controlled release of the active ingredient in vivo. The tablet package may contain one tablet to be taken each month.

  In addition, when the unit dosage form is a capsule, a liquid carrier such as oil can be further included in addition to the aforementioned ingredients.

  Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or dry products for dissolution with water or other suitable solvent before use May be provided as Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous solvents (which may include edible oils), or preservatives.

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

  In addition, sterile composites for injection can be formulated according to conventional drug production using a solvent such as distilled water suitable for injection. Saline, glucose, and other isotonic liquids, including adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used as an aqueous solution for injection. These may be used in combination with suitable solubilizers such as alcohols such as ethanol; polyhydric alcohols such as propylene glycol and polyethylene glycol; and nonionic surfactants such as polysorbate 80 ™ and HCO-50. it can.

  Sesame oil or soybean oil can be used as an oily liquid, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent, such as phosphate buffer and sodium acetate buffer. It may be formulated with buffers, analgesics such as procaine hydrochloride, stabilizers such as benzyl alcohol and phenol, and / or antioxidants. The prepared injection can be filled into an appropriate ampoule.

  Formulations for rectal administration include suppositories with standard carriers such as cocoa butter or polyethylene glycol. Formulations for topical administration in the mouth, eg, buccal or sublingual, include sucrose and a lozenge containing the active ingredient in a flavored base such as gum arabic or gum tragacanth and gelatin, glycerin, sucrose, or Included are pastilles containing the active ingredient in a base such as rubber. When the active ingredient is administered intranasally, it can be used in the form of a liquid spray or dispersible powder, or drops. Drops can also be formulated with an aqueous or non-aqueous base containing 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. 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 can be determined by providing a valve that delivers a metered 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. The powder composition may be provided in unit dosage forms, such as capsules, cartridges, gelatin or blister packs, from which powders may be administered with the aid of an inhaler or insufflator.

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

  If desired, the aforementioned formulations adapted to give sustained release of the active ingredient can be used. The pharmaceutical composition may also contain other active ingredients such as antibacterial agents, immunosuppressive agents, or preservatives. In addition to the ingredients specifically mentioned above, the formulations of the present invention may contain other agents customary in the art, taking into account the type of formulation concerned, such as those suitable for oral administration It should be understood that a flavoring agent may be included.

  Preferred dosage unit formulations are those containing an effective amount of each active ingredient of the present invention or an appropriate divided amount thereof, as mentioned in the section “Methods for treating or preventing cancer” below.

IV-1. Pharmaceutical Composition Containing Screened Substance The present invention provides a composition for treating or preventing cancer, comprising any of the substances selected by the aforementioned screening method of the present invention.

  Substances screened by the methods of the present invention may be administered directly or may be formulated into dosage forms according to any conventional pharmaceutical formulation method detailed above.

IV-2. Pharmaceutical composition containing siRNA
The siRNA against the RASGEF1A gene (hereinafter also referred to as “RASGEF1A siRNA”) can be used to reduce the expression level of this gene.In this specification, the term “siRNA” refers to the two that prevent translation of the target mRNA. In the context of the present invention, siRNA comprises a sense nucleic acid sequence and an antisense nucleic acid sequence for the up-regulated marker gene RASGEF1 A. The siRNA comprises a target gene sense sequence and a complementary antisense sequence. In other words, that is, a nucleotide containing a hairpin structure.

  The siRNA of the RASGEF1A gene hybridizes to the target mRNA, i.e. normally binds to a single-stranded mRNA transcript, thereby preventing translation of the mRNA and ultimately producing the polypeptide encoded by that gene ( Expression) is reduced or inhibited. Thus, the siRNA molecules of the present invention can be defined by their ability to specifically hybridize to the mRNA of the RASGEF1A gene under stringent conditions.

  In the context of the present invention, the length of the siRNA is preferably less than 500, 200, 100, 50, or 25 nucleotides. More preferably, the siRNA length is 19-25 nucleotides. Exemplary target nucleic acid sequences for RASGEF1A siRNA include the nucleotide sequence of SEQ ID NO: 9 or 13. Nucleotide “t” in the sequence should be replaced with “u” in RNA or its derivatives. Thus, for example, the present invention provides a double-stranded RNA molecule comprising the nucleotide sequence 5′-gagaauggcacagugaaga-3 ′ (SEQ ID NO: 9) or 5′-caucuacuuccugcacaaa-3 ′ (SEQ ID NO: 13). In order to enhance the inhibitory activity of siRNA, nucleotide “u” can be added to the 3 ′ end of the antisense strand of the target sequence. The number of “u” added is at least 2, generally 2 to 10, preferably 2 to 5. The added “u” forms a single strand at the 3 ′ end of the antisense strand of the siRNA.

A loop sequence consisting of an arbitrary nucleotide sequence can be arranged between the sense sequence and the antisense sequence to form a hairpin loop structure. Accordingly, the present invention also provides siRNA having the general formula 5 ′-[A]-[B]-[A ′]-3 ′, wherein [A] is specific for the mRNA or cDNA of the RASGEF1A gene The ribonucleotide sequence corresponding to the sequence that hybridizes to In a preferred embodiment, [A] is a ribonucleotide sequence corresponding to the sequence of the RASGEF1A gene, [B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides, and [A ′] is [A ′] ] Is a ribonucleotide sequence consisting of a complementary sequence. Region [A] hybridizes to [A ′], and then a loop consisting of region [B] is formed. The length of the loop sequence may preferably be 3-23 nucleotides. For example, the loop sequence can be selected from the group consisting of the following sequences (http://www.ambion.com/techlib/tb/tb_506.html):
CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002, 418: 435-8.
UUCG: Lee NS et al., Nature Biotechnology 2002, 20: 500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003, 100 (4): 1639-44.
UUCAAGAGA: Dykxhoorn DM et al., Nature Reviews Molecular Cell Biology 2003, 4: 457-67.

  “UUCAAGAGA (“ ttcaagaga ”in DNA)” is a particularly suitable loop sequence. In addition, a loop sequence of 23 nucleotides also provides an active siRNA (Jacque J-M et al., Nature 2002, 418: 435-8).

Exemplary hairpin siRNAs suitable for use in the context of the present invention include, in the case of RASGEF1A-siRNA:
5'-gagaauggcacagugaaga- [B] -ucuucacugugccauucuc-3 '(to the target sequence of SEQ ID NO: 9); and
5'-caucuacuuccugcacaaa- [B] -uuugugcaggaaguagaug-3 '(to the target sequence of SEQ ID NO: 13) is included.

  A suitable siRNA nucleotide sequence can be designed using the siRNA design computer program available from the Ambion website (http://www.ambion.com/techlib/misc/siRNA_finder.html). The computer program selects nucleotide sequences for siRNA synthesis based on the following protocol.

siRNA target site selection:
1． Starting from the AUG start codon of the transcript of interest, the AA dinucleotide sequence is scanned downstream. Record the occurrence of individual AA and 3 ′ adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al. Identified siRNA against these because the 5 'and 3' untranslated regions (UTR) and the region near the start codon (within 75 nucleotides) may be richer in regulatory protein binding sites. It is recommended not to design. UTR-binding proteins and / or translation initiation complexes can interfere with the binding of the siRNA endonuclease complex.
2． Potential target sites are compared to the human genome database and any target sequences that have significant homology to other coding sequences are excluded from consideration. Homology searches can be performed using BLAST (Altschul SF et al., Nucleic Acids Res 1997, 25: 3389-402; J Mol Biol 1990, 215: 403-10.) On the NCBI server. At www.ncbi.nlm.nih.gov/BLAST/.
3． A target sequence suitable for synthesis is selected. In Ambion, preferably several target sequences can be selected and evaluated along the length of the gene.

  Standard techniques for introducing siRNA into cells can be used. For example, RASGEF1A siRNA can be introduced directly into cells in a form that can bind to mRNA transcripts. In these embodiments, the siRNA molecules of the invention are typically modified as described above for antisense molecules. Other modifications are also possible. For example, cholesterol conjugated siRNAs showed improved pharmacological properties (Song et al., Nature Med 2003, 9: 347-51).

