EP1907547A2 - Genes cst6 et gabrp associes au cancer du pancreas - Google Patents

Genes cst6 et gabrp associes au cancer du pancreas

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Publication number
EP1907547A2
EP1907547A2 EP06781399A EP06781399A EP1907547A2 EP 1907547 A2 EP1907547 A2 EP 1907547A2 EP 06781399 A EP06781399 A EP 06781399A EP 06781399 A EP06781399 A EP 06781399A EP 1907547 A2 EP1907547 A2 EP 1907547A2
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European Patent Office
Prior art keywords
cst6
gabrp
pancreatic cancer
expression
cell
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EP06781399A
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German (de)
English (en)
Inventor
Yusuke c/o THE UNIVERSITY OF TOKYO NAKAMURA
Hidewaki c/o THE UNIVERSITY OF TOKYO NAKAGAWA
Shuichi c/o ONCOTHERAPY SCIENCE INC. NAKATSURU
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Oncotherapy Science Inc
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Oncotherapy Science Inc
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Publication of EP1907547A2 publication Critical patent/EP1907547A2/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to methods of detecting and diagnosing pancreatic cancer as well as methods of treating and preventing pancreatic cancer.
  • Pancreatic ductal adenocarcinoma is the fifth leading cause of cancer death in the western world and shows the worst mortality among malignancies, with a 5 -year survival rate of only 4% (DiMagno EP 3 et. al, Gastroenterology 1999; 117: 1464-84., Zervos EE, et. al, Cancer Control 2004; 11: 23-31.). Approximately 30,700 patients are diagnosed with pancreatic cancer in the United States, and nearly 30,000 of them will die of the disease (Jemal A, et. al, CA Cancer J Clin 2003; 53: 5-26.).
  • Cystatins are specific inhibitors of lysomal cysteine proteinases, such as cathepsins B, L, H and S, and they function both intracellularly and extracellularly (Barrett AJ. Trends Biochem. Sci., 1987; 12: 193-6., Keppler D. Cancer Letter 2005). They control the catalytic function of target proteases by forming reversible high-affinity complexes. Cystatin superfamilies are classified into three distinct subfamilies, and the family 1 cystatins represented by cystatin A (stefin A) and cystatin B (stefin B) are not glycosylated, lack disulf ⁇ te bonds, and function only intracellularly.
  • the family 2 cystatins C, D, S, SA, SN, E/M, and F are mainly secreted proteins composed of 115-120 amino acids with two interchain disulfide bonds.
  • the family 3 cystatins are composed of L- and H-kininogen, which are complex glycosylated cytoplasmic proteins with two cystatin domains and the bradykinin moiety (Barrett AJ. Trends Biochem. Sci., 1987; 12: 193-6., Keppler D. Cancer Letter 2005 May 10).
  • Cystatin E/M (CST6), belonging to the family 2, is a N-glycosylated secreted protein with 20-22kDa and CST6 was identified as a down-regulated gene in breast cancer independently by two groups comparing the differential transcripts between primary and metastatic breast cancers (Sotiropoulou G, et. al., J Biol. Chem. 1997, 272: 903-10., Ni J, et. al., J Biol Chem. 1997; 272:10853-8.), and they considered that this molecule suppressed proliferation, metastasis or invasion of breast cancer by modulating proteolysis of cell matrix or other mechanism (Shridhar R, et. al., Oncogene. 2004; 23: 2206-15., Zhang J, et. ah, Cancer Res. 2004; 64: 6957-64.).
  • GABAA receptor is a multi-subunit chloride channel that mediates the fastest inhibitory synaptic transmission in the central nervous system (Macdonald RL, Olsen RW., Annu Rev Neurosci 1994; 17: 569-602., Owens DF, Kriegstein AR., Nature Rev Neurosci 2002; 3: 715-27.). It consists mainly of ⁇ ⁇ ⁇ units and there are ⁇ l-6 subunits, ⁇ l-3 subunits, and ⁇ l-3 subunits reported so far (Macdonald RL, Olsen RW., Annu Rev Neurosci 1994; 17: 569-602., Owens DF, Kriegstein AR., Nature Rev Neurosci 2002; 3: 715-27.).
  • GABA receptor ⁇ (GABRP) subunit can assemble with these known GABAA receptor subunits and the incorporation of this subunit into GABAA receptor could alter the sensitivity of GABA receptors to GABA or modulating agents (Hedblom E, Kirkness EF., J Biol Chem 1997; 272: 15346-50., Neelands TR , Macdonald RL , MoI Pharmacol 1999; 56: 598-610.).
  • GABA functions primarily as an inhibitory neurotransmitter, but it can also act as a trophic factor during nervous system development to influence proliferation, migration and differentiation of neural cells and others (Macdonald RL, Olsen RW., Annu RevNeurosci 1994; 17: 569-602., Fiszman ML, Brain Res Dev Brain Res 1999; 115: 1-8.).
  • GABA and GABA receptors express and function in other peripheral tissues than the central nervous system (Macdonald RL, Olsen RW., Annu Rev Neurosci 1994; 17: 569-602.), but their precise function and distributions in non-neuronal cells are presently ill-defined.
  • Gene-expression profiles generated by cDNA microarray analysis can provide considerably more detail about the nature of individual cancers than traditional histopathological methods are able to supply.
  • the promise of such information lies in its potential for improving clinical strategies for treating neoplastic diseases and developing novel drugs (Petricoin, E. Y.et ⁇ /.,Nat Genet, 32 Suppl: 474-479, 2002.).
  • the present inventors have analyzed the expression profiles of tumor or tumors from various tissues by cDNA microarrays (Okabe, ⁇ ..et ah, Cancer Res, 61: 2129-2137, 2001.; Hasegawa, S.et ah, Cancer Res, 62: 7012-7017, 2002.; Kaneta, Y.et al.,. Jpn J Cancer Res, 93: 849-856, 2002.; Kaneta, Y.et ah, Int J Oncol, 23: 681-691, 2003.; Kitahara, O.et ah, Cancer Res, 61: 3544-3549, 2001.; Lin, YM. et al.
  • pancreatic cancers Studies into gene-expression profiles in pancreatic cancers have resulted in the identification of genes that may serve as candidates for diagnostic markers or prognosis profiles.
  • these data derived primarily from tumor masses, cannot adequately reflect expressional changes during pancreatic carcinogenesis, because pancreatic cancer cells exist as a solid mass with a highly inflammatory reaction and containing various cellular components. Therefore, previously published microarray data is likely to reflect heterogenous profiles.
  • FTIs farnesyltransferase
  • Agents of these kinds are designed to suppress oncogenic activity of specific gene products (Fang G.et ah, (2000). Blood, 96, 2246- 2253). Accordingly, it is apparent that gene products commonly up-regulated in cancerous cells may serve as potential targets for developing novel anti-cancer agents.
  • CTLs cytotoxic T lymphocytes
  • TAAs tumor-associated antigens
  • TAAs are currently undergoing clinical development as targets of immunotherapy. TAAs discovered so far include MAGE (van der Bruggen et al., Science 254: 1643-7 (1991)), gplOO (Kawakami et al., J Exp Med 180: 347-52 (1994)), SART (Shichijo et al., J Exp Med 187: 277-88 (1998)), and NY-ESO-I (Chen et al., Proc Natl Acad Sci USA 94: 1914-8 (1997)).
  • gene products demonstrated to be specifically over-expressed in tumor cells have been shown to be recognized as targets inducing cellular immune responses.
  • Such gene products include p53 (Umano et ah, Brit J Cancer 84: 1052-7 (2001)), HER2/neu (Tanaka et al., Brit J Cancer 84: 94-9 (2001)), CEA (Nukaya et al., Int J Cancer 80: 92-7 (1999)), and so on.
  • PBMCs peripheral blood mononuclear cells
  • HLA-A24 and HLA-A0201 are popular HLA alleles in the Japanese, as well as the Caucasian populations (Date et al, Tissue Antigens 47: 93-101 (1996); Kondo et al, J Immunol 155: 4307-12 (1995); Kubo et al, J Immunol 152: 3913-24 (1994); Imanishi et al, Proceeding of the eleventh International Histocompatibility Workshop and Conference Oxford University Press, Oxford, 1065 (1992); Williams et al, Tissue Antigen 49: 129 (1997)).
  • antigenic peptides of carcinomas presented by these HLAs may be especially useful for the treatment of carcinomas among Japanese and Caucasians.
  • CST6 cystatin E/M
  • GABA receptor ⁇ GABA receptor ⁇
  • Cystatin E/M belongs to the cystatin superfamily of proteinase inhibitors, which control the catalytic function of target proteases such as cathepsin M, and functions intracellularly and extracellularly.
  • Constitutive expression of CST6 in CST6-null cells promoted cell growth in vitro and the presence of mature recombinant CST6 in culture medium also promoted cell proliferation dose-dependently, suggesting that secreted CST6 is likely to involve PDAC cell proliferation in the autocrine/ paracrine manner.
  • GABA receptor ⁇ (GABRP) is one of the subunits of GAB A A receptor complex and its incorporation into GABAA receptor can modulate sensitivity to GABA. It was observed that GABA stimulation promoted the proliferation of PDAC cells that expressed GABRP and GABAA receptor antagonist abolished this promoting effect. On the other hand, GABA stimulation did not affect the proliferation of PDAC cells with no GABRP expression. These findings imply that CST6, GABA and GABA receptor ⁇ (GABRP) are the promising molecular targets for development of new therapeutic strategies for PDACs. hi the present invention, contrary to previous reports, the present inventors report CST6 over-expression in the some proportions of PDACs and demonstrate that CST6 promoted PDAC cell growth in the autocrine /paracrine matter, and GABA receptor ⁇
  • GABARP GABA receptor ⁇ subunit promoted PDAC cell growth, implicating that they are promising targets for PDAC treatment.
  • CST6 and GABRP are set forth in SEQ ID NO:41 and 42, 43 and 44, respectively. These sequences are also availabale from Genbank Accession NO.NM_001323 and NM_014211.
  • the present invention provides a method of diagnosing or determining a predisposition to pancreatic cancer in a subject by detennining an expression level of CST ⁇ or GABRP in a patient-derived biological sample, such as tissue sample.
  • a normal cell is one obtained from pancreatic tissue.
  • An alteration e.g., an increase in the level of expression of a gene as compared to a normal control level of the gene, indicates that the subject suffers from or is at risk of developing PDAC.
  • pancreatic cancer encompasses a state of a subject of being predisposed to, having a tendency, prevalence, inclination or susceptibility to pancreatic cancer. Moreover, said term also encompasses that a subject is at a risk of acquiring pancreatic cancer.
  • control level refers to a protein expression level detected in a control sample and includes both a normal control level and a pancreatic cancer control level.
  • a control level can be a single expression pattern derived from a single reference population or from a plurality of expression patterns.
  • the control level can be a database of expression patterns from previously tested cells.
  • a "normal control level” refers to a level of gene expression detected in a normal, healthy individual or in a population of individuals known not to be suffering from pancreatic cancer. A normal individual is one with no clinical symptoms of pancreatic cancer.
  • PDAC control level refers to an expression profile of CST6 or GABRP found in a population suffering from PDAC.
  • expression of a panel of CST6 or GABRP in a sample can be compared to a PDAC control level of the same panel of genes.
  • a similarity between a sample expression and PDAC control expression indicates that the subject (from which the sample was obtained) suffers from or is at risk of developing PDAC.
