WO2016003231A1 - Composition pour le diagnostic ou le traitement du cancer du pancréas utilisant kiaa1199 - Google Patents

Composition pour le diagnostic ou le traitement du cancer du pancréas utilisant kiaa1199 Download PDF

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WO2016003231A1
WO2016003231A1 PCT/KR2015/006855 KR2015006855W WO2016003231A1 WO 2016003231 A1 WO2016003231 A1 WO 2016003231A1 KR 2015006855 W KR2015006855 W KR 2015006855W WO 2016003231 A1 WO2016003231 A1 WO 2016003231A1
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pancreatic cancer
protein
seq
cells
antibody
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PCT/KR2015/006855
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Korean (ko)
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송시영
장찬영
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연세대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • 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
    • 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

Definitions

  • the present invention relates to a composition for diagnosing or treating pancreatic cancer using KIAA1199.
  • Cancer stem cells that maintain and regenerate cancerous tissues, like normal organs, are believed to be involved in the initial onset of cancer, as well as reduced cancer cells after cancer treatment. Involvement in regeneration has been reported to have a profound effect on cancer recurrence or metastasis and induction of chemotherapy resistance (BB Zhou et al. Nature Reviews Drug Discovery , 8: 806-823 (2009)). Therefore, in order to fundamentally diagnose and treat cancer, it is necessary to focus on cancer stem cells that occupy only a small part of cancer tissues and play a key role in the development, maintenance, and recurrence of cancer. It will also help to develop diagnostic markers.
  • Pancreatic cancer is a fatal cancer with a 5-year survival rate of 1-4% and a median survival of 5 months. It has the poorest prognosis among human cancers. Since the prognosis is poor and treatment is mainly dependent on chemotherapy, 80-90% of patients are found in a state where curative resection is not possible.
  • Cancer stem cells refer to cancer cells capable of self-renewal and have recently emerged as a major target for cancer treatment and conquest.
  • cancers occurring in each organ of living body are known to have molecular patterns distinguished according to the producing organs.
  • the present inventors have reported and characterized the molecular markers of new pancreatic cancer stem cells in pancreatic carcinoma that does not have clear molecular markers.
  • the present inventors have made diligent research efforts to discover novel biomarkers for pancreatic cancer stem cells and targets for the treatment of pancreatic cancer based on pancreatic cancer stem cell characteristics.
  • KIAA1199 which is specifically expressed in pancreatic cancer stem cells, may be a molecular target for pancreatic cancer treatment.
  • the biomarkers discovered are diagnostic markers, particularly early diagnosis of pancreatic cancer based on cancer stem cell biological characteristics, markers for determining prognosis, and markers for future therapeutic targets. The invention was completed.
  • Another object of the present invention to provide a pancreatic cancer stem cell detection kit.
  • Still another object of the present invention is to provide a kit for diagnosing or prognosticting pancreatic cancer.
  • Another object of the present invention to provide a composition for inhibiting pancreatic cancer stem cell formation.
  • Another object of the present invention to provide a pharmaceutical composition for treating or inhibiting metastasis of pancreatic cancer.
  • the present invention provides a method for screening a substance for preventing or treating pancreatic cancer, comprising the following steps:
  • step (b) measuring the expression level of the protein or polynucleotide in step (a), wherein when the sample reduces the expression of the protein or polynucleotide, the sample is a substance for preventing or treating pancreatic cancer. It is determined.
  • the present inventors have made diligent research efforts to discover novel biomarkers for cancer stem cells and therapeutic targets for cancer based on pancreatic cancer stem cell characteristics. As a result, the present inventors found that KIAA1199, which is specifically expressed in pancreatic cancer stem cells, may be a molecular target for pancreatic cancer treatment. In addition, the inventors have found that the biomarkers discovered are diagnostic markers, particularly early diagnosis of pancreatic cancer, markers for determining prognosis and markers for future therapeutic targets based on cancer stem cell biological properties.
  • a protein specifically expressed in pancreatic cancer stem cells isolated from a pancreatic cancer cell line may be a therapeutic target for pancreatic cancer, and thus, the pancreatic cancer may be diagnosed early and analyze the prognosis very early.
  • the marker of the present invention is specifically expressed in pancreatic cancer stem cells. Moreover, the marker is a marker showing an expression pattern that is highly expressed in pancreatic cancer stem cells compared with pancreatic cancer cells, that is, the ability to distinguish between pancreatic cancer cells and pancreatic cancer stem cells.
  • the expression "cell or tissue” used for the screening of a substance for preventing or treating pancreatic cancer is preferably pancreatic cancer stem cells or pancreatic cancer tissue.
  • pancreatic cancer refers to a cancer that originates in pancreatic cells.
  • pancreatic adenocarcinoma of the pancreatic ducts accounts for about 90% of the pancreatic cancer.
  • cystic cancer cystic adenocarcinoma
  • endocrine tumors and the like.
  • pancreatic cancer patients have hereditary predisposition, and in the case of pancreatic cancer patients, family history of pancreatic cancer is about 7.8%, which is more frequent than the general incidence of pancreatic cancer of 0.6%.
  • Pancreatic cancer has a very poor prognosis with a 5 year survival rate of less than 5%.
  • pancreatic cancer stem cells compared to pancreatic cancer cells.
  • pancreatic cancer stem cells the specific remarkable increase in the expression of a target protein or polynucleotide encoding the same in only pancreatic cancer stem cells indicates that it is an essential factor for the survival of pancreatic cancer stem cells.
  • a pancreatic cancer therapeutic agent By inducing growth inhibition and death of stem cells, it is determined to be a substance useful for the fundamental treatment of pancreatic cancer, that is, a pancreatic cancer therapeutic agent.
  • pancreatic cancer therapeutic agent identified by the screening method of the present invention does not target only general pancreatic cancer cells, which occupy most of the cancer tissue, but occupies only a small portion of the cancer tissue, but also plays a key role in the development, maintenance, and recurrence of pancreatic cancer.
  • Targeting the stromal cells enables the fundamental treatment of pancreatic cancer.
  • the sample used in step (a) is a single compound or a mixture of compounds (eg a natural extract or a cell or tissue culture).
  • Test substances can be obtained from libraries of synthetic or natural compounds. Methods of obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA). ) And MycoSearch (USA).
  • Samples can be obtained by a variety of combinatorial library methods known in the art, for example biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution required By a synthetic library method, a “1-bead 1-compound” library method, and a synthetic library method using affinity chromatography screening.
  • Methods of synthesizing molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem.
  • the present invention provides a method for screening a substance for preventing or treating pancreatic cancer, comprising the following steps:
  • test substance binds to the protein or whether the test substance inhibits the function of the protein; When the test substance binds to the protein or inhibits the function of the protein, it is determined as a substance for preventing or treating pancreatic cancer.
  • a test substance is contacted with a protein of SEQ ID NO: 1.
  • the protein used in the present invention may be in a form displayed on a cell surface, a form displayed on a virus (eg, bacteriophage) surface, an isolated form, or a purified form.
  • a virus eg, bacteriophage
  • the cell or virus In the case of using a protein exhibited on the cell surface