  Alternatively, DNA encoding siRNA may be retained in a vector (hereinafter also referred to as “siRNA vector”). Such vectors are functionally linked regulatory sequences (e.g., small nuclear RNA (snRNA) adjacent to the sequence in a manner that allows expression of both strands (e.g., by transcription of the DNA molecule). It can be made by cloning the target RASGEF1A gene sequence into an expression vector with RNA polymerase III transcription unit from U6 or human H1 RNA promoter) (Lee NS et al., Nature Biotechnology 2002, 20: 500). -Five). For example, an RNA molecule that is antisense to the mRNA of the RASGEF1A gene is transcribed by a first promoter (for example, the promoter sequence 3 ′ of the cloned DNA), and an RNA molecule that is the sense strand for the mRNA of the RASGEF1A gene is Is transcribed by a promoter (eg, promoter sequence 5 ′ of cloned DNA). The sense and antisense strands hybridize in vivo to yield an siRNA construct for silencing the expression of the RASGEF1A gene. Alternatively, the two constructs can be used to create the sense and antisense strands of a single stranded siRNA construct. In this case, a construct having a secondary structure, such as a hairpin, is made as a single transcript containing both the sense sequence and the complementary antisense sequence of the target gene.

  Thus, a pharmaceutical composition of the invention for treating or preventing cancer comprises either siRNA or a vector that expresses siRNA in vivo.

  To introduce the siRNA vector into the cell, a transfection facilitating agent can be used. FuGENE6 (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are useful as transfection promoters. Accordingly, the pharmaceutical composition of the present invention may further comprise such a transfection facilitating agent.

IV-3. Pharmaceutical composition comprising antisense nucleic acid
Antisense nucleic acids corresponding to the nucleotide sequence of the RASGEF1A gene can be used to reduce the expression level of genes that are up-regulated in various cancer cells and are useful in the treatment of cancer, and thus are useful in the present invention. Is included. An antisense nucleic acid binds to the nucleotide sequence of the RASGEF1A gene or its corresponding mRNA, thereby inhibiting transcription or translation of that gene, promoting mRNA degradation, and / or expression of the protein encoded by this gene It works by inhibiting Thus, as a result, the antisense nucleic acid inhibits the RASGEF1A protein from functioning in cancer cells. As used herein, the phrase “antisense nucleic acid” refers to a nucleotide that specifically hybridizes to a target sequence, and one or more nucleotide mismatches, as well as nucleotides that are fully complementary to the target sequence. Also included are nucleotides containing For example, the antisense nucleic acid of the invention includes at least 70% or more, preferably at least 80% or more, more preferably over a range of at least 15 contiguous nucleotides of the RASGEF1A gene or its complementary sequence. Includes polynucleotides having a homology of at least 90% or more, even more preferably at least 95% or more. Such homology can be determined using algorithms known in the art.

  The antisense nucleic acid of the present invention is a protein encoded by the RASGEF1A gene by binding to the DNA or mRNA of the gene, inhibiting their transcription or translation, promoting mRNA degradation, and inhibiting protein expression. Acts on the cells that produce the protein, and eventually inhibits the protein from functioning.

  The antisense nucleic acid of the present invention can be formulated into an external preparation such as a liniment or bup agent by mixing with an appropriate base material that is inactive to the nucleic acid.

  If necessary, the antisense nucleic acid of the present invention can be added to excipients, isotonic agents, solubilizers, stabilizers, preservatives, analgesics, etc., to form tablets, powders, granules. , Capsules, liposome capsules, injections, solutions, nasal drops, and lyophilizates. Antisense encapsulants can also be used to increase durability and membrane permeability. Examples include, but are not limited to, liposomes, poly-L-lysine, lipids, cholesterol, lipofectin, or derivatives thereof. These can be prepared by the following known methods.

  The antisense nucleic acid of the present invention is useful for inhibiting the expression of RASGEF1A protein and suppressing the biological activity of this protein. Furthermore, the expression inhibitor containing the antisense nucleic acid of the present invention is useful in that it can inhibit the biological activity of the RASGEF1A protein.

  The antisense nucleic acid of the present invention includes a modified oligonucleotide. For example, thioated oligonucleotides can be used to confer nuclease resistance to oligonucleotides.

IV-4. Pharmaceutical Compositions Containing Antibodies The function of the gene product of the RASGEF1A gene that is overexpressed in various cancers is administered with a compound that binds to this gene product or otherwise inhibits its function Can be inhibited. Antibodies against the RASGEF1A polypeptide can be mentioned as such compounds and can be used as an active ingredient in a pharmaceutical composition for treating or preventing cancer.

  The present invention relates to the use of antibodies against proteins encoded by the RASGEF1A gene, or fragments of these antibodies. As used herein, the term `` antibody '' only interacts with the antigen used to synthesize the antibody (i.e., the gene product of the up-regulated marker) or an antigen closely related thereto ( That is, it means an immunoglobulin molecule having a specific structure that binds). Molecules containing the antigen used to synthesize the antibody and molecules containing the epitope of the antigen recognized by the antibody can be cited as closely related antigens.

Furthermore, the antibody used in the pharmaceutical composition of the present invention may be an antibody fragment or a modified antibody (eg, an immunologically active fragment of an anti-RASGEF1A antibody) as long as it binds to the protein encoded by the RASGEF1A gene. For example, antibody fragments can be Fab, F (ab ′) ₂ , Fv, or single chain Fv (scFv) in which Hv and L chain derived Fv fragments are linked by a suitable linker (Huston JS et al. Proc Natl Acad Sci USA 1988, 85: 5879-83). Such antibody fragments may be generated by treating the antibody with an enzyme such as papain or pepsin. Alternatively, a gene encoding the antibody fragment may be constructed, inserted into an expression vector, and expressed in a suitable host cell (eg, Co MS et al., J Immunol 1994, 152: 2968-76; Better M et al., Methods Enzymol 1989, 178: 476-96; Pluckthun A et al., Methods Enzymol 1989, 178: 497-515; Lamoyi E, Methods Enzymol 1986, 121: 652-63; Rousseaux J et al. , Methods Enzymol 1986, 121: 663-9; Bird RE et al., Trends Biotechnol 1991, 9: 132-7).

  An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG). The present invention includes such modified antibodies. Modified antibodies can be obtained by chemically modifying antibodies. Such modification methods are common in the art.

  Alternatively, the antibody used for the present invention is a chimeric antibody having a variable region derived from a non-human antibody and a constant region derived from a human antibody against the RASGEF1A polypeptide, or a complementarity determining region (CDR) derived from a non-human antibody, It may be a humanized antibody comprising a framework region (FR) derived from a human antibody and a constant region. Such antibodies can be prepared using known techniques. Humanization can be performed by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence (see, eg, Verhoeyen et al., Science 1988, 239: 1534-6). ). Accordingly, such humanized antibodies are chimeric antibodies in which substantially non-complete human variable domains are replaced by corresponding sequences from non-human species.

  Fully human antibodies that contain human variable regions in addition to the human framework and constant regions can also be used. Such antibodies can be generated using various techniques known in the art. For example, in vitro methods include the use of recombinant libraries of human antibody fragments displayed on bacteriophages (eg, Hoogenboom et al., J Mol Biol 1992, 227: 381). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, eg, mice in which endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, for example, in US Pat. Nos. 6,150,584, 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016.

  When the resulting antibody is administered to the human body (antibody therapy), human or humanized antibodies are preferred in order to reduce immunogenicity.

  The antibody obtained as described above may be purified to homogeneity. For example, antibody separation and purification can be performed according to separation and purification methods used for general proteins. For example, the antibody is not limited, but column chromatography such as affinity chromatography, filtration, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. are appropriately selected and used in combination. Can be separated and isolated (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)). Protein A columns and protein G columns can be used as affinity columns. Exemplary protein A columns used include, for example, Hyper D, POROS, and Sepharose F.F (Pharmacia).