  • gene expression level is deemed “altered” when gene expression is increased or decreased 10%, 25%, 50% as compared to the control level.
  • an expression level is deemed “increased” or “decreased” when gene expression is increased or decreased by at least 0.1, at least 0.2, at least 1, at least 2, at least 5, or at least 10 or more fold as compared to a control level.
  • Expression is determined by detecting hybridization, e.g., CST6 or GABRP probe to a gene transcript of the patient- derived tissue sample.
  • the patient-derived tissue sample is any tissue obtained from a test subject, e.g., a. patient known to or suspected of having PDAC.
  • the tissue may contain an epithelial cell.
  • the tissue may be an epithelial cell from a pancreatic ductal adenocarcinoma.
  • the present invention further provides methods of identifying an agent that inhibits or enhances the expression or activity of CST6 or GABRP, by contacting a test cell expressing 44
  • the test cell may be an epithelial cell, such as an epithelial cell obtained from a pancreatic ductal adenocarcinoma.
  • a decrease in the expression level of CST6 or GABRP or the activity of its gene product as compared to a control level or activity of the gene or gene product indicates that the test agent is an inhibitor of CST6 or GABRP and may be used to reduce a symptom of PDAC.
  • the present invention also provides a kit comprising a detection reagent which binds to CST6 or GABRP nucleic acids or polypeptides.
  • Therapeutic methods of the present invention include a method of treating or preventing PDAC in a subject including the step of administering to the subject an antagonist or inhibitor of CST6 or GABRP which is, for example, an antisense composition or an antibody composition.
  • the antagonist or inhibitor can either act on the nucleic acid or protein level so as to reduce or inhibit CST6 or GABRP expression or activity.
  • the antisense composition reduces the expression of the specific target gene.
  • the antisense composition may contain a nucleotide which is complementary to CST6 or GABRP sequence.
  • the present method may include the steps of administering to a subject a small interfering RNA (siRNA) composition.
  • siRNA small interfering RNA
  • the siRNA composition reduces the expression of CST6 or GABRP.
  • the treatment or prevention of PDAC in a subject may be carried out by administering to a subject a ribozyme composition, hi the context of the present invention, the nucleic acid-specific ribozyme composition reduces the expression of CST6 or GABRP.
  • the inhibition effect of the siRNA for CST6 or GABRP was confirmed.
  • the siRNA for CST6 or GABRP inhibit cell proliferation of pancreatic cancer cells in the examples section.
  • CST6 or GABRP are preferable therapeutic target of pancreatic cancer.
  • the present invention also includes vaccines and vaccination methods.
  • a method of treating or preventing PDAC in a subject may involve administering to the subject a vaccine containing a polypeptide encoded by a nucleic acid of CST6 or GABRP or an immunologically active fragment of such a polypeptide, hi the context of the present invention, an immunologically active fragment is a polypeptide that is shorter in length than the full-length naturally-occurring protein yet which induces an immune response analogous to that induced by the full-length protein.
  • an immunologically active fragment should be at least 8 residues in length and capable of stimulating an immune cell such as a T cell or a B cell.
  • Immune cell stimulation can be measured by detecting cell proliferation, elaboration of cytokines (e.g., IL-2), or production of an antibody.
  • Fig. 1 shows the over-expression of Cystatin E/M (CST6) in PDAC cells.
  • CST6 Cystatin E/M
  • Fig. 2 shows the effect of CST ⁇ 5-siRNAs on growth of PDAC cells.
  • Fig. 3 shows the over-expression of CST6 promoted cell proliferation.
  • X-, and Y-axis represent day point after seeding and relative growth rate that was calculated in absorbance of the diameter by comparison with the absorbance value of day 1 as a control. Each average is plotted with error bars representing SD.
  • FIG. 4 shows cell proliferation stimulated by the recombinant CST6 protein.
  • COS7 cells were incubated with mature recombinant CST6 at serial concentration (0, 0.02, 0.2, and 2 ng/ml), supplied with 2%FBS.
  • Y-axis represent relative growth-promoting rate at day 6 that was calculated by comparison with the absorbance value of 0 ng/ml CST6 as a control. Each average is plotted with error bars representing SD. These experiences were hi triplicate altogether. ** Means p value ⁇ 0.01 (Students' t-test).
  • Fig. 5 shows the over-expression of GABA receptor ⁇ subunit (GABRP) and GADl in PDAC cells.
  • GABRP GABA receptor ⁇ subunit
  • Fig. 6 shows the effect of GABRP-siKNAs on growth of PDAC cells.
  • RNA-expressing vectors were transfected with each of siRNA-expressing vectors to GABRP (si6, si7, si8, and si 10) as well as a negative control vector (siEGFP), which confirmed the knockdown effect at si6, si7, si8, and silO, but not at a negative control siEGFP.
  • ⁇ 2-MG was used to quantify RNAs.
  • Fig. 7 shows the influence of GABA stimulation on PDAC cell proliferation and modulation by GABA receptor antagonists.
  • Y-axis represent relative growth-promoting rate at day 6 which was calculated by comparison with the absorbance value of 0 ng/ml GABA as a control. Each average is plotted with error bars representing SD. These experiences were in triplicate altogether. ** Means p value ⁇ 0.01 (Students' t-test).
  • GABAA receptor antagonist BMI 250 ⁇ M
  • GABA B receptor antagonist CGP-35348 1 mM with or without 100 ⁇ M GABA.
  • Cell viability was measured after 6 day exposure of these drugs, and Y-axis represent relative growth-promoting rate at day 6 which was calculated by comparison with the absorbance value of 0 ng/ml GABA and no drugs as a control. Each average is plotted with error bars representing SD. These experiences were in triplicate altogether. ** Means p value ⁇ 0.01 (Students' t-test).
  • pancreatic cancer cells exist as a solid mass having a highly inflammatory reaction and containing various cellular components. Therefore, previous published microarray data are likely to reflect heterogenous profiles.
  • the present invention is based in part of the discovery of elevated expression of CST6 or GABRP in cells from patients with pancreatic cancer.
  • CST6 and GABRP identified herein find diagnostic utility as markers of PDAC and as
  • PDAC gene target the expression of which may be altered to treat or alleviate a symptom of PDAC.
  • PDAC By measuring expression of CST6 or GABRP in a sample of cells, PDAC can be diagnosed. Similarly, measuring the expression of CST6 or GABRP in response to various agents can identify agents for treating PDAC.
  • the present invention involves determining (e.g., measuring) the expression of CST6 or GABRP.
  • CST6 or GABRP can be detected and measured using techniques well known to one of ordinary skill in the art.
  • sequences within the sequence database entries corresponding to CST6 or GABRP can be used to construct probes for delecting RNA sequences corresponding to CST6 or GABRP in, e.g., Northern blot hybridization analyses.
  • the sequences can be used to construct primers for specifically amplifying the PDAC nucleic acid in, e.g., amplification-based detection methods, such as reverse-transcription based polymerase chain reaction.
  • Expression level of CST6 or GABRP in a test cell population is then compared to the expression level of CST6 or GABRP in a reference population.
  • the reference cell population includes one or more cells for which the compared parameter is known, i.e., pancreatic ductal adenocarcinoma cells (e.g., PDAC cells) or normal pancreatic ductal epithelial cells (e.g., non-PDAC cells).
  • pancreatic ductal adenocarcinoma cells e.g., PDAC cells
  • normal pancreatic ductal epithelial cells e.g., non-PDAC cells.
  • the reference cell population is composed of non-PDAC cells
  • a similarity in gene expression pattern between the test cell population and the reference cell population indicates the test cell population is non-PDAC.
  • the reference cell population is made up of PDAC cells
  • a similarity in gene expression profile between the test cell population and the reference cell population indicates that the test cell population includes PDAC cells.
  • a level of expression of a PDAC marker gene in a test cell population is considered “altered” if it varies &om the expression level of the corresponding PDAC marker gene in a reference cell population by more than 1.1, more than 1.5, more than 2.0, more than 5.0, more than 10.0 or more fold.
  • Differential gene expression between a test cell population and a reference cell population can be normalized to a control nucleic acid, e.g. a housekeeping gene.
  • a control nucleic acid is one which is known not to differ depending on the cancerous or non-cancerous state of the cell.
  • the expression level of a control nucleic acid can be used to normalize signal levels in the test and reference populations.
  • Exemplary control genes include, but are not limited to, e.g., ⁇ -actin, glyceraldehyde 3- phosphate dehydrogenase and ribosomal protein P 1.
  • the test cell population can be compared to multiple reference cell populations. Each of the multiple reference populations may differ in the known parameter. Thus, a test cell population may be compared to a first reference cell population known to contain, e.g., PDAC cells, as well as a second reference population known to contain, e.g., non-PDAC cells (normal cells).
  • the test cell may be included in a tissue type or cell sample from a subject known to contain, or suspected of containing, PDAC cells.
  • the test cell is obtained from a bodily tissue or a bodily fluid, e.g., biological fluid (such as blood or sputum, for example).
  • the test cell may be purified from pancreatic tissue.
  • the test cell population comprises an epithelial cell.
  • the epithelial cell is preferably from a tissue known to be or suspected to be a pancreatic ductal adenocarcinoma.
  • Cells in the reference cell population should be derived from a tissue type similar to that of the test cell.
  • the reference cell population is a cell line, e.g. a PDAC cell line (i.e., a positive control) or a normal non-PDAC cell line (i.e., a negative control).
  • the control cell population may be derived from a database of molecular information derived from cells for which the assayed parameter or condition is known.
  • the subject is preferably a mammal.
  • exemplary mammals include, but are not limited to, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow.
  • CST6 or GABRP disclosed herein can be determined at the protein or nucleic acid level, using methods known in the art. For example, Northern hybridization analysis, using probes which specifically recognize the sequence can be used to determine gene expression. Alternatively, gene expression may be measured using reverse- transcription-based PCR assays, e.g., using primers specific for the CST6 or GABRP sequence. Expression may also be determined at the protein level, i.e., by measuring the level of a polypeptide encoded by a gene described herein, or the biological activity thereof. Such methods are well known in the art and include, but are not limited to, e.g., immunoassays that utilize antibodies to protein encoded by the gene. The biological activities of the protein encoded by the gene are generally well known. Diagnosing pancreatic cancer:
  • PDAC is diagnosed by measuring the expression level of CST6 or GABRP from a test population of cells, (i. e. , a patient-derived biological sample).
  • the test cell population contains an epithelial cell, e.g., a cell obtained from pancreatic tissue.
  • Gene expression can also be measured from blood or other bodily fluids such as urine.
  • Other biological samples can be used for measuring protein levels.
  • the protein level in blood or serum derived from a subject to be diagnosed can be measured by immunoassay or other conventional biological assay.
  • CST6 or GABRP is determined in the test cell or biological sample and compared to the normal control expression level associated with CST6 or GABRP assayed.
  • a normaTcontrol level is an expression profile of CST6 or GABRP typically found in a population known not to be suffering from PDAC.
  • An alteration e.g., an increase
  • an increase in the expression of CST6 or GABRP in the test population as compared to the normal control level indicates that the subject is suffering from or is at risk of developing PDAC.
  • the expression levels of PDAC-associated genes in a biological sample can be estimated by quantifying mRNA corresponding to or protein encoded by PDAC-associated genes. Quantification methods for mRNA are known to those skilled in the art. For example, the levels of mRNAs corresponding to PDAC-associated genes can be estimated by Northern blotting or RT-PCR. Since the nucleotide sequences of PDAC-associated genes are known, anyone skilled in the art can design the nucleotide sequences for probes or ' primers to quantify PDAC-associated genes.
  • the expression level of the PDAC-associated genes can be analyzed based on the activity or quantity of protein encoded by the genes.
  • a method for determining the quantity of the protein encoded by PDAC-associated genes is shown in below.
  • immunoassay methods are useful for the determination of the proteins in biological materials. Any biological materials can be used as the biological sample for the determination of the protein or it's activity so long as the marker gene (PDAC-associated genes) is expressed in the sample of a pancreatic cancer patient. However, bodily fluids such as blood and urine may be also analyzed.
  • a suitable method can be selected for the determination of the activity of a protein encoded by PDAC-associated genes according to the activity of a protein to be analyzed.
  • Expression levels of PDAC-associated genes in a biological sample are estimated and compared with those in a normal sample (e.g., a sample derived from a non-diseased subject). When such a comparison shows that the expression level of the genes is higher than those in the normal sample, the subject is judged to be affected with PDAC.
  • the expression level of PDAC-associated genes in the biological samples from a normal subject and subject to be diagnosed may be determined at the same time.
  • normal ranges of the expression levels can be determined by a statistical method based on the results obtained by analyzing the expression level of the genes in samples previously collected from a control group. A result obtained by comparing the sample of a subject is compared with the normal range; when the result does not fall within the normal range, the subject is judged to be affected with or is at risk of developing PDAC.
  • a diagnostic agent for diagnosing cell proliferative disease such as PDAC
  • the diagnostic agent of the present invention comprises a compound that binds to a polynucleotide or a polypeptide of PDAC-associated genes.
  • an oligonucleotide that hybridizes to the polynucleotide of PDAC-associated genes or an antibody that binds to the polypeptide encoded by PDAC-associated genes may be used as such a compound.
  • a aptamers such as a RNA, DNA or peptide aptamer may be used as such a compound.
  • An agent that inhibits the expression of CST6 or GABRP or the activity of its gene product can be identified by contacting a test cell population expressing CST6 or GABRP with a test agent and then determining the expression level of CST6 or GABRP or the activity of its gene product.
  • a decrease in the level of expression of CST6 or GABRP or in the level of activity of its gene product in the presence of the agent as compared to the expression or activity level in the absence of the test agent indicates that the agent is an inhibitor of CST6 or GABRP and useful in inhibiting PDAC.
  • the test cell population may be any cell expressing CST6 or GABRP.
  • the test cell population may contain an epithelial cell, such as a cell derived from pancreatic tissue.
  • test cell may be an immortalized cell line derived from a adenocarcinoma cell.
  • test cell may be a cell which has been transfected with CST6 or GABRP or which has been transfected with a regulatory sequence (e.g. promoter sequence) from CST6 or GABRP operably linked to a reporter gene.
  • a regulatory sequence e.g. promoter sequence
  • the differentially expressed CST6 or GABRP identified herein also allow for the course of treatment of PDAC to be monitored.
  • a test cell population is provided from a subject undergoing treatment for PDAC. If desired, test cell populations are obtained from the subject at various time points, before, during, and/or after treatment. Expression of CST6 or GABRP in the cell population is then determined and compared to a reference cell population which includes cells whose PDAC state is known. In the context of the present invention, the reference cells should have not been exposed to the treatment of interest.
  • the reference cell population contains no PDAC cells, a similarity in the expression of CST6 or GABRP in the test cell population and the reference cell population indicates that the treatment of interest is efficacious. However, an increase in the expression of CST6 or GABRP in the test population compared to a normal control reference cell population indicates a less favorable clinical outcome or prognosis. Similarly, if the reference cell population contains PDAG cells, a decrease in the expression of CST6 or GABRP in the test cell population compared to the reference cell population indicates that the treatment of interest is efficacious, while a similarity in the expression of CST6 or GABRP in the test population and a cancer control reference cell population indicates a less favorable clinical outcome or prognosis.
  • the expression level of CST6 or GABRP determined in a subject-derived biological sample obtained after treatment can be compared to the expression level of CST6 or GABRP determined in a subject-derived biological sample obtained prior to treatment onset (i.e., pre-treatment levels).
  • a decrease in the expression level of CST6 or GABRP in a post-treatment sample indicates that the treatment of interest is efficacious while an increase or maintenance in the expression level in the post-treatment sample indicates a less favorable clinical outcome or prognosis.
  • the term "efficacious" indicates that the treatment leads to a reduction in the expression of a pathologically up-regulated gene, an increase in the expression of a pathologically down-regulated gene or a decrease in size, prevalence, or metastatic potential of pancreatic ductal adenocarcinoma in a subject.
  • the term “efficacious” means that the treatment retards or prevents a pancreatic tumor from forming or retards, prevents, or alleviates a symptom of clinical PDAC. Assessment of pancreatic tumors can be made using standard clinical protocols.
  • efficaciousness can be determined in association with any known method of diagnosing or treating PDAC.
  • PDAC can be diagnosed, for example, by identifying symptomatic anomalies, e.g., weight loss, abdominal pain, back pain, anorexia, nausea, vomiting and generalized malaise, weakness, and j aundice.
  • An agent that is metabolized in a subject to act as an anti-PDAC agent can manifest itself by inducing a change in a gene expression pattern in the subject's cells from that characteristic of a cancerous state to a gene expression pattern characteristic of a non-cancerous state.
  • the differentially expressed CST6 or GABRP disclosed herein allow for a putative therapeutic or prophylactic inhibitor of PDAC to be tested in a test cell population from a selected subject in order to determine if the agent is a suitable inhibitor of PDAC in the subject.
  • a test cell population from the subject is exposed to a therapeutic agent, and the expression of CST6 or GABRP is determined.
  • the test cell population contains a PDAC cell expressing CST6 or GABRP.
  • the test cell is an epithelial cell.
  • a test cell population may be incubated in the presence of a candidate agent and the pattern of gene expression of the test cell population may be measured and compared to one or more reference profiles, e.g., a PDAC reference expression profile or a non-PDAC reference expression profile.
  • a decrease in expression of CST6 or GABRP in a test cell population relative to a reference cell population containing PDAC indicates that the agent has therapeutic potential.
  • test agent can be any compound or composition.
  • exemplary test agents include, but are not limited to, immunomodulatory agents.
  • proteins encoded by the gene or transcriptional regulatory region of the gene proteins encoded by the gene or transcriptional regulatory region of the gene, compounds can be screened that alter the expression of the gene or the biological activity of a polypeptide encoded by the gene. Such compounds are used as pharmaceuticals for treating or preventing PDAC.
  • the present invention provides a method of screening for a compound for treating or preventing PDAC using the CST6 or GABRP polypeptide.
  • An embodiment of this screening method comprises the steps of: a) contacting a test compound with a polypeptide encoded by a polynucleotide of
  • the CST6 or GABRP polypeptide to be used for screening may be a recombinant polypeptide or a protein derived from the nature or a partial peptide thereof.
  • the polypeptide to be contacted with a test compound can be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides.
  • a method of screening for proteins for example, that bind to the CST6 or GABRP polypeptide using the CST6 or GABRP polypeptide
  • many methods well known by a person skilled in the art can be used.
  • Such a screening can be conducted by, for example, immunoprecipitation method, specifically, in the following manner.
  • the gene encoding the CST6 or GABRP polypeptide is expressed in host (e.g., animal) cells and so on by inserting the gene to an expression vector for foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8.
  • the promoter to be used for the expression may be any promoter that can be used commonly and include, for example, the SV40 early promoter (Rigby in
  • the introduction of the gene into host cells to express a foreign gene can be performed according to any methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 15: 1311-26 (1987)), the calcium phosphate method (Chen and Okayama, MoI Cell Biol 7: 2745-52 (1987)), the DEAE dextran method (Lopata et al., Nucleic Acids Res 12: 5707-17 (1984); Sussman and Milman, MoI Cell Biol 4: 1641-3 (1984)), the Lipofectin method (Derijard B., Cell 76: 1025-37 (1994); Lamb et al, Nature Genetics 5: 22-30 (1993):
  • the polypeptide encoded by CST6 or GABRP gene can be expressed as a fusion protein comprising a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C- terminus of the polypeptide.
  • a commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (1995)).
  • Vectors which can express a fusion protein with, for example, ⁇ -galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP) and so on by the use of its multiple cloning sites are commercially available.
  • a fusion protein prepared by introducing only small epitopes consisting of several to a dozen amino acids so as not to change the property of the CST6 or GABRP polypeptide by the fusion is also reported.
  • Epitopes such as polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on . monoclonal phage) and such, and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the CST6 or GABRP polypeptide (Experimental Medicine 13 : 85-90 (1995)).
  • an immune complex is formed by adding these antibodies to cell lysate prepared using an appropriate detergent.
  • the immune complex consists of the CST6 or GABRP polypeptide, a polypeptide comprising the binding ability with the polypeptide, and an antibody.
  • Immunoprecipitation can be also conducted using antibodies against the CST6 or GABRP polypeptide, besides using antibodies against the above epitopes, which antibodies can be prepared as described above.
  • An immune complex can be precipitated, for example by Protein A sepharose or Protein G sepharose when the antibody is a mouse IgG antibody.
  • an immune complex can be formed in the same manner as in the use of the antibody against the CST6 or GABRP polypeptide, using a substance specifically binding to these epitopes, such as glutathione-Sepharose 4B. Immunoprecipitation can be performed by following or according to, for example, the methods in the literature (Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)).
  • SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Since the protein bound to the CST6 or GABRP polypeptide is difficult to detect by a common staining method, such as Coomassie staining or silver staining, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, 35 S-methionine or 35 S-cystein, labeling proteins in the cells, and detecting the proteins. The target protein can be purified directly from the SDS- polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.
  • a protein binding to the CST6 or GABRP polypeptide can be obtained by preparing a cDNA library from cells, tissues, organs (for example, tissues such as a placenta or thyroid for CST6, or trachea, prostate or stomach for GABRP), or cultured cells (e.g., PK-59 or PK-I for CST6, or KLM-I or PK-45P for GABRP) expected to express a protein binding to the CST6 or GABRP polypeptide using a phage vector (e.g., ZAP), expressing the protein on LB-agarose, fixing the protein expressed on a filter, reacting the purified and labeled CST6 or GABRP polypeptide with the above filter, and detecting the
  • the polypeptide of the invention may be labeled by utilizing the binding between biotin and avidin, or by utilizing an antibody that specifically binds to the CST6 or GABRP polypeptide, or a peptide or polypeptide (for example, GST) that is fused to the CST6 or GABRP polypeptide. Methods using radioisotope or fluorescence and such may be also used.
  • a two-hybrid system utilizing cells may be used ("MATCHMAKER Two-Hybrid system", “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene); the references “Dalton and Treisman, Cell 68: 597-612 (1992)", “Fields and Sternglanz, Trends Genet 10: 286-92 (1994)”).
  • MATCHMAKER Two-Hybrid system Mammalian MATCHMAKER Two-Hybrid Assay Kit
  • MATCHMAKER one-Hybrid system (Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene); the references “Dalton and Treisman, Cell 68: 597-612 (1992)", “Fields and Sternglanz, Trends Genet 10: 286
  • the polypeptide of the invention is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells.