  Exemplary chromatography, excluding affinity chromatography, includes, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). Chromatographic methods can be performed by liquid phase chromatography such as HPLC and FPLC.

V. Methods for Treating or Preventing Cancer Cancer treatments that target specific molecular changes that occur in cancer cells include trastuzumab (Herceptin) for the treatment of advanced cancer, and imatinib mesylate (Gleevec) for chronic myeloid leukemia ), Gefitinib (Iressa) for non-small cell lung cancer (NSCLC), and clinical development and regulatory approval of antitumor drugs such as rituximab (anti-CD20 mAb) for B-cell and mantle cell lymphoma (Ciardiello F et al., Clin Cancer Res 2001, 7: 2958-70, Review; Slamon DJ et al., N Engl J Med 2001, 344: 783-92; Rehwald U et al., Blood 2003, 101: 420-4 Fang G et al., Blood 2000, 96: 2246-53). These drugs are better tolerated than conventional anti-tumor agents because they are clinically effective and target only transformed cells. Thus, such drugs not only improve the survival and quality of life of cancer patients, but also demonstrate the concept of molecular cancer therapy. Furthermore, targeted drugs can increase their effectiveness when used in combination with standard chemotherapy (Gianni L, Oncology 2002, 63 Suppl 1: 47-56; Klejman A et al., Oncogene 2002, 21: 5868-76). Thus, future cancer treatments are likely to involve combining conventional drugs with target-specific agents that target various characteristics of tumor cells such as angiogenesis and invasiveness.

  These regulatory methods can be performed ex vivo or in vitro (e.g., by culturing cells with the agent) or alternatively, in vivo (by administering the agent to a subject). . These methods administer a protein or a combination of proteins, or a nucleic acid molecule or a combination of nucleic acid molecules as a treatment to counteract abnormal expression of differentially expressed genes or abnormal activity of their gene products. Including doing.

  Diseases and disorders characterized by increased expression levels or biological activity of genes and gene products, respectively (compared to subjects not afflicted with the disease or disorder) are those of overexpressed genes Can be treated with therapeutic agents that antagonize (ie, reduce or inhibit). A therapeutic agent that antagonizes activity can be administered therapeutically or prophylactically.

  Thus, therapeutic agents that can be used in the context of the present invention include, for example: (i) a polypeptide of the overexpressed RASGEF1A gene, or an analog, derivative, fragment or homologue thereof; (ii) an overexpressed gene Or an antibody against a gene product; (iii) a nucleic acid encoding an overexpressed gene; (iv) an antisense nucleic acid or a nucleic acid that is “dysfunctional” (ie, for heterologous insertion of an overexpressed gene into a nucleic acid) (V) a small interfering RNA (siRNA); or (vi) a modulator (ie, an inhibitor, an antagonist that alters the interaction of an overexpressed polypeptide with its binding partner). A dysfunctional antisense molecule is used to “knock out” the endogenous function of a polypeptide by homologous recombination (see, eg, Capecchi, Science 1989, 244: 1288-92).

  Increased levels are obtained by taking a patient tissue sample (e.g., from a biopsy tissue) and in vitro for RNA or peptide levels, structure and / or activity of expressed peptides (or mRNAs of genes with altered expression) Can be easily detected by quantifying peptides and / or RNA. Methods well known in the art include immunoassay methods (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and / or mRNA. Hybridization assays that detect the expression of (eg, Northern blot assay, dot blot, in situ hybridization, etc.), but are not limited thereto.

  Prophylactic administration is performed before overt clinical symptoms of the disease have developed so that the disease or disorder is prevented or its progression is delayed.

  The therapeutic methods of the invention can include contacting a cell with a substance that modulates one or more activities of the RASGEF1A gene product. Examples of substances that modulate protein activity include, but are not limited to, nucleic acids, proteins, natural cognate ligands of such proteins, peptides, peptidomimetics, and other small molecules.

  Accordingly, the present invention provides a method for treating or alleviating cancer symptoms or preventing cancer in a subject by reducing the expression of the RASGEF1A gene or the activity of its gene product. The method of the present invention is particularly suitable for treating or preventing ICC, bladder cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, chronic myelogenous leukemia, kidney cancer, soft tissue sarcoma, pancreatic cancer, and seminoma. ing.

  An appropriate therapeutic agent can be administered prophylactically or therapeutically to a subject suffering from or at risk of (or susceptible to) developing cancer. Such subjects are identified by using standard clinical methods or by detecting abnormal expression levels of the RASGEF1A gene ("upregulation" or "overexpression") or abnormal activity of its gene product. can do.

  According to one aspect of the present invention, a substance screened by the method of the present invention may be used for treating or preventing cancer. Administration of the substance to a patient using methods well known to those skilled in the art, for example, as intraarterial injection, intravenous injection, or intradermal injection, or as intranasal, transbronchial, intramuscular, or oral administration May be. If the substance can be encoded by DNA, the therapy can be performed by inserting the DNA into a vector for gene therapy and administering the vector to a patient.

  The dosage and method of administration will vary depending on the weight, age, sex, symptoms, condition and method of administration of the patient to be treated. However, one skilled in the art can routinely select appropriate dosages and methods of administration.

  For example, the dose of a substance that binds to and modulates the activity of the RASGEF1A polypeptide depends on various factors as described above, but the dose is generally administered orally to a normal human adult (body weight 60 kg) About 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day, and more preferably about 1.0 mg to about 20 mg per day.

  When the agent is administered parenterally in the form of injections into normal human adults (body weight 60 kg), there are some differences depending on the patient, target organ, symptoms, and method of administration, but from about 0.01 mg to about Conveniently, a dose of 30 mg, preferably about 0.1 mg to about 20 mg per day, and more preferably about 0.1 mg to about 10 mg per day is injected intravenously. For other animals, the appropriate dosage may be calculated as prescribed by converting to a body weight of 60 kg.

  Similarly, the pharmaceutical compositions of the invention can be used to treat or prevent cancer. Administration of the composition to a patient using methods well known to those skilled in the art, for example, as intraarterial injection, intravenous injection, or intradermal injection, or as intranasal, transbronchial, intramuscular, or oral administration May be.

  For each of the aforementioned conditions, the compositions, such as polypeptides and organic compounds, can be administered orally or by injection at a dose ranging from about 0.1 mg / kg to about 250 mg / kg per day. The dose range for human adults is generally about 5 mg / day to about 17.5 g / day, preferably about 5 mg / day to about 10 g / day, and most preferably about 100 mg / day to about 3 g / day. is there. A tablet, or other unit dosage form in a form provided in discrete units, may conveniently include an amount that is effective at such dosages, or as multiples thereof, eg, each unit is About 5 mg to about 500 mg, usually about 100 mg to about 500 mg.

  The dose used will depend on a number of factors, including the age, weight, and sex of the subject, the exact disease being treated and its severity. Similarly, the route of administration may vary depending on the condition and its severity. In any event, the appropriate dosage and optimal dosage may be calculated as prescribed by one of ordinary skill in the art, taking into account the factors discussed above.

  Specifically, an antisense nucleic acid against the RASGEF1A gene can be given to a patient by applying it directly to the affected area or by injecting it into a blood vessel so as to reach the disease site.

  The dosage of the antisense nucleic acid derivative of the present invention can be appropriately adjusted according to the patient's condition and used in a desired amount. For example, a dose range of 0.1 mg / kg to 100 mg / kg, preferably 0.1 mg / kg to 50 mg / kg can be administered.

VI. Vaccination against cancer The present invention relates to a polypeptide encoded by the RASGEF1A gene (ie a RAS polypeptide), an immunologically active fragment of said polypeptide, or a polynucleotide encoding such a polypeptide or a fragment thereof. It relates to a method for treating or preventing cancer in a subject comprising administering to the subject a vaccine comprising. In the present invention, a vaccine against cancer means a substance having the ability to induce anti-tumor immunity when inoculated into an animal.