  • a cDNA library is prepared from cells expected to express a protein binding to the polypeptide of the invention, such that the library, when expressed, is fused to the VP 16 or GAL4 transcriptional activation region.
  • the cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the polypeptide of the invention is expressed in yeast cells, the binding of the two activates a reporter gene, making positive clones detectable).
  • a protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E. coli and expressing the protein.
  • a reporter gene for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.
  • a compound binding to the polypeptide encoded by CST6 or GABRP gene can also be screened using affinity chromatography.
  • the polypeptide of the invention may be immobilized on a carrier of an affinity column, and a test compound, containing a protein capable of binding to the polypeptide of the invention, is applied to the column.
  • a test compound herein may be, for example, cell extracts, cell lysates, etc. After loading the test compound, the column is washed, and compounds bound to the polypeptide of the invention can be prepared.
  • test compound When the test compound is a protein, the amino acid sequence of the obtained protein is analyzed, an oligo DNA is synthesized based on the sequence, and cDNA libraries are screened using the oligo DNA as a probe to obtain a DNA encoding the protein.
  • a biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound compound in the present invention.
  • a biosensor When such a biosensor is used, the interaction between the polypeptide of the invention and a test ( compound can be observed real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide of the invention and a test compound using a biosensor such as BIAcore.
  • the present invention provides a method of screening for a compound for treating or preventing PDAC using the polypeptide encoded by CST6 or GABRP gene comprising the steps as follows: a) contacting a test compound with a polypeptide encoded by a polynucleotide of CST6 or GABRP; b) detecting the biological activity of the polypeptide of step (a); and c) selecting the test compound that suppresses the biological activity of the polypeptide encoded by the polynucleotide of CST6 or GABRP as compared to the biological activity of said polypeptide detected in the absence of the test compound.
  • the CST6 or GABRP protein have the activity of promoting cell proliferation of PDAC cells, a compound which inhibits this activity of this protein can be screened using this activity as an index.
  • Any polypeptides can be used for screening so long as they comprise the biological activity of the CST6 or GABRP protein.
  • Such biological activity include cell-proliferating activity of the human CST6 or GABRP protein.
  • a human CST6 or GABRP protein can be used and polypeptides functionally equivalent to these proteins can also be used.
  • Such polypeptides may be expressed endogenously or exogenously by cells.
  • the compound isolated by this screening is a candidate for agonists or antagonists of the polypeptide encoded by CST6 or GABRP gene.
  • agonist refers to molecules that activate the function of the polypeptide by binding thereto. Said term, however, also refers to molecules that activate or enhance expression of the gene endoding CST6 or GABRP. Likewise, the term “antagonist” refers to molecules that inhibit the function of the polypeptide by binding thereto. Yet, said term also refers to molecules that reduce or inhibit expression of the gene encoding CST6 or GABRP. Moreover, a compound isolated by this screening is a candidate for compounds which inhibit the in vivo interaction of the CST6 or GABRP polypeptide with molecules (including DNAs and proteins).
  • the biological activity to be detected in the present method is cell proliferation, it can be detected, for example, by preparing cells which express the CST6 or GABRP polypeptide, culturing the cells in the presence of a test compound, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by measuring the colony forming activity as described in the Examples.
  • the present invention provides methods of screening for compounds, for treating or preventing PDAC. As discussed in detail above, by controlling the expression levels of the CST6 or GABRP 3 one can control the onset and progression of PDAC. Thus, compounds that may be used in the treatment or prevention of PDAC can be identified through screenings that use the expression levels of CST6 or GABRP as indices.
  • such screening may comprise, for example, the following steps: a) contacting a candidate compound with a cell expressing CST6 or GABRP; and b) selecting the candidate compound that reduces the expression level of CST6 or GABRP as compared to a control.
  • Cells expressing the CST6 or GABRP include, for example, cell lines established from PDAC; such cells can be used for the above screening of the present invention (e.g., PK-59 or PK-I for CST6, or KLM-I or PK-45P for GABRP).
  • the expression level can be estimated by methods well known to one skilled in the art.
  • a compound that reduces the expression level of CST6 or GABRP can be selected as candidate agents to be used for the treatment or prevention of PDAC.
  • the screening method of the present invention may comprise the following steps: a) contacting a candidate compound with a cell into which a vector, comprising the transcriptional regulatory region of CST6 or GABRP and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; b) measuring the expression or activity of said reporter gene; and c) selecting the candidate compound that reduces the expression or activity of said reporter gene.
  • Suitable reporter genes and host cells are well known in the art.
  • the reporter construct required for the screening can be prepared by using the transcriptional regulatory region of a marker gene.
  • a reporter construct can be prepared by using the previous sequence information.
  • a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library based on the nucleotide sequence information of the marker gene.
  • Exan ⁇ ples of supports that may be used for binding proteins include insoluble polysaccharides, such as agarose, cellulose and dextran; and synthetic resins, such as polyacrylamide, polystyrene and silicon; preferably commercial available beads and plates (e.g., multi-well plates, biosensor chip, etc.) prepared from the above materials may be used. When using beads, they may be filled into a column.
  • binding of a protein to a support may be conducted according to routine methods, such as chemical bonding and physical adsorption.
  • a protein may be bound to a support via antibodies specifically recognizing the protein.
  • binding of a protein to a support can be also conducted by means of avidin and biotin.
  • the binding between proteins is carried out in buffer, for example, but are not limited to, phosphate buffer and Tris buffer, as long as the buffer does not inhibit the binding between the proteins.
  • a biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound protein.
  • the interaction between the proteins can be observed real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide and without labeling (for example, BIAcore, Pharmacia).
  • CST6 or GABRP polypeptide may be labeled, and the label of the bound protein may be used to detect or measure the bound protein. Specifically, after pre- labeling one of the proteins, the labeled protein is contacted with the other protein in the presence of a test compound, and then bound proteins are detected or measured according to the label after washing.
  • Labeling substances such as radioisotope (e.g., 3 H, 14 C, 32 P 3 33 P, 35 S, 125 1, 131 I), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, ⁇ -glucosidase), fluorescent substances (e.g., fluorescein isothiosyanete (FITC), rhodamine) and biotin/avidin, may be used for the labeling of a protein in the present method.
  • radioisotope e.g., 3 H, 14 C, 32 P 3 33 P, 35 S, 125 1, 131 I
  • enzymes e.g., alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, ⁇ -glucosidase
  • fluorescent substances e.g., fluorescein isothiosyanete (FITC), rho
  • proteins labeled with enzymes can be detected or measured by adding a substrate of the enzyme to detect the enzymatic change of the substrate, such as generation of color, with absorptiometer. Further, in case where a fluorescent substance is used as the label, the bound protein may be detected or measured using fluorophotometer.
  • the antibody is preferably labeled with one of the labeling substances mentioned above, and detected or measured based on the labeling substance.
  • the antibody against the CST6 or GABRP polypeptide may be used as a primary antibody to be detected with a secondary antibody that is labeled with a labeling substance.
  • the antibody bound to the protein in the screening of the present invention may be detected or measured using protein G or protein A column.
  • a two-hybrid system utilizing cells may be used ("MATCHMAKER Two-Hybrid system", “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene); the references “Dalton and Treisman, Cell 68: 597-612 (1992)", “Fields and Sternglanz, Trends Genet 10: 286-92 (1994)”).
  • the CST6 or GABRP polypeptide of the invention is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells.
  • the present invention provides method of treating or preventing pancreatic cancer in a subject, said method comprising the step of administering a pharmaceutically effective amount of a GABAA receptor (GABRA) antagonist.
  • GABAA receptor GABAA receptor
  • the present invention also provides composition for treating or preventing pancreatic cancer, said composition comprising as an active ingredient a pharmaceutically effective amount of a GABAA receptor (GABRA) antagonist, and a pharmaceutically acceptable carrier.
  • GABA A receptor (GABRA) antagonist is Bicuculline or pharmaceutically acceptable quaternary ammonium salts thereof.
  • methiodide, methobromide, methochloride or methoxide of Bicuculline may be included in the pharmaceutically acceptable quaternary ammonium salts of Bicuculline.
  • Bicuculline (CAS Registration number 19730-80-4) is well known type-A gamma-aminobutyric acid receptor antagonist (Kardos J. et al., Eur. J. Pharmacol. 337/1, 83-86, 1997, Oct. 15).
  • suppression of cell proliferation of PDAC by Bicuculline is novel approach.
  • GABAA receptor antagonist may be used for treating or preventing PDAC. Therefore the present invention provides method of screening for a compound for treating or preventing PDAC.
  • An embodiment of this screening method comprises the step of:
  • step (b) selecting the compound which decreases the binding activity detected in step (a) compared to that detected in the absence of the test compound.
  • GABA indicates a neurotransmitter gamma aminobutyric acid. GABA functions primarily as an inhibitory neurotransmitter in the mature brain. It can also act as a trophic factor during nervous system development to influence proliferation, migration and differentiation of neural cells and others.
  • Amalogue herein refers to a derivative of the ligand having the same physiological activity as the ligand, or inhibiting a physiological activity of the ligand, and contains both natural and artificially synthesized compounds.
  • GABRP can be prepared not only as a natural protein but also as a recombinant protein prepared by the gene recombination technique.
  • the natural protein can be prepared, for example, by affinity chromatography.
  • the recombinant protein may be prepared by culturing cells transformed with DNA encoding the GABRP to express the protein therein and then recovering it.
  • the binding activity of GABRP with GABA or an analog thereof can be detected using a label attached to the GABA or analog thereof (for example, the amount of bound is determined by the radioactivity or fluorescence intensity).
  • a label attached to the GABA or analog thereof for example, the amount of bound is determined by the radioactivity or fluorescence intensity.
  • changes in the cell resulting from the binding of a test compound to GABRP on the cell surface is used as an indicator.
  • test compound for example, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds and natural compounds can be used in the screening methods of the present invention.
  • the test compound can be also obtained using any of the numerous approaches in combinatorial library methods known in. the art, including (1) biological libraries, (2) spatially addressable parallel solid phase or solution phase libraries, (3) synthetic library methods requiring deconvolution, (4) the "one-bead one-compound” library method and (5) synthetic library methods using affinity chromatography selection.
  • a compound isolated by the screening methods of the present invention is a candidate for drugs which inhibit the activity of the CST6 or GABRP polypeptide, for treating or preventing diseases attributed to, for example, cell proliferative diseases, such as PDAC.
  • Pharmaceutical compositions for treating or preventing PDAC The present invention provides compositions for treating or preventing pancreatic cancer comprising any of the compounds selected by the screening methods of the present invention.
  • the isolated compound When administrating a compound isolated by the method of the present invention as a pharmaceutical for humans and other mammals, such as mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees, the isolated compound can be directly administered or can be formulated into a dosage form using known pharmaceutical preparation methods.
  • the drugs can be taken orally, as sugar-coated tablets, capsules, elixirs and microcapsules, or non-orally, in the form of injections of sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid.
  • the compounds can be mixed with pharmaceutically acceptable carriers or media, specifically, sterilized water, physiological saline, plant-oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and such, in a unit dose form required for generally accepted drug implementation.
  • pharmaceutically acceptable carriers or media specifically, sterilized water, physiological saline, plant-oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and such, in a unit dose form required for generally accepted drug implementation.
  • the amount of active ingredient contained in such a preparation makes a suitable dosage within the indicated range acquirable.
  • additives that can be admixed into tablets and capsules include, but are not limited to, binders, such as gelatin, corn starch, tragacanth gum and arabic gum; excipients, such as crystalline cellulose; swelling agents, such as corn starch, gelatin and alginic acid; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose or saccharin; and flavoring agents, such as peppermint, Gaultheria adenothrix oil and cherry.
  • a liquid carrier such as an oil
  • Sterile composites for injection can be formulated following normal drug implementations using vehicles, such as distilled water, suitable for injection.
  • Physiological saline, glucose, and other isotonic liquids, including adjuvants, such as D-sorbitol, D-mannnose, D-mannitol, and sodium chloride can be used as aqueous solutions for injection.
  • adjuvants such as D-sorbitol, D-mannnose, D-mannitol, and sodium chloride
  • suitable solubilizers such as alcohol, for example, ethanol
  • polyalcohols such as propylene glycol and polyethylene glycol
  • non- ionic surfactants such as Polysorbate 80 (TM) and HCO-50.
  • Sesame oil or soy-bean oil can be used as an oleaginous liquid, may be used in conjunction with benzyl benzoate or benzyl alcohol as a solubilizer, and may be formulated with a buffer, such as phosphate buffer and sodium acetate buffer; a pain-killer, such as procaine hydrochloride; a stabilizer, such as benzyl alcohol and phenol; and/or an anti-oxidant.
  • a prepared injection may be filled into a suitable ampoule.
  • Methods well known to those skilled in the art may be used to administer the pharmaceutical composition of the present invention to patients, for example as an intraarterial, intravenous, or percutaneous injection or as an intranasal, transbronchial, intramuscular or oral adn ⁇ nistration.
  • the dosage and method of administration vary according to the body- weight and age of a patient and the administration method; however, one skilled in the art can routinely select a suitable method of administration. If said compound is encodable by a DNA, the DNA can be inserted into a vector for gene therapy and the vector administered to a patient to perform the therapy.
  • the dosage and method of administration vary according to the body- weight, age, and symptoms of the patient; however, one skilled in the art can suitably select them.
  • the dose of a compound that binds to a protein of the present invention and regulates its activity depends on the symptoms, the dose is generally about 0.1 nig to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day and more preferably about 1.0 mg to about 20 mg per day, when administered orally to a normal adult human (weight 60 kg).
  • the present invention also provides a method of assessing the prognosis of a subject with PDAC including the step of comparing the expression of CST6 or GABRP in a test cell population to the expression of CST6 or GABRP in a reference cell population derived from patients over a spectrum of disease stages.
  • a method of assessing the prognosis of a subject with PDAC including the step of comparing the expression of CST6 or GABRP in a test cell population to the expression of CST6 or GABRP in a reference cell population derived from patients over a spectrum of disease stages.
  • an increase in the expression of CST6 or GABRP as compared to a normal control indicates less favorable prognosis.
  • a similarity in the expression of CST6 or GABRP as compared to normal control indicates a more favorable prognosis for the subject.
  • the prognosis of a subject can be assessed by comparing the expression profile of CST6 or GABRP. Kits:
  • the present invention also includes a PDAC-detection reagent, e.g., a. nucleic acid that specifically binds to or identifies CST6 or GABRP nucleic acids, such as oligonucleotide sequences which are complementary to a portion of CST6 or GABRP nucleic acid, a DNA, RNA or peptide aptamer or an antibody that bind to proteins encoded by CST6 or GABRP nucleic acid.
  • the detection reagents may be packaged together in the form of a kit.
  • the detection reagents may be packaged in separate containers, e.g., a nucleic acid or antibody (either bound to a solid matrix or packaged separately with reagents for binding them to the matrix), a control reagent (positive and/or negative), and/or a detectable label.
  • Instructions e.g., written, tape, VCR, CD-ROM, etc.
  • the assay format of the kit may be a Northern hybridization or a sandwich ELISA, both of which are known in the art.
  • a PDAC detection reagent may be immobilized on a solid matrix, such as a porous strip, to form at least one PDAC detection site.
  • the measurement or detection region of the porous strip may include a plurality of sites, each containing a nucleic acid.
  • a test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a separate strip from the test strip.
  • the different detection sites may contain different amounts of immobilized nucleic acids, i.e., a higher amount in the first detection site and lesser amounts in subsequent sites.
  • the number of sites displaying a detectable signal provides a quantitative indication of the amount of PDAC present in the sample.
  • the detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
  • the present invention further provides a method of treating or alleviating a symptom of PDAC in a subject by decreasing the expression of CST6 or GABRP (or the activity of its gene product).
  • Suitable therapeutic compounds can be administered prophylactically or therapeutically to a subject suffering from or at risk of (or susceptible to) developing PDAC. Such subjects can be identified using standard clinical methods or by detecting an aberrant level of expression of CST6 or GABRP or aberrant activity of its gene product.
  • suitable therapeutic agents include, for example, inhibitors of cell cycle regulation and cell proliferation.
  • the therapeutic method of the present invention may include the step of decreasing the expression, function, or both, of gene products of CST6 or GABRP whose expression is aberrantly increased ("up-regulated” or "over-expressed” gene) in pancreatic cells.
  • Expression may be inhibited in any of several ways known in the art. For example, expression can be inhibited by administering to the subject a nucleic acid that inhibits, or antagonizes the expression of the over-expressed gene, e.g., an antisense oligonucleotide or small interfering RNA which disrupts expression of the over-expressed gene.
  • a compound which decreases the expression of CST6 or GABRP (or the activity of its gene product) as described herein for the preparation of a pharmaceutical composition for treating or alleviating a symptom of PDAC in a subject.
  • a compound could be, for exmple, an antisense, siRNA, antibody, aptamers or ribozyme composition.
  • antisense nucleic acids corresponding to the nucleotide sequence of CST6 or GABRP can be used to reduce the expression level of the gene.
  • Antisense nucleic acids corresponding to CST6 or GABRP that are up-regulated in pancreatic cancer are useful for the treatment of pancreatic cancer.
  • the antisense nucleic acids of the present invention may act by binding to nucleotide sequence of CST6 or GABRP, or mRNAs corresponding thereto, thereby inhibiting the transcription or translation of the genes, promoting the degradation of the mRNAs, and/or inhibiting the expression of proteins encoded by CST6 or GABRP, thereby, inhibiting the function of the proteins.
  • antisense nucleic acids encompasses both nucleotides that are entirely complementary to the target sequence and those having a mismatch of one or more nucleotides, so long as the antisense nucleic acids can specifically hybridize to the target sequences.
  • the antisense nucleic acids of the present invention include polynucleotides that have a homology of at least 70% or higher, preferably at least 80% or higher, more preferably at least 90% or higher, even more preferably at least 95% or higher over a span of at least 15 continuous nucleotides. Algorithms known in the art can be used to determine the homology.
  • the antisense nucleic acid of the present invention act on cells producing the proteins encoded by CST6 or GABRP by binding to the DNA or mRNA encoding the protein, inhibiting their transcription or translation, promoting the degradation of the mRNA, and inhibiting the expression of the protein, thereby resulting in the inhibition of the protein function.
  • An antisense nucleic acid of the present invention can be made into an external preparation, such as a liniment or a poultice, by admixing it with a suitable base material which is inactive against the nucleic acid.
  • the antisense nucleic acids of the present invention can be formulated into tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops and freeze-drying agents by adding excipients, isotonic agents, solubilizers, stabilizers, preservatives, pain-killers, and such. These can be prepared by following known methods.
  • the antisense nucleic acids of the present invention can be given to the patient by direct application onto the ailing site or by injection into a blood vessel so that it will reach the site of ailment.
  • An antisense-mounting medium can also be used to increase durability and membrane-permeability. Examples include, but are not limited to, liposomes, poly-L- lysine, lipids, cholesterol, lipofectine or derivatives of these.
  • the dosage of the antisense nucleic acid derivative of the present invention can be adjusted suitably according to the patient's condition and used in desired amounts. For example, a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered.
  • antisense nucleic acids of the present invention inhibit the expression of a protein of the present invention and are thereby useful for suppressing the biological activity of the protein of the invention.
  • expression-inhibitors comprising antisense nucleic acids of the present invention, are useful in that they can inhibit the biological activity of a protein of the present invention.
  • the antisense nucleic acids of present invention include modified oligonucleotides.
  • thioated oligonucleotides may be used to confer nuclease resistance to an oligonucleotide.
  • siRNA against CST6 or GABRP can be used to reduce the expression level of CST6 or GABRP.
  • siRNA refers to a double stranded RNA molecule which prevents translation of a target mRNA. Standard techniques for introducing siRNA into the cell may be used, including those in which DNA is a template from which RNA is transcribed.
  • the siRNA comprises a sense nucleic acid sequence and an anti-sense nucleic acid sequence against an up-regulated marker gene, such as CST6 or GABRP.
  • the siRNA is constructed such that a single transcript has both the sense and complementary antisense sequences of the target gene, e.g., a hairpin.
  • siRNA molecules of the invention can be defined by their ability to hybridize specifically to mRNA of CST6 or GABRP under stringent conditions.
  • hybridize or “hybridize specifically” are used to refer the ability of two nucleic acid molecules to hybridize under “stringent hybridization conditions.”
  • stringent hybridization conditions refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • stringent conditions are selected to be about 5-1O 0 C lower than the thermal melting point (T n ,) for the specific sequence at a defined ionic strength pH.
  • T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times of background, preferably 10 times of background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 0 C, or, 5x SSC, 1% SDS, incubating at 65 0 C, with wash in 0.2x SSC, and 0.1% SDS at 5O 0 C.
  • an siRNA is preferably less than 500, 200, 100, 50, or 25 nucleotides in length. More preferably an siRNA is 19-25 nucleotides in length.
  • Exemplary nucleic acid sequence for the production of CST6 or GABRP. siRNA includes the sequences of nucleotides of SEQ ID NO: 15, 19, 23, 27 or 31 as the target sequence. In RNA or derivatives thereof, base "t" shoulde be replaced with "u" in the nucleotide sequences.
  • the present invention provides a double stranded RNA molecule comprising nucleotide sequence 5'- gugguucccuggcagaacu-3' (SEQ ID NO: 15), 5'- gaugggcaggauuguugau-3' (SEQ ID NO: 19), 5'- uaucaucaacagcuccauc-3' (SEQ ID NO: 23), 5 1 - ccccaguaauguugaucac-3' (SEQ ID NO: 27) or 5'-aggaaguagaagaagucag-3' (SEQ ID NO: 31).
  • nucleotide "u” can be added to 3 'end of the antisense strand of the target sequence.
  • the number of "u"s to be added is at least 2, generally 2 to 10, preferably 2 to 5.
  • the added "u”s form single strand at the 3 'end of the antisense strand of the siRNA.
  • siRNA of CST6 or GABRP can be directly introduced into the cells in a form that is capable of binding to the mRNA transcripts.
  • 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 have shown improved pharmacological properties (Song et al. Nature Med. 9:347-351 (2003)).
  • a DNA encoding the siRNA may be carried in a vector.
  • Vectors may be produced, for example, by cloning CST6 or GABRP target sequence into an expression vector having operatively-linked regulatory sequences flanking the sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands (Lee, N.S.et al., (2002) Nature Biotechnology 20 : 500-505.).