  Administration of such a vaccine induces anti-tumor immunity in the subject. According to the present invention, the RASGEF1A polypeptide and immunologically active fragments thereof are capable of inducing a strong and specific immune response against cancer cells that express the RASGEF1A gene, and are HLA-A24 or HLA-A * 0201 restricted. It was suggested to be a sex epitope peptide. Accordingly, the present invention also provides a vaccine comprising a polypeptide encoded by the RASGEF1A gene (ie, a RAS polypeptide), an immunologically active fragment of the polypeptide, or a polynucleotide encoding such a polypeptide or fragment thereof. It also relates to a method for inducing it in a subject in need of anti-tumor immunity by administering to the subject.

  These methods are particularly suitable for treating or preventing ICC, bladder cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, chronic myelogenous leukemia, kidney cancer, soft tissue sarcoma, pancreatic cancer, and seminoma. Yes.

  In some cases, the RASGEF1A protein or immunologically active fragment thereof is presented by a T cell receptor (TCR) bound form or by an APC such as a macrophage, dendritic cell (DC), B cell, or PBMC. Can be administered in different forms. The use of DC is most preferred among APCs because of its strong antigen presentation ability.

In general, anti-tumor immunity includes the following immune responses:
-Induction of cytotoxic lymphocytes against the tumor,
-Induction of antibodies that recognize tumors, and
-Induction of anti-tumor cytokine production.

  Thus, if a certain protein induces any one of these immune responses when inoculated into an animal, it is determined that the protein has the effect of inducing anti-tumor immunity. Any protein fragment of the RASGEF1A polypeptide can be used as an immunologically active fragment of the methods of the invention.

  Induction of anti-tumor immunity by a protein can be detected in vivo or in vitro by observing the immune system response in the host against the protein.

  For example, a method for detecting the induction of CTL is well known. Specifically, a foreign substance that enters the living body is presented to T cells and B cells by the action of APC. T cells that respond in an antigen-specific manner to the antigen presented by APC differentiate into CTLs upon stimulation with the antigen and then proliferate (this is called T cell activation). Therefore, CTL induction by a certain peptide can be evaluated by presenting the peptide to T cells via APC and detecting the induction of CTL. Furthermore, APC has the effect of activating CD4 + T cells, CD8 + T cells, macrophages, eosinophils, and NK cells. Since CD4 + T cells and CD8 + T cells are also important in antitumor immunity, the antitumor immunity-inducing action of peptides can be evaluated using the activation effect of these cells as an index.

Methods for evaluating the inducing action of CTL using DC as APC are well known in the art. DC is a representative APC having the strongest CTL inducing action among APCs. To determine whether a fragment of a RASGEF1A polypeptide is immunologically active and can be used in the methods of the invention, a test polypeptide (i.e., any fragment of the RASGEF1A polypeptide) is selected. First contact with DC, then DC with T cells. If a T cell is detected that has a cytotoxic effect on the cell of interest after contact with the DC, it indicates that the test polypeptide has activity to induce cytotoxic T cells. The activity of CTL against a tumor can be detected using, for example, lysis of tumor cells labeled with ⁵¹ Cr as an indicator. Alternatively, a method for evaluating the degree of tumor cell damage using ³ H-thymidine uptake activity or LDH (lactose dehydrogenase) release as an index is also well known.

  Other than DC, PBMC may be used as APC. Induction of CTL has been reported to be enhanced by culturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL was also shown to be induced by culturing PBMC in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

  A test polypeptide that is confirmed to have CTL-inducing activity by these methods is considered to be a polypeptide having a DC activation effect and subsequent CTL-inducing activity. Therefore, polypeptides that induce CTL against tumor cells are useful as vaccines against tumors. Furthermore, APCs that have acquired the ability to induce CTLs against tumors through contact with the polypeptides are also useful as vaccines against tumors. Furthermore, CTLs that have acquired cytotoxicity due to presentation of polypeptide antigens by APC can also be used as vaccines against tumors. Such a method of treating tumors using anti-tumor immunity resulting from APC and CTL is called cellular immunotherapy.

  In general, when using polypeptides for cellular immunotherapy, it is known that the efficiency of CTL induction is increased by combining multiple polypeptides having different structures and contacting them with DC. Therefore, when stimulating DC with protein fragments, it is advantageous to use a mixture of multiple types of fragments.

  Alternatively, the induction of anti-tumor immunity by a polypeptide can be confirmed by observing the induction of antibody production against tumors. For example, if an antibody against a polypeptide is induced in a laboratory animal immunized with that polypeptide, and if growth of tumor cells is suppressed by these antibodies, the polypeptide induces anti-tumor immunity Is considered to have the ability to

  Anti-tumor immunity is induced by administering the vaccine of the present invention, and induction of anti-tumor immunity allows the treatment and prevention of cancer. Therapy for cancer or prevention of cancer development includes any of the following stages, for example, inhibition of cancer cell proliferation, cancer regression, and suppression of cancer pathogenesis. Decreased mortality and morbidity in individuals with cancer, reduced levels of tumor markers in blood, alleviation of 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, in observation, the therapeutic or prophylactic effect of a vaccine against cell proliferative disease is at a significant level of 5% or less when compared to a control without vaccine administration. For example, Student's t test, Mann-Whitney U test, or ANOVA may be used for statistical analysis.

  The aforementioned RASGEF1A polypeptide or a fragment thereof having immunological activity, or a vector encoding them may be combined with an adjuvant. By adjuvant is meant a compound that enhances the immune response to the RASGEF1A polypeptide when administered together (or sequentially) with this polypeptide or fragment having immunological activity. Exemplary adjuvants include, but are not limited to, cholera toxin, salmonella toxin, alum and the like. Furthermore, the vaccine of the present invention may be appropriately combined with a pharmaceutically acceptable carrier. Examples of such carriers include sterilized water, physiological saline, phosphate buffer, culture solution, and the like. Furthermore, the vaccine may contain a stabilizer, a suspending agent, a preservative, a surfactant and the like as required. The vaccine can be administered systemically or locally. Vaccine administration may be performed by a single dose or boosted by multiple doses.

  When using APC or CTL as the vaccine of the present invention, the tumor can be treated or prevented by, for example, an ex vivo method. More specifically, PBMCs of the subject to be treated or prevented are collected, the cells are contacted with the RASGEF1A polypeptide or a fragment thereof ex vivo, APC or CTL is induced, and then the cells are administered to the subject. Also good. APC can also be induced by introducing a vector encoding the polypeptide into PBMC ex vivo. In vitro derived APCs or CTLs can be administered after cloning. Cellular immunotherapy can be more effectively performed by cloning and proliferating cells with high activity that damage target cells. Furthermore, APCs and CTLs isolated in this manner can be used not only for individuals from which those cells are derived, but also for cellular immunotherapy against similar types of tumors from other individuals .

  Further provided is a pharmaceutical composition for treating or preventing a cell proliferative disease, such as cancer, comprising a pharmaceutically effective amount of the RASGEF1A polypeptide or immunologically active fragment thereof. Such pharmaceutical compositions can be used to increase anti-tumor immunity.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the following materials, methods, and examples are merely illustrative of aspects of the invention and are in no way intended to limit the scope of the invention. Accordingly, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

Example
I. Materials and methods
1. Cell lines and tissue specimens The monkey kidney cell line COS7 and the mouse fibroblast cell line NIH3T3 were obtained from the American Type Culture Collection (ATCC). Human cholangiocarcinoma cell line SSP25 was obtained from Japanese Collection of Research Bioresources (JCRB). All cells were cultured as monolayers in appropriate media as follows: Dulbecco's Modified Eagle Medium (Sigma-Aldrich Corporation, St. Louis, MO) for COS7 and NIH3T3; and RPMI1640 for SSP25 (Sigma-Aldrich Corporation, St. Louis, MO). Clinical tissue was obtained from surgical specimens of patients who received informed consent and underwent hepatectomy.