  • RNA molecule that is antisense to mRNA of CST6 or GABRP is transcribed by a first promoter (e.g., a promoter sequence 3' of the cloned DNA) and an RNA molecule that is the sense strand for the mRNA of CST6 or GABRP is transcribed by a second promoter (e.g., a promoter sequence 5' of the cloned DNA).
  • the sense and antisense strands hybridize in vivo to generate siRNA constructs for silencing of CST6 or GABRP.
  • the two constructs can be utilized to create the sense and anti-sense strands of a siRNA construct.
  • Cloned CST6 or GABRP can encode a construct having secondary structure, e.g., hairpins, wherein a single transcript has both the sense and complementary antisense sequences from the target gene.
  • a loop sequence consisting of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form the hairpin loop structure.
  • the present invention also provides siRNA having the general formula 5'-[A]-[B]-[A']-3', wherein [A] is a ribonucleotide sequence corresponding to a sequence that specifically hybridizes to an mRNA or a cDNA of CST6 or GABRP.
  • [A] is a ribonucleotide sequence corresponding a sequence of CST6 or GABRP
  • [B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides
  • [A'] is a ribonucleotide sequence consisting of the complementary sequence of [A].
  • the region [A] hybridizes to [A'], and then a loop consisting of region [B] is formed.
  • the loop sequence may be preferably 3 to 23 nucleotide in length.
  • the loop sequence for example, can be selected from group consisting of following sequences (http://www.ambion.com/techlib/tb/tb_506.html).
  • loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque, J.-M., et.al, (2002) Nature 418 : 435- 438.).
  • CCC, CCACC or CCACACC Jacque, J. M, et.al, (2002) Nature, Vol. 418: 435-438.
  • UUCG Lee, N.S., et.al, (2002) Nature Biotechnology 20 : 500-505.
  • UUCAAGAGA Dykxhoorn, D. M., et.al, (2003) Nature Reviews Molecular Cell
  • the loop sequence can be selected from group consisting of, CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA.
  • Preferable loop sequence is UUCAAGAGA ("ttcaagaga" in DNA).
  • Exemplary hairpin siRNA suitable for use in the context of the present invention include: for CST6-siRNA
  • nucleotide sequence of suitable siRNAs can be designed using an siRNA design computer program available from the Ambion website (http://www.ambion.com/techlib/ misc/siRNA_fmder.html).
  • the computer program selects nucleotide sequences for siRNA synthesis based on the following protocol.
  • siRNA Target Sites 1. Beginning with the AUG start codon of the object transcript, scan downstream for AA dinucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Tuschl, et al. don't recommend against designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex.
  • the homology search can be performed using BLAST, which can be found on the NCBI server at: www.ncbi.nhn.nih.gov/BLAST/(Altschul SF 3 et. ah, J MoI Biol.
  • siRNAs are transcribed intracellularly by cloning CST6 or GABRP templates, respectively, into a vector containing, e.g., a RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human Hl RNA promoter.
  • snRNA small nuclear RNA
  • transfection-enhancing agent can be used for introducing the vector into the cell.
  • the antisense oligonucleotide or siRNA of the present invention inhibits the expression of a polypeptide of the present invention and is thereby useful for suppressing the biological activity of a polypeptide of the invention.
  • expression-inhibitors comprising the antisense oligonucleotide or siRNA of the invention, are useful in the point that they can inhibit the biological activity of the polypeptide of the invention. Therefore, a composition comprising an antisense oligonucleotide or siRNA of the present invention is useful for treating a pancreatic cancer.
  • function of gene product of CST6 or GABRP which is over-expressed in PDAC can be inhibited by administering a compound that binds to or otherwise inhibits the function of the gene products.
  • the compound is an antibody which binds to the gene product of CST6 or GABRP.
  • the present invention refers to the use of antibodies, particularly antibodies against a protein encoded by CST6 or GABRP, or a fragment of such an antibody.
  • antibody refers to an immunoglobulin molecule having a specific structure, that interacts (i.e., binds) only with the antigen that was used for synthesizing the antibody (i.e., the gene product of an up-regulated marker) or with an antigen closely related thereto.
  • an antibody may be a fragment of an antibody or a modified antibody, so long as it binds to the protein encoded by CST6 or GABRP.
  • the antibody fragment may be Fab, F(ab') 2 , Fv, or single chain Fv (scFv), in which Fv fragments ' from H and L chains are ligated by an appropriate linker (Huston J. S. et al. Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883 (1988)). More specifically, an antibody fragment may be generated by treating an antibody with an enzyme, such as papain or pepsin. Alternatively, a gene encoding the antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co M. S. et al. J. Immunol. 152:2968-2976 (1994); Better M.
  • An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the present invention provides such modified antibodies.
  • the modified antibody can be obtained by chemically modifying an antibody. Such modification methods are conventional in the field.
  • an antibody may comprise a chimeric antibody having a variable region derived from a nonhuman antibody and a constant region derived from a human antibody, or a humanized antibody, comprising a complementarity determining region (CDR) derived from a nonhuman antibody, a frame work region (FR) and a constant region derived from a human antibody.
  • CDR complementarity determining region
  • FR frame work region
  • Such antibodies can be prepared by using known technologies.
  • Humanization can be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (see e.g., Verhoeyen et al, Science 239:1534-1536 (1988)). Accordingly, such humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. Fully human antibodies comprising human variable regions in addition to human framework and constant regions can also be used. Such antibodies can be produced using various techniques known in the art. For example in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage (e.g., Hoogenboom & Winter, J. MoI. Biol. 227:381 (1992).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, e.g., in U.S. Patent Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016.
  • the antbody used in the context of the present invention for treating or alleviating a symptom of PDAC may also be a single chain antibody.
  • Techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies to polypeptide(s) or portions thereof of this invention.
  • Cancer therapies directed at specific molecular alterations that occur in cancer cells have been validated through clinical development and regulatory approval of anti-cancer drugs such as trastuzumab (Herceptin) for the treatment of advanced breast cancer, imatinib mesylate (Gleevec) for chronic myeloid leukemia, gefitinib (Iressa) for non-small cell lung cancer (NSCLC), and rituximab (anti-CD20 mAb) for B-cell lymphoma and mantle cell lymphoma (Ciardiello F.et al, Clin Cancer Res. 2001 Oct;7(10):2958-70. Review.; Slamon DJ.et al, N Engl J Med.
  • modulatory methods can be performed ex vivo or in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the methods involve administering a protein or combination of proteins or a nucleic acid molecule or combination of nucleic acid molecules as therapy to counteract aberrant expression of the differentially expressed genes or aberrant activity of their gene products.
  • Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) expression levels or biological activities of genes and gene products, respectively may be treated with therapeutics that antagonize (i.e., reduce or inhibit) activity of the over-expressed gene or genes.
  • Therapeutics that antagonize activity can be administered therapeutically or prophylactically.
  • therapeutics that may be utilized in the context of the present invention include, e.g., (i) a polypeptide of the over-expressed gene or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to the over-expressed gene or gene products; (iii) nucleic acids encoding the over-expressed gene; (iv) antisense nucleic acids or nucleic acids that are "dysfunctional" (i. e., due to a heterologous insertion within the nucleic acids of over- expressed gene); (v) small interfering RNA (siRNA); or (vi) modulators (i.e., inhibitors, antagonists that alter the interaction between an over-expressed polypeptide and its binding partner).
  • the dysfunctional antisense molecules are utilized to "knockout" endogenous function of a polypeptide by homologous recombination (see, e.g., Capecchi, Science 244: 1288-1292 1989).
  • Increased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a gene whose expression is altered).
  • Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, irnmunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
  • immunoassays e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, irnmunocytochemistry, etc.
  • hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
  • Prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • Therapeutic methods of the present invention may include the step of contacting a cell with an agent that modulates one or more of the activities of the gene products of the differentially expressed genes.
  • agent that modulates protein activity include, but are not limited to, nucleic acids, proteins, naturally-occurring cognate ligands of such proteins, peptides, peptidomimetics, and other small molecule.
  • the present invention also relates to a method of treating or preventing pancreatic cancer in a subject comprising the step of administering to said subject a vaccine comprising a polypeptide encoded by a nucleic acid of CST6 or GABRP, an immunologically active fragment of said polypeptide, or a polynucleotide encoding such a polypeptide or fragment thereof.
  • the present invention also relates to a use a polypeptide encoded by a nucleic acid of CST6 or GABRP, an immunologically active fragment of said polypeptide, or a polynucleotide encoding such a polypeptide or fragment thereof for the preparation of a vaccine composition for treating or preventing pancreatic cancer in a subject.
  • polypeptide induces an anti-tumor immunity in a subject.
  • a polypeptide encoded by a nucleic acid of CST6 or GABRP an immunologically active fragment of said polypeptide, or a polynucleotide encoding such a polypeptide or fragment thereof is administered to subject in need thereof.
  • the polypeptide encoded by a nucleic acid of CST6 or GABRP may induce anti-tumor immunity against invasion of pancreatic cancer.
  • the polypeptide or the immunologically active fragments thereof are useful as vaccines against PDAC.
  • the proteins or fragments thereof may be administered in a form bound to the T cell receptor (TCR) or presented by an antigen presenting cell (APC), such as macrophage, dendritic cell (DC), or B- cells. Due to the strong antigen presenting ability of DC, the use of DC is most preferable among the APCs.
  • TCR T cell receptor
  • APC antigen presenting cell
  • DC dendritic cell
  • B- cells B- cells. Due to the strong antigen presenting ability of DC, the use of DC is most preferable among the APCs.
  • a vaccine against PDAC refers to a substance that has the ability to induce anti-tumor immunity upon inoculation into animals.
  • polypeptides encoded by CST6 or GABRP or fragments thereof were suggested to be HLA-A24 or HLA-A* 0201 restricted epitopes peptides that may induce potent and specific immune response against PDAC cells expressing CST6 or GABRP.
  • the present invention also encompasses a method of inducing anti-tumor immunity using the polypeptides.
  • anti-tumor immunity includes immune responses such as follows: induction of cytotoxic lymphocytes against tumors, - induction of antibodies that recognize tumors, and - induction of anti-tumor cytokine production.
  • the protein when a certain protein induces any one of these immune responses upon inoculation into an animal, the protein is determined to have anti-tumor immunity inducing effect.
  • the induction of the anti-tumor immunity by a protein can be detected by observing in vivo or in vitro the response of the immune system in the host against the protein.
  • cytotoxic T lymphocytes For example, a method of detecting the induction of cytotoxic T lymphocytes is well known. Specifically, a foreign substance that enters the living body is presented to T cells and B cells by the action of antigen presenting cells (APCs). T cells that respond to the antigen presented by the APCs in an antigen specific manner differentiate into cytotoxic T cells (or cytotoxic T lymphocytes; CTLs) due to stimulation by the antigen, and then proliferate (this is referred to as activation of T cells). Therefore, CTL induction by a certain peptide can be evaluated by presenting the peptide to a T cell via an APC, and detecting the induction of CTLs.
  • APCs antigen presenting cells
  • APCs have 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 anti-tumor immunity, the anti-tumor immunity-inducing action of the peptide can be evaluated using the activation effect of these cells as indicators.
  • DCs dendritic cells
  • a method of evaluating the inducing action of CTLs using dendritic cells (DCs) as the APC is well known in the art.
  • DCs are a representative APCs having the strongest CTL- inducing action among APCs.
  • the test polypeptide is initially contacted with DCs 3 and then the DCs are contacted with T cells.
  • Detection of T cells having cytotoxic effects against the cells of interest after the contact with DC shows that the test polypeptide has an activity of inducing the cytotoxic T cells.
  • Activity of CTLs against tumors can be detected, for example, using the lysis of 51 Cr-labeled tumor cells as the indicator.
  • PBMCs peripheral blood mononuclear cells
  • LDH lactose dehydrogenase
  • APC peripheral blood mononuclear cells
  • CTLs have been reported to be enhanced by culturing PBMCs in the presence of GM-CSF and IL-4.
  • CTLs have been shown to be induced by culturing PBMCs in the presence of keyhole limpet hemocyanin (KLH) and IL-7.
  • KLH keyhole limpet hemocyanin
  • Test polypeptides confirmed to possess CTL-inducing activity by these methods are deemed to be polypeptides having DC activation effect and subsequent CTL-inducing activity. Therefore, polypeptides that induce CTLs against tumor cells are useful as vaccines against tumors. Furthermore, APCs that have acquired the ability to induce CTLs against rumors through contact with the polypeptides are also useful as vaccines against tumors. Furthermore, CTLs that have acquired cytotoxicity due to presentation of the polypeptide antigens by APCs can be also used as vaccines against tumors. Such therapeutic methods of tumors, using antitumor immunity due to APCs and CTLs, are referred to as cellular immunotherapy. Generally, when using a polypeptide for cellular immunotherapy, efficiency of the
  • CTL-induction is known to be increased by combining a plurality of polypeptides having different structures and contacting them with DCs. Therefore, when stimulating DCs with protein fragments, it is advantageous to use a mixture of multiple types of fragments.
  • the induction of anti-tumor immunity by a polypeptide can be confirmed by observing the induction of antibody production against tumors. For example, when antibodies against a polypeptide are induced in a laboratory animal immunized with the polypeptide, and when growth of tumor cells is suppressed by those antibodies, the polypeptide is deemed to have the ability to induce anti-tumor immunity.
  • Anti-tumor immunity is induced by administering the vaccine of this invention, and the induction of anti-tumor immunity enables treatment and prevention of PDAC.
  • Therapy against cancer or prevention of the onset of cancer includes any of the following steps, such as inhibition of the growth of cancerous cells, involution of cancer, and suppression of the occurrence of cancer.
  • a decrease in mortality and morbidity of individuals having cancer, decrease in the levels of tumor markers in the blood, alleviation of detectable symptoms accompanying cancer, and such are also included in the therapy or prevention of cancer.
  • Such therapeutic and preventive effects are preferably statistically significant. For example, in observation, at a significance level of 5% or less, wherein the therapeutic or preventive effect of a vaccine against cell proliferative diseases is compared to a control without vaccine administration.
  • Student's t-test, the Mann- Whitney U-test, or ANOVA may be used for statistical analysis.
  • the above-mentioned protein having immunological activity or a vector encoding the protein may be combined with an adjuvant.
  • An adjuvant refers to a compound that enhances the immune response against the protein when administered together (or successively) with the protein having immunological activity.
  • Exemplary adjuvants include, but are not limited to, cholera toxin, salmonella toxin, alum, and such, but are not limited thereto.
  • the vaccine of this invention may be combined appropriately with a pharmaceutically acceptable carrier. Examples of such carriers include sterilized water, physiological saline, phosphate buffer, culture fluid, and such.
  • the vaccine may contain as necessary, stabilizers, suspensions, preservatives, surfactants, and such.
  • the vaccine can be administered systemically or locally. Vaccine administration can be performed by single administration, or boosted by multiple administrations.
  • tumors can be treated or prevented, for example, by the ex vivo method. More specifically, PBMCs of the subject receiving treatment or prevention are collected, the cells are contacted with the polypeptide ex vivo, and following the induction of APCs or CTLs, the cells may be administered to the subject.
  • APCs can be also induced by introducing a vector encoding the polypeptide into PBMCs ex vivo.
  • APCs or CTLs induced in vitro can be cloned prior to administration. By cloning and growing cells having high activity of damaging target cells, cellular immunotherapy can be performed more effectively.
  • APCs and CTLs isolated in this manner may be used for cellular immunotherapy not only against individuals from whom the cells are derived, but also against similar types of tumors from other individuals.
  • a pharmaceutical composition for treating or preventing a cell proliferative disease, such as cancer comprising a pharmaceutically effective amount of the polypeptide of the present invention is provided.
  • the pharmaceutical composition may be used for raising ami tumor immunity.
  • Pharmaceutical compositions for inhibiting PDAC include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration, or for administration by inhalation or insufflation. Preferably, administration is intravenous.
  • the formulations are optionally packaged in discrete dosage units.
  • compositions suitable for oral administration include capsules, cachets or tablets, each containing a predetermined amount of active ingredient. Suitable formulations also include powders, granules, solutions, suspensions and emulsions. The active ingredient is optionally administered as a bolus electuary or paste. Tablets and capsules for oral administration may contain conventional excipients, such as binding agents, fillers, lubricants, disintegrant and/or wetting agents. A tablet may be made by compression or molding, optionally with one or more formulational ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active and/or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be coated according to methods well known in the art. Oral fluid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives, such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), and/or preservatives.
  • the tablets may optionally be formulated so as to provide slow or controlled release of the active ingredient therein.
  • a package of tablets may contain one tablet to be taken on each of the month.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, optionally contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; as well as aqueous and non-aqueous sterile suspensions including suspending agents and/or thickening agents.
  • the formulations may be presented in unit dose or multi-dose containers, for example as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition, requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use.
  • the formulations may be presented for continuous infusion.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Formulations suitable for rectal administration include suppositories with standard carriers such as cocoa butter or polyethylene glycol.
  • Formulations suitable for topical administration in the mouth include lozenges, containing the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles, comprising the active ingredient in a base such as gelatin and glycerin or sucrose and acacia.
  • the compounds of the invention may be used as a liquid spray, a dispersible powder, or in the form of drops. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents and/or suspending agents.
  • the compounds can be conveniently delivered from an insufflator, nebulizer, pressurized packs or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichiorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the compounds may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base, such as lactose or starch.
  • a powder composition may be presented in unit dosage form, for example, as capsules, cartridges, gelatin or blister packs, from which the powder may be administered with the aid of an inhalator or insufflators.
  • formulations include implantable devices and adhesive patches which release a therapeutic agent.
  • compositions adapted to give sustained release of the active ingredient, may be employed.
  • the pharmaceutical compositions may also contain other active ingredients, such as antimicrobial agents, immunosuppressants and/or preservatives.
  • formulations of this invention may include other agents conventional in the art with regard to the type of formulation in question.
  • formulations suitable for oral adtninistration may include flavoring agents.
  • Preferred unit dosage formulations contain an effective dose, as recited below, or an appropriate fraction thereof, of the active ingredient.
  • the compositions e.g., polypeptides and organic compounds
  • the dose range for adult humans is generally from about 5 mg to about 17.5 g/day, preferably about 5 mg to about 10 g/day, and most preferably about 100 mg to about 3 g/day.
  • Tablets or other unit dosage forms of presentation provided in discrete units may conveniently contain an amount which is effective at such dosage or as a multiple of the same, for instance, units containing about 5 mg to about 500 mg, usually from about 100 mg to about 500 mg.
  • the dose employed will depend upon a number of factors, including the age and sex of the subject, the precise disorder being treated, and its severity. Also the route of administration may vary depending upon the condition and its severity. In any event, appropriate and optimum dosages may be routinely calculated by those skilled in the art, taking into consideration the above-mentioned factors.
  • PDAC cell lines KLM-I, SUTT-?, KP-IN, PK-I PK-45P and PK-59 were provided from Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan), Cos7 was purchased from the American Type Culture Collection (ATCC 5 Rockville, MD). MIAPaCa-2 and Panc-1 were purchased from the American Type Culture Collection (ATCC, Rockville, MD). All cell lines were grown in RPMIl 640 (Sigma- Aldorich, St. Louis, MO), supplemented with 10% fetal bovine serum (Cansera International, Ontario, Canada) and 1% antibiotic/antimycotic solution (Sigma- Aldorich).
  • RNA from the purified PDAC cells and normal pancreatic ductal epithelial cells were subjected to two rounds of RNA amplification using T7-based in vitro transcription (Epicentre Technologies, Madison, WI) and synthesized to single-strand cDNA.
  • Total RNA from human pancreatic cancer cell lines was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommended procedures.
  • the present inventors prepared appropriate dilutions of each single-stranded cDNA for subsequent PCR amplification by monitoring a-tubulin (TUBA) as a quantitative control.
  • the primer sequences used were 5'-AAGGATTATGAGGAGGTTGGTGT -3' (SEQ ID NO.l) and 5'-CTTGGGTCTGTAACAAAGCATTC -3' (SEQ ID NO.2) for TUBA, 5'- GGCAGCAACAGCATCTACTACTT-3' (SEQ ID NO.3) and
  • MIAPaCa-2, Panc-1, PK-I, and SUIT-2) and several adult normal tissues were blotted to the membrane.
  • Probe cDNA of CST6 was prepared as a 255-bp PCR product by using primers
  • probe cDNA of GABRP was prepared as a 958-bp PCR product by using primers
  • a cDNA fragment encoding the human CST6 protein lacking its signal peptide was amplified by PCR using primers 5'-CGC GGA TCC GCC GCA GGA GCG CAT GGT COGS' (SEQ ID NO.13) and 5'-CCG GAA TTC TCA CAT CTG CAC ACA GTT GTG -3' (SEQ ID NO.14), which contained BamHI and EcoRI restriction sites indicated by the first and second underlines, respectively.
  • the product was cloned into pET28b vector (Novagen, Madison, WI) to produce a fusion protein, bearing an N-terminal 6-His tag, which was purified with TALONTM Superflow Metal Affinity Resin (BD Biosciences, Franklin Lakes, NJ) under native condition according to the supplier's protocol.
  • This recombinant CST6-6His was used to immunize rabbits; the resulting polyclonal antibody was affinity-purified using Affi-gel 10 (Bio-Rad Laboratories, Hercules, CA) conjugated with the 6-histidine fused CST6 protein. Immunohistochemical staining.
  • tissue sections from PDACs were obtained from surgical specimens that were resected under the appropriate informed consent.
  • the sections were deparaffinized and autoclaved at 108 0 C in citrate buffer, pH6.0 for 15 min. Endogenous peroxidase activity was quenched by incubation in Peroxidase Blocking Reagent (Dako Cytomation, Carpinteria, CA) for 30 min. After incubated with fetal bovine serum for blocking, the sections were incubated with rabbit anti-CST6 polyclonal antibody (dilution 1:5000) at room temperature for 1 hour.
  • RNA-expressing constructs specific to CST6 or GABRP Small interfering RNA (siRNA)-expressing constructs specific to CST6 or GABRP.
  • siRNA small interfering RNA
  • psiU6BX3.0 vector was used for expression of short hairpin RNA against a target gene as described previously (Taniucbi K, et ah, Cancer Res 2005; 65: 105-12.).
  • the U6 promoter was cloned upstream of the gene-specific sequence (19-nt sequence from the target transcript, separated from the reverse complement of the same sequence by a short spacer, TTCAAGAGA), with five thymidines as a termination signal and a neo cassette for selection by Geneticin (Invitrogen).