2. RNA preparation and semi-quantitative RT-PCR
The inventors collected pure populations of cholangiocarcinoma cells as well as non-cancerous bile duct epithelium from surgical specimens using laser microbeam microdissection techniques. Section preparation, laser microbeam microdissection, total RNA extraction, T7-based amplification were performed as previously described (Nakamura T et al., Oncogene 2004, 23: 2385-400). A mixture of normal intrahepatic bile duct epithelial cells in liver tissue from 10 patients without ICC was prepared as a universal control. Total RNA was extracted from cultured cells using TRIZOL reagent (Invitrogen) according to the manufacturer's protocol. The extracted RNA was reverse transcribed into single stranded cDNA using poly dT _12-18 primer (Amersham Biosciences) with Superscript II reverse transcriptase (Life Technologies). Each single stranded cDNA was diluted for subsequent PCR amplification. Standard RT-PCR is performed in GeneAmp PCR system 9700 (Perkin-Elmer) in a volume of 12 μl PCR buffer (TAKARA) for 4 min at 94 ° C for denaturation followed by 20 sec at 94 ° C. Was amplified at 25 cycles (for ACTB) or 30 cycles (for RASGEF1A), 53 ° C (for ACTB) or 60 ° C (for RASGEF1A) for 30 seconds and 72 ° C for 30 seconds. The primer sequences were as follows:
For ACTB, 5′-CAAGATGAGATTGGCATGG-3 ′ (SEQ ID NO: 3) and 5′-TCTCCTTAGAGAGAAGTGG-3 ′ (SEQ ID NO: 4); and
For RASGEF1A, 5′-TTTGCACTTTCTGAGGTTCTAGC-3 ′ (SEQ ID NO: 5) and 5′-GTCCTGCATTATGGTACAGCTTC-3 ′ (SEQ ID NO: 6).

3. Northern Blot Analysis Human multi-tissue Northern blot (Clontech) was hybridized with ³² P-labeled RASGEF1A cDNA. Prehybridization, hybridization, and washing were performed according to the supplier's recommendations. Using an intensifying screen, the autoradiograph of the blot was photographed at -80 ° C for 120 hours.

4. Intracellular localization of RASGEF1A
The entire coding region of RASGEF1A (GenBank accession number AK095136 (SEQ ID NO: 1), encoding the amino acid sequence of SEQ ID NO: 2) is used as a primer set, 5'-ATTGAATTCCGGCCAGAATGTTCCTGGAGC-3 '(SEQ ID NO: 7 ) And 5′-AATCTCGAGGGCTCTGTTCAGAAGGGTGGTC-3 ′ (SEQ ID NO: 8) and cloned into the assigned enzyme site of the pCAGGS-n3Fc vector (pCAGGS-n3Fc-RASGEF1A). NIH3T3 cells transiently transfected with a plasmid expressing RASGEF1A tagged with FLAG were transferred onto a chamber slide. Twenty-four hours after transfection, cultured cells were fixed with PBS containing 4% paraformaldehyde for 15 minutes and then permeabilized with PBS containing 0.1% Triton X-100 for 2.5 minutes at room temperature. These cells were covered with 3% BSA in PBS for 24 hours at 4 ° C. to block non-specific hybridization and subsequently incubated with anti-FLAG antibody (SIGMA F-3165) as the primary antibody. The antibody was fluorescently stained with anti-mouse IgG secondary antibody conjugated with substrate (ICN / Cappel and Jackson Immuno Research). The nuclei were counterstained with 4 ', 6-diamidino-2-phenylindole dihydrochloride (DAPI). Fluorescence images were obtained using a TCS-SP2 spectral confocal scanning system (Leica).

5. Colony formation assay
COS7 cells were transfected with a plasmid expressing RASGEF1A (pCAGGS-n3Fc-RASGEF1A). Cells expressing antisense RASGEF1A plasmid (pCAGGS-n3Fc-RASGEF1A (−)) or mock plasmid (pCAGGS-n3Fc-mock) were incubated for 14 days with the appropriate concentration of geneticin. These cells were fixed with 100% methanol and stained with Giemsa solution. Cell viability was analyzed in triplicate using a cell counting kit (DOJINDO, kumamoto, Japan) according to the manufacturer's protocol.

6. Construction of siRNA expression vector for RASGEF1A Cloning double-stranded oligonucleotide into BbsI site of psiH1BX3.0 vector as previously described (Shimokawa T et al., Cancer Res 2003, 63: 6116-20) Thus, a plasmid expressing siRNA was prepared. The sequence of the paired oligonucleotides was as follows:
For siRNA-B, 5'-TCCCGAGAATGGCACAGTGAAGATTCAAGAGATCTTCACTGTGCCATTCTC-3 '(SEQ ID NO: 10) and 5'-AAAAGAGAATGGCACAGTGAAGATCTCTTGAATCTTCACTGTGCCATTCTC-3' (SEQ ID NO: 11);
for siRNA-E, 5'-TCCCCATCTACTTCCTGCACAAATTCAAGAGATTTGTGCAGGAAGTAGATG-3 '(SEQ ID NO: 14) and 5'-AAAACATCTACTTCCTGCACAAATCTCTTGAATTTGTGCAGGAAGTAGATG-3' (SEQ ID NO: 15);
For siRNA-EGFP, 5'-TCCCGAAGCAGCACGACTTCTTCTTCAAGAGAGAAGAAGTCGTGCTGCTTC-3 '(SEQ ID NO: 18) and 5'-AAAAGAAGCAGCACGACTTCTTCTCTCTTGAAGAAGAAGTCGTGCTGCTTC-3' (SEQ ID NO: 19). After phosphorylation with T4-polynucleotide kinase, the paired oligonucleotides were boiled for 5 minutes and then annealed by slow cooling to create double stranded oligonucleotides. The expression of RASGEF1A was examined by semi-quantitative RT-PCR 24 hours after transfection. The siRNA sequences used for the present invention are shown below.

7. Cell viability assay
SSP25 cells (1 × 10 ⁶ cells / dish) seeded in 10 cm dishes were transfected with plasmids expressing siRNA using either Nucleofector (AMAXA) or FuGENE6 reagent (Roche Diagnostics). Transfected cells were maintained for 2 weeks in medium containing 10% fetal calf serum supplemented with 0.9 mg / ml geneticin. Viable cells were fixed with 100% methanol and stained with Giemsa solution. In another experiment, these live cells were measured using a cell counting kit (DOJINDO, Kumamoto, Japan). To analyze the effect on RASGEF1A expression, total RNA was extracted from cells 24 hours after transfection and subjected to semi-quantitative RT-PCR analysis.

8. Ras nucleotide dissociation assay
The entire coding region of the catalytic domain of K-Ras (amino acids 1-188), Ha-Ras (amino acids 1-188), N-RAS (amino acids 1-189), RASGEF1A, or Sos (amino acids 564-1049) is expressed in pGEX It was cloned into the appropriate cloning sites (BamHI and XhoI) of the -6P bacterial expression vector (Amersham Pharmacia Biotech). The GST fusion protein was expressed in the BL21 bacterial strain of Escherichia coli and the recombinant protein was purified from the whole cell lysate using glutathione-Sepharose 4B resin (Amersham Pharmacia Biotech) according to the manufacturer's protocol. The Ras nucleotide dissociation assay was performed as described elsewhere (Hall BE et al., J Biol Chem 2001, 276: 27629-37). GST-Ras fusion protein was dissolved in 100 ml buffer containing 20 mM Tris (pH 8.0), 50 mM NaCl, 1 mg / ml bovine serum albumin (BSA), 1 mM dithiothreitol, and 1 mM EDTA. the 40 pmol of Ras, the 400 pmol [8- ³ H] labeled GDP; with (12.4 Ci / mmol Amersham Biosciences) , in a constant temperature shaking water tank, by incubating 15 minutes at 30 ℃, [8- ³ H] Conjugated with labeled guanosine 5′-diphosphate (GDP). Recombinant 20 ml of this mixture in 20 ml buffer containing 20 mM Tris (pH 8.0), 50 mM NaCl, 1 mg / ml BSA, 1 mM dithiothreitol, 5 mM MgCl ₂ , and 0.8 mM GTPgS. Further incubation for 15 minutes at 30 ° C. with or without RASGEF1A. The catalytic domain of Sos served as a positive control. The reaction was terminated by adding 40 ml of ice-cold stop solution (20 mM Tris (pH 8.0), 50 mM NaCl, and 15 mM MgCl ₂ ), and the mixture was washed with square P81 phosphocellulose paper (Upstate Spotted on catalog number 20-134). After washing twice with 3 ml of ice-cold stop solution, the filter was placed on an Aloka LSC-5100 liquid scintillation counter, and the radioactivity of [ ³ H] -GDP bound to Ras was measured.

9. Analysis of Ras activity
Ras activation was measured by Raf-1 pulldown assay using Ras activation assay kit (Upstate, catalog number 17-218, lot number 26220) according to the supplier's protocol. Specifically, agarose bead-bound GST fusion protein containing Raf-1 RBD was prepared and incubated at 4 ° C for 45 minutes with lysates from COS7 cells transfected with a plasmid expressing RASGEF1A or a control plasmid (mock) did. Proteins bound to Raf-1 were precipitated, separated by SDS-PAGE, and subsequently analyzed by Western blotting using an anti-pan-Ras antibody (Upstate).

10. Wound healing and Matrigel invasion assay
Using the entire coding region of RASGEF1A (GenBank accession number; AK095136), primer set, 5'-ATTGAATTCCGGCCAGAATGTTCCTGGAGC-3 '(SEQ ID NO: 7) and 5'-AATCTCGAGGGCTCTGTTCAGAAGGGTGGTC-3' (SEQ ID NO; 8) Were amplified by RT-PCR and cloned into the appropriate enzyme sites of the pCAGGS-n3Fc vector (pCAGGS-n3Fc-RASGEF1A). COS7 cells expressing RASGEF1A (COS7-RASGEF1A) and control cells (COS7-sham) were established by transfecting COS7 cells with pCAGGS-n3Fc-RASGEF1A plasmid or pCAGGS-pseudoplasmid. These cells were maintained in medium supplemented with 0.9 mg / ml geneticin and single colonies were selected 2 weeks after transfection. The expression of RASGEF1A was confirmed by Western blotting using an anti-FLAG antibody. Cell proliferation was analyzed in triplicate using a cell counting kit (DOJINDO, Kumamoto, Japan) according to the manufacturer's protocol. For the wound healing migration assay, cells were grown to confluence in 6-well plates for 2 days and the confluent monolayer was scratched into a cruciform shape with the tip of a plastic pipette. Several injured areas were marked and marked for orientation and then photographed with a phase-contact microscope a specified number of times after scratching. Cell invasion was examined using a BD BioCoat ™ Matrigel ™ infiltration chamber (BD Biosciences, San Jose, Calif.) With a pore size of 8 μm. After 36 hours incubation, the number of COS7-RASGEF1A cells and COS7-pseudocells that migrated and passed through the chamber were counted in five independent fields under the microscope. Statistical analysis. Statistical significance was determined by Student's t test.

II. Results
1. Increased RASGEF1A expression in a wide range of human cancers
Using a genome-wide cDNA microarray containing 27,648 genes, we analyzed the expression profile of 25 ICCs, and expression was often up-regulated in tumors that passed a set cutoff filter. 52 genes have been identified. In addition to 52 genes, the gene of institutional accession number D4223 corresponding to the EST of Hs.125293 in the Unigene database (creation number 183) of the National Center for Biotechnology Information is compared with normal intrahepatic bile duct epithelium. Of the 14 ICCs that passed through the cut-off filter, 11 cases (78.6%) were found to be significantly up-regulated more than 2-fold. In the microarray data obtained by the inventors, D4223 expression was also observed in 8 out of 9 cases (88.9%) of bladder cancer, 16 out of 26 cases of lung cancer (61.5%), and 4 out of 5 cases of gastric cancer (80%). 3 out of 5 colorectal cancers (60%), 1 out of 3 prostate cancers (33.3%), 8 out of 44 cases of chronic myeloid leukemia (18.2%), all 5 cases of renal cancer, soft tissue sarcoma 5 All cases (100%), all three pancreatic cancers (100%), and all three seminoma (100%) increased by more than 2-fold compared to the corresponding non-cancerous controls. Subsequent semi-quantitative RT-PCR analysis revealed that D4223 expression was increased in 9 out of 13 ICCs subjected to microarray analysis (FIG. 1A). In order to assess the expression level of D4223 in normal human adult tissues, we performed multi-tissue northern blot analysis using D4223 cDNA as a probe, expressed moderately in the brain, spinal cord, and lymph nodes An approximately 3.3 kb transcript was identified that was weakly expressed in the adrenal gland but not expressed in any of the 19 other tissues examined (FIG. 1B). This gene was recently named RASGEF1A because it contains the RAS-GEF domain. Homology search with the estimated RASGEF1A amino acid sequence showed 30% match with PDZ-GEF2 and 29% match with PDZ-GEF1.

2. Intracellular localization of RASGEF1A
To investigate the subcellular localization of RASGEF1A, we expressed RASGEF1A tagged with FLAG in NIH3T3 cells by transfection of a plasmid (p3Xflag-RASGEF1A) (Figure 2B), and immunocytochemical Staining was performed. Immunocytochemical staining revealed that RASGEF1A protein was localized in the cytoplasm of the transfected cells (FIG. 2A).

3. Association between RASGEF1A and ICC cell proliferation A colony formation assay was performed to examine the carcinogenic activity of RASGEF1A in COS7 cells. As a result, when these cells were transfected with a plasmid expressing RASGEF1A (pCAGGS-n3Fc-RASGEF1A), compared to transfection with a control plasmid, pCAGGS-n3Fc-mock, or pCAGGS-n3Fc-RASGEF1A (-) A significant increase in the number of colonies was observed (FIGS. 3A and 3B). To further evaluate the potential role in cell proliferation, plasmid psiH1BX-RASGEF1A-siB and plasmid psiH1BX-RASGEF1A-siE were prepared that express siRNA specific for RASGEF1A with a neomycin resistance gene. Either of these plasmids or a control plasmid (psiH1BX-siEGFP, psiH1BX-mock) was transfected into SSP25 ICC cells. Semi-quantitative RT-PCR showed that RASGEF1A expression was suppressed by psiRNA-BX-RASGEF1A-siB and psiH1BX-RASGEF1A-siE transfections compared to transfection of control plasmids (Figure 3C). ). In particular, the significant growth delay of the transfected cells was inhibited by siRNA-B and siRNA-E compared to cells transfected with the control plasmid (FIG. 3D). These data suggest that the expression of RASGEF1A is very important for ICC cell proliferation and / or survival.

4. Ras nucleotide dissociation activity of RASGEF1A
Since RASEGF1A contained a putative RasGEF domain, its GDP dissociation activity was examined. Either the reaction mixture with or without the recombinant RASGEF1A protein was subsequently added and the GDP-bound RAS was measured with a scintillation counter. SOS, a known RASGEF protein, was used as a positive control. SOS reduced GDP-bound K-RAS by approximately 40%. Similarly, RASGEF1A reduced GDP-bound K-RAS by approximately 50% (FIG. 4A, upper left panel). GDP bound to H-RAS and N-RAS also dissociated about 40% and about 80%, respectively (FIG. 4A, upper right panel and lower panel). These data suggest that RASEGF1A exhibits GDP dissociation activity against all three RAS proteins studied.

5. Activation of RAS by RASGEF1A
Since RASGEF1A contained a putative RASGEF domain, it was further investigated whether RASGEF1A had activity to stimulate RAS. This activity was measured using COS7 cells expressing exogenous RASGEF1A and control cells. Extracts from cells transfected with the pxFLAG-RASGEF1A plasmid were analyzed by immunoblot using anti-FLAG antibody, confirming exogenous RASGEF1A expression in the transfected cells (FIG. 4B, upper left panel). Extracts from these cells or control cells were subjected to a pull-down assay of recombinant Raf-1, an effector of RAS. As a result, the amount of Ras that interacted with Raf-1 RBD as measured by immunoblot analysis with anti-pan-Ras antibody increased with RAGEEF1A expression (Figure 4B, upper right panel) and RASGEF1A caused RAS It was suggested that the activity of is enhanced.