  • the target sequences were 5'-GTGGTTCCCTGGCAGAACT-3 ' (SEQ ID NO.15) for CST6-#448, 5'-GATGGGCAGGATTGTTGAT-S' (SEQ ID NO.19) for GABRP- ⁇ , 5'-TATCATCAACAGCTCCATC-S' (SEQ ID NO.23) for GABRP-s ⁇ , 5'-CCCCAGTAATGTTGATCAC-3' (SEQ ID NO.27) for GABRP-siS, 5'-AGGAAGTAGAAGAAGTCAG-S' (SEQ ID NO.31) for GABRP-silO, and 5'-GAAGCAGCACGACTTCTTC-S' (SEQ ID NO.35) for EGFP as a negative control.
  • the siRNA sequences used for the experiment are shown below:
  • Cells were selected by 0.15 mg/ml (PK-59), 0.2 mg/ml (PK-I) or 500 ⁇ g/ml (PK-45P, KLM- 1) Geneticin for 9 days. Preliminarily, cells from 10-cm dishes were harvested 3 days to analyze knockdown effect on CST6 and 7 days on GABRP by RT-PCR using the above primers.
  • the cDNA encoding an open reading frame of CST6 was amplified by PCR using the primer pair with restriction enzyme sites; 5'-GGGGTACCGAATGGCGCGTTCGAACCTCC -3' (SEQ ID NO.39) and 5'- CCGGAATTCCATCTGCACACAGTTGTGCT -3' (SEQ ID NO.40) (Kpnl and EcoRI sites shown by underlines, respectively).
  • the PCR-amplified product was cloned into pcDNA3.1/myc-His A(+) vector (Invitrogen).
  • the plasmids were transfected into the CST6-null PDAC cell line KLM-I using FuGENE ⁇ (Roche) according to the manufacturer's recommended procedures.
  • a population of cells was selected with 0.5 mg/ml Geneticin (Invitrogen), and clonal KLM-I cells were sub-cloned by limiting dilution.
  • Myc-tagged CST6 expression in these clonal cells was assessed by Western blotting using anti-myc antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and anti- ⁇ -actin antibody (Sigma), and three clones that expressed CST6 constitutively were established (KLMl -CST6).
  • Control KLM-I cells transfected with pcDNA3.1/myc-His A(+) vector was also established (KLMl -Mock). The growth curve of these established clones were measured by using Cell- counting kit-8 (DOJINDO). CST6 autocrine/paracrine assay.
  • Mature recombinant human CST6 was purchased from R&D system (Minneapolis, MN), which was generated in mammalian cells. Mature recombinant CST6 was added to culture medium of COS7 cells at several concentration (0, 0.02, 0.2, and 2 ng/ml), supplied with 2 %FBS. The growth curves of cells in the presence of each concentration of CST6 were measured by using Cell-counting kit-8 (DOJINDO).
  • GABRP-positive cell lines KLM-I and PK-45P
  • GABRP-negative cell lines PK-
  • GABA GABA A receptor antagonist BMI (Bicuculline methiodide, Sigma) at 250 ⁇ M or GABA B receptor antagonist CGP-35348 (Sigma) at 1 mM.
  • BMI Bicuculline methiodide, Sigma
  • CGP-35348 GABA B receptor antagonist CGP-35348
  • the present inventors constructed several siRNA-expression vectors specific to CST6 mRNA sequences and transfected them into PK-I and PK-59, PDAC cell lines that endogenously express high levels of CST6.
  • a knockdown effect was confirmed by RT-PCR when #448 (Fig. 2A) was used.
  • Colony-formation assays (Fig. 2B) and MTT assays (Fig. 2C) using PK-59 revealed a drastic reduction in the number of cells transfected with #448, compared to a negative control EGFP for which no knockdown effect was apparent. Similar effects were obtained with the PK-I cell line (Fig. 2A, B, and C).
  • the effective siRNA did not affect the cell viability of other PDAC cell line KLM-I that did not express CST6 (data not shown). Over-expressed CST6 promoted PDCA cell growth.
  • the present inventors established cell lines that expressed wild-type CST6 stably and constitutively using KLM-I cells in which CST6 expression was hardly detectable by RT- PCR. As shown in Fig. 3 A, a high level of expression of three KLM-I clones (CST6-1, -2 and —3) was confirmed by Western blot analysis using anti-myc antibody, but no expression was detected in three KLMl -Mock clones (Mock-1, -2 and -3). The present inventors also confirmed CST6 protein abundant in culture medium from each of theses KLM1-CST6 clone (data not shown).
  • CST6 is a secreted protein and is likely to function mainly extracellularly.
  • cell growth assay was performed in the presence of several concentration (0.02-2 ng/ml) of mature human recombinant CST6. This recombinant CST6 protein was generated in mammalian cells and was validated to be N-glycosylated. Fig.
  • the present inventors also analyzed GABRP expression of several PDCA cell lines and Fig. 5B showed GABRP was expressed evidently in KLM-I, PK-45P and PK-I, but not in vital organs including heart, lung, liver, kidney, and brain. Furthermore, to investigate local GABA production in pancreatic cancer tissues, the expression of glutamate decarboxylase 1 (GADl) was also analyzed, which mainly in neurons catalyzes the production of GABA, in PDAC cells, normal pancreatic ductal cells, and normal pancreas by RT-PCR. Fig. 5 C showed GADl expression was up-regulated in PDAC cells as well as GABRP, suggesting that PDAC cells produce GABA by themselves. Effect of GABRP -siRNAs on growth of PDAC cells. The present inventors constructed several siRNA-expression vectors specific to
  • GABRP-positive or -negative PDAC cell lines were incubated with GABA at several concentrations.
  • GABA in the culture medium dose-dependently promoted the proliferation of GABRP-positive PDAC cell line KLM-I and PK-45P (upper panel) with approximately 50% promoting effect on cell proliferation, at most, but not GABRP-negative PDAC cell line, PK-59 and KP-IN (lower panel).
  • GABA antagonists were analyzed.
  • GABA A receptor antagonist BMI
  • GABA B receptor antagonist BMI
  • GABA A receptor antagonist BMI
  • GABA B receptor antagonist did not affect GABA-stimulated cell proliferation
  • GABRP-negative PDAC cell lines did not respond to GABA or GABA receptor antagonists (Fig. 7B lower panel)
  • the solo treatment with GABAA receptor antagonist BMI suppressed cell proliferation in KLM-I and PK-59 cells (Fig.
  • the present inventors focused on the CST6 gene among dozens of genes that were identified to be over-expressed in PDACs through previous genome-wide gene expression analysis (Nakamura T, et. ah, Oncogene 2004; 23: 2385-400.). Immunohistochemical analysis showed that CST6 was over-expressed in some populations of PDACs (60%), and expression analysis in RNA and protein level showed that CST6 was expressed at very low level in adult normal vital organs (lung, heart, liver, and kidney). These findings would be essential to select a molecular target for a novel therapeutic approach with minimal side effect, and CST6 is likely to be the promising molecular target for PDCA treatment in the aspect of its expression pattern.
  • CST6 secreted CST6 or overexpressed CST6 promoted cell proliferation in vitro and, inversely, depletion of CST6 by siRNA in PDAC cells attenuated their growth or viability.
  • CST6 was up-regulated in head and neck cancers and provided anti-apoptotic property to cancer cells and promoted metastasis (VigneswaranN, et. al, Oral Oncology 2003; 39: 559-68.), and up-regulation of rat CST6 was suggested to involve in neural cell differentiation and development also (Hong J, et. al, J Neurochem. 2002; 81: 922-34.)- Functionally, CST6 is an inhibitor of cysteine proteinases and indeed it has a potential to inhibit cathepsin B (Ni J 3 et. al, J Biol Chem. 1997; 272:10853-8., Hong J, et. al, J Neurochem.
  • cystatin family member cystatin C (CST3) stimulated cell growth (Tavera C, et. al., Biochem Biophys Res Commun. 1992; 182: 1082-8.) and can act as a TGF-beta receptor antagonist (Sokol JP, et. al., MoI Cancer Res. 2004; 2:183-95.), involving in TGF-beta pathway in cell proliferation and cancer invasion.
  • Cystatin C stimulated the proliferation of neural stem cells (Taupin P, et. al., Neuron. 2000; 28: 385-97) as an autocrine / paracrine cofactor with FGF-2, and its proteinase inhibitory activity is not essential to its growth effect on neural stem cells. This indicates that other functions than proteinase inhibitor of cystatin family may involve cell growth promotion.
  • GABA receptor ⁇ GABA receptor ⁇
  • GABRP GABA receptor ⁇
  • GABA and GABA receptors function primarily as an inhibitory neurotransmitter, and it can also act as a trophic factor during nervous system development to influence proliferation, migration and differentiation of neural stem cells and others (Macdonald RL, Olsen RW., Amu Rev Neurosci 1994; 17: 569-602., Fiszman ML, Brain Res Dev Brain Res 1999; 115: 1-8.).
  • Macdonald RL Olsen RW.
  • Amu Rev Neurosci 1994; 17: 569-602. Fiszman ML, Brain Res Dev Brain Res 1999; 115: 1-8.
  • GABA and GABA receptors are expressed in non-neural tissues, and their precise function in non-neuronal cells is presently unknown (Macdonald RL, Olsen RW., Annu Rev Neurosci 1994; 17: 569-602.).
  • GABA and GABA B receptor pathway could involve prostate cancer metastasis or invasion through regulation of MMP production.
  • GABA could inhibit colon cancer migration associated with nor-epinepherin-induced pathway (Joseph J, et. ⁇ /.,Cancer Res.
  • GABA pathway can act positively or negatively on cancer cell behavior, but the findings apparently showed GABA and GABA A receptor complex incorporated by ⁇ subunit should promote PDAC cell proliferation.
  • GABA-producing enzyme GADl was also up-regulated in PDAC cells, as we showed here, and the local concentration of GABA is expected to be at high level in PDAC tissues. GABA is also likely to function in the autocrine/ paracrine manner in pancreatic cancer tissues to promote cancer cell growth.
  • GABRP GABA A receptor ⁇ subunit
  • pancreatic cancer described herein ? obtained through " a genome- wide cDNA microarray, has identified specific genes as targets for cancer prevention and therapy. Based on the expression of a subset of these differentially expressed genes, the present invention provides molecular diagnostic markers for identifying and detecting pancreatic cancer.
  • the methods described herein axe also useful in the identification of additional molecular targets for prevention, diagnosis and treatment of pancreatic cancer.
  • the data reported herein add to a comprehensive understanding of pancreatic cancer, facilitate development of novel diagnostic strategies, and provide clues for identification of molecular targets for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of pancreatic tumorigenesis, and provide indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of pancreatic cancer.

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Abstract

L'invention concerne des méthodes objectives de détection et de diagnostic du cancer du pancréas (PDAC). Dans un mode de mise en oeuvre, cette méthode de diagnostic consiste à mesurer le niveau d'expression de CST6 ou de GABRP, qui diffère entre les cellules du cancer pancréatique et les cellules normales. Enfin, cette invention concerne des méthodes de criblage permettant la sélection d'agents thérapeutiques qui peuvent servir au traitement du cancer du pancréas, des méthodes de traitement du cancer du pancréas, et une méthode permettant de vacciner un sujet contre le cancer du pancréas.
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