6. Effects of RASGEF1A on cancer cell migration
RAS family proteins have been reported to play important roles in cell migration as well as cell proliferation. Therefore, we investigated the involvement of RASGEF1A in cancer cell migration and invasion. COS7-RASGEF1A cells expressing exogenous RASGEF1A and control cells (COS7-sham) were established for wound healing assays. As shown in FIG. 5A, COS7-RASGEF1A cells migrated rapidly and closed the wound significantly faster than COS7-pseudocells, suggesting that RASGEF1A is associated with cell migration. Furthermore, in order to investigate the effect of RASGEF1A on infiltration, a transwell assay using Matrigel was performed. Consistent with the results of the wound healing assay, the number of COS7-RASGEF1A cells that passed through the chamber pores increased compared to the number of COS7-pseudocells (FIG. 5B). Since both cells had similar growth rates, these data suggest the involvement of RASGEF1A in cell migration.

III. Discussion of Results We have demonstrated herein increased expression of RASGEF1A in human ICC and its guanine nucleotide exchange activity against K-RAS, H-RAS, and N-RAS, which is expressed Implying that this increase plays a very important role in ICC carcinogenesis. To date, approximately 20 members belonging to the Ras-GEF family sharing a core catalytic domain with five structurally conserved regions have been reported (Boguski MS and McCornick F, Nature 1993, 366: 643). -54; Rebhun JF et al., J Biol Chem 2000, 275: 13406-10). Like other Ras-GEF members, RASGEF1A contained these five conserved regions. Furthermore, it had a REM (Ras exchange motif) or RasGEFN domain between codon 49 and codon 178. This is expected to stabilize large helical hairpin structures that pry up the GTP binding pocket. However, RASGEF1A lacks any additional conserved domains such as the Dbl homology (DH) domain, the pleckstrin homology (PH) domain, the EF hand, the cysteine-rich C1 domain, and the PDZ domain, so SOS, GRF1, RasGRP, or It may function differently from other members such as PDZ-GEF (Quilliam LA et al., Progress in Nucleic Acid Research and Molecular Biology 2002, 71: 391-444). In the present invention, it was revealed that RASGEF1A has guanine-nucleotide exchange activity for K-RAS, H-RAS, and N-RAS. Other Ras-GEF members have a different range of substrates; for example, SOS1 regulates K-RAS, H-RAS, N-RAS, R-RAS2, R-RAS3, and Rac1, while R- Ras-GRP2 stimulates K-RAS, H-RAS, N-RAS, R-RAS, R-RAS2, Rap1A, Rap2A but not H-RAS (Mitin N et al. , Current Biology 2005, 15: R563-74). Therefore, RASGEF1A may specifically activate a substrate that has not been studied in this study, and may be involved in crosstalk in the Ras signaling pathway.

  Recent studies have shown that Ras signaling is involved not only in cell proliferation and survival, but also in cell motility and cell adhesion. Active Ras interacts with multiple downstream effectors that stimulate the Raf / MEK / ERK pathway and diverse signaling pathways including the phosphoinositide 3 kinase (PI3K) / Akt, RalGDS / Ral, and Tiam1 / Rac1 pathways Act and regulate them. There is increasing evidence suggesting the involvement of Raf / MEK / ERK in Ras-mediated cell proliferation. Raf binding assays demonstrate an increase in activated RAS in the presence of RASGEF1A, and therefore downstream Raf / MEK / ERK is expected to be enhanced in RASGEF1A expressing cells. Consistently, suppression of RASGEF1A by the introduction of specific siRNA reduced ICC cell proliferation, consistent with the view that enhanced RAF / MEK / ERK signaling has a proliferative effect on cancer cells . In the colony formation assay using COS7 cells and NIH3T3 fibroblasts to test the oncogenic activity of RASGEF1A, the number of colonies was not different between cells expressing exogenous RASGEF1A and controls. Therefore, it is considered that introduction of RASGEF1A alone may not be sufficient to cause transformation of COS7 cells or NIH3T3 fibroblasts. However, the oncogenic activity of RASGEF1A was suggested to be tissue and / or cell type dependent, and siRNA experiments indicated that RASGEF1A is essential to maintain ICC cell growth or proliferation . Thus, inhibition of its guanine nucleotide exchange activity may be a promising therapeutic option for ICC. In addition to its growth promoting effect, Ras has been reported to play a role in cell motility by various mechanisms. Enhanced PI3 kinase activity increased breast cancer cell invasion mediated through β4 integrin in a Rac1-dependent manner. PI3K also activates RacGEF and induces actin remodeling and membrane roughing associated with cell motility. In addition, Ras potentiates Tiam1, resulting in an active form of Rac1 that is involved in migration and the actin cytoskeleton. In fibroblasts, H-RAS activation suppresses the activity of integrin, a cell surface receptor that plays an important role in cell-extracellular matrix adhesion (Paul EH et al., Mol Biol Cell 2002, 13: 2256-65; Sechler JL et al., J Cell Sci 2000, 113: 1491-8). The present invention discloses that RASGEF1A activates RAS protein and that exogenous expression of RASGEF1A improves cell motility in Matrigel assay. Although further investigation is necessary to clarify the mechanism by which RASGEF1A enhances motility, it was suggested that RASGEF1A improves cell motility through activation of PI3K or Tiam1.

  We further investigated the correlation between K-Ras mutation status and RASGEF1A expression level, but we did not find any relationship between them (data not shown). Increased expression of RASGEF1A activates H-Ras and N-Ras in addition to K-Ras, so accumulation of RASGEF1A may have an additional role in ICC carcinogenesis compared to mutations in K-Ras There is. Since suppression of RASGEF1A by siRNA results in inhibition of cancer cells, RASGEF1A may be a promising therapeutic target for ICC.

INDUSTRIAL APPLICABILITY Cancer gene expression analysis described herein using a combination of laser capture dissection and genome-wide cDNA microarrays identified specific genes as targets for cancer prevention and treatment. It was. Based on the expression of a subset of these differentially expressed genes, the present invention provides molecular diagnostic markers for identifying and detecting cancer.

  The methods described herein are also useful for identifying additional molecular targets for cancer prevention, diagnosis, and treatment. The data provided herein aids a comprehensive understanding of cancer, facilitates the development of new diagnostic strategies, and provides clues for identifying molecular targets for therapeutic and prophylactic agents . Such information contributes to a better understanding of tumorigenesis and provides an indicator for developing new strategies for diagnosing, treating, and ultimately preventing cancer.

  All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

  Further, while the invention has been described in detail with reference to specific embodiments thereof, it is understood that the foregoing description is illustrative and explanatory in nature and is intended to illustrate the invention and preferred embodiments thereof. Should. Those skilled in the art will readily recognize that routine experimentation may make various changes and modifications therein without departing from the spirit and scope of the invention. Accordingly, the invention is intended to be defined not by the above description, but by the following claims and their equivalents.

FIG. 1A shows the expression of the D4223 (RASGEF1A) gene in 13 ICC tissues, a mixture of non-cancerous bile duct cells, and 5 normal tissues. Semi-quantitative RT-PCR was performed using a set of primers specific for the RASGEF1A gene. β-actin (ACTB) gene expression served as a quantitative control. FIG. 1B shows a multi-tissue northern blot analysis of the D4223 (RASGEF1A) gene. The intracellular localization of RASGEF1A is shown. FIG. 2A shows the results of immunocytochemical fluorescence staining of FLAG-tagged RASGEF1A using NIH3T3 cells transfected with a plasmid expressing RASGEF1A tagged with FLAG. Proteins tagged with FLAG were stained with anti-FLAG mouse antibody and subsequently visualized using a goat anti-mouse secondary antibody conjugated to FITC (upper right panel). DAPI was used to counterstain nuclei (upper left panel). The fused image is shown in the lower left panel. In FIG. 2B, expression of Flag-tagged proteins in these cells was analyzed by Western blot analysis. FIG. 3A shows a colony formation assay of COS7 cells transfected with a plasmid expressing RASGEF1A. Cells transfected with pCAGGS-n3Fc-RASGEF1A, pCAGGS-n3Fc-RASGEF1A (−), or pCAGGS-n3Fc-sham were stained with Giemsa solution. FIG. 3B shows cell viability as measured by MTT assay using cell counting kit. FIG. 3C shows the expression of the RASGEF1A gene in cells transfected with plasmids expressing siRNA (si-B and si-E). A plasmid expressing EGFP (siEGFP) and a mock plasmid (mock) were used as controls. Semiquantitative RT-PCR was performed to examine the gene silencing effect of siRNA. ACTB gene expression served as a quantitative control. FIG. 3D shows the growth inhibitory effect of siRNA designated to inhibit RASGEF1A. Viability of SSP25 cells in response to siRNA was measured using a cell counting kit. FIG. 4A shows the results of nucleotide dissociation assays for K-Ras (upper left panel), Ha-Ras (upper right panel), and N-RAS (lower panel). [ ³ H] -GDP interacted with Ras after treatment with recombinant GST (white bars), SOS (hatched bars), or RASGEF1A (black bars) was obtained without such treatment Relative amount compared to the result (shaded bar). FIG. 4B shows the RAS activation activity of RASGEF1A. Extracts from cells transfected with pCAGGS-n3Fc-RASGEF1A or cells transfected with mock plasmids were analyzed by GST-RafRBD pull-down assay. The expression of RASGEF1A in these cells was examined by immunoblot analysis using an anti-FLAG antibody (left panel). Active GTP-bound Ras was precipitated using recombinant Raf-1 protein and analyzed by Western blot analysis using anti-pan-Ras antibody (right panel). FIG. 5A shows the results of a wound healing assay using COS7 cells transfected with RASGEF1A or mock vector. Representative images of migrating cells were taken 12 hours and 24 hours after scratching. FIG. 5B shows the results of a Matrigel invasion assay using the same cells. The average number of cells migrating through Matrigel was determined by counting in 5 different fields. Similar results were obtained when the experiment was repeated.

Claims (28)

  A method for diagnosing cancer or a predisposition to develop cancer in a subject, comprising determining the expression level of a RASGEF1A gene in a biological sample derived from the subject, the expression relative to a normal control level of the gene A method wherein the level is elevated suggests that the subject is suffering from or at risk of developing cancer.   2. The method of claim 1, wherein the expression level is at least 10% higher than the normal control level. 2. The method of claim 1, wherein the expression level is determined by any of the methods selected from the group consisting of:
(a) detecting mRNA of RASGEF1A gene;
(b) detecting a protein encoded by the RASGEF1A gene; and
(c) detecting the biological activity of the protein encoded by the RASGEF1A gene.   2. The method of claim 1, wherein the biological sample from the subject comprises epithelial cells.   2. The method of claim 1, wherein the biological sample derived from the subject comprises cancer cells.   2. The method of claim 1, wherein the biological sample derived from the subject comprises cancer epithelial cells.   2. The cancer is selected from the group consisting of ICC, bladder cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, chronic myelogenous leukemia, kidney cancer, soft tissue sarcoma, pancreatic cancer, and seminoma. the method of.   A kit containing a detection reagent that binds to the transcription product or translation product of the RASGEF1A gene. A method of screening for a substance for treating or preventing cancer comprising the following steps:
(a) contacting a test substance with a RASGEF1A polypeptide or fragment thereof;
(b) detecting binding of the polypeptide to the test substance; and
(c) selecting a test substance that binds to the polypeptide. A method of screening for a substance for treating or preventing cancer comprising the following steps:
(a) contacting a test substance with a RASGEF1A polypeptide or fragment thereof;
(b) detecting the biological activity of the polypeptide; and
(c) selecting a test substance that inhibits the biological activity of the polypeptide as compared to the biological activity detected in the absence of the test substance. A method of screening for a substance for treating or preventing cancer comprising the following steps:
(a) contacting RASGEF1A polypeptide or a fragment thereof with RAS in the presence of a test substance;
(b) detecting RAS activity; and
(c) selecting a test substance that reduces the RAS activity as compared to the activity detected in the absence of the test substance. A method of screening for a substance for treating or preventing cancer comprising the following steps:
(a) contacting the test substance with a cell expressing the RASGEF1A gene;
(b) detecting the expression level of the RASGEF1A gene; and
(c) selecting a test substance that reduces the expression level of the gene compared to the level detected in the absence of the test substance.   13. The method of claim 12, wherein the cell is derived from ICC, bladder cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, chronic myelogenous leukemia, kidney cancer, soft tissue sarcoma, pancreatic cancer, or seminoma. A method of screening for a substance for treating or preventing cancer comprising the following steps:
(a) contacting a test substance with a cell into which a vector containing a transcriptional regulatory region of the RASGEF1A gene and a reporter gene expressed under the control of the transcriptional regulatory region is introduced;
(b) measuring the expression or activity of the reporter gene; and
(c) selecting a test substance that reduces the expression or activity of the reporter gene compared to the expression or activity detected in the absence of the test substance.   The cancer is selected from the group consisting of ICC, bladder cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, chronic myelogenous leukemia, kidney cancer, soft tissue sarcoma, pancreatic cancer, and seminoma. 14. The method according to any one of 14.   A composition for treating or preventing cancer comprising, as an active ingredient, a pharmaceutically effective amount of an agent selected by the method according to any one of claims 9 to 15 and a pharmaceutically acceptable carrier. .   A composition for treating or preventing cancer, comprising a pharmaceutically effective amount of an antisense polynucleotide or siRNA against a polynucleotide encoded by the RASGEF1A gene.   18. The composition of claim 17, wherein the siRNA comprises the sense strand of the RASGEF1A gene comprising the nucleotide sequence of SEQ ID NO: 9 or 13.   The siRNA has the general formula 5 ′-[A]-[B]-[A ′]-3 ′, where [A] is a ribonucleotide sequence corresponding to the sequence of SEQ ID NO: 9 or 13 19. The composition according to claim 18, wherein [B] is a ribonucleotide loop sequence consisting of 3 to 23 nucleotides, and [A ′] is a ribonucleotide sequence complementary to [A].   A composition for treating or preventing cancer, comprising a pharmaceutically effective amount of an antibody or fragment thereof that binds to a RASGEF1A polypeptide.   The cancer is selected from the group consisting of ICC, bladder cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, chronic myelogenous leukemia, kidney cancer, soft tissue sarcoma, pancreatic cancer, and seminoma. 21. The composition according to any one of 20.   16. A method for treating or preventing cancer in a subject comprising administering a substance obtained by the method of any one of claims 9-15.   21. A method for treating or preventing cancer in a subject comprising administering to the subject a composition according to any one of claims 16-20.   A method for treating or preventing cancer in a subject comprising administering to the subject a pharmaceutically effective amount of an antibody or immunologically active fragment thereof that binds to a RASGEF1A polypeptide.   A method for treating or preventing cancer in a subject comprising administering to the subject a vaccine comprising: (a) a RASGEF1A polypeptide, (b) an immunologically active fragment of the polypeptide, or (c) the A polynucleotide encoding a polypeptide or a fragment thereof.   A method for inducing anti-tumor immunity comprising the step of contacting APC with a RASGEF1A polypeptide or a fragment thereof, a polynucleotide encoding the polypeptide or the fragment, or a vector containing the polynucleotide.   27. The method for inducing anti-tumor immunity according to claim 26, further comprising administering APC to the subject.   The cancer is selected from the group consisting of ICC, bladder cancer, lung cancer, gastric cancer, colorectal cancer, prostate cancer, chronic myelogenous leukemia, kidney cancer, soft tissue sarcoma, pancreatic cancer, and seminoma. 28. The method according to any one of 27.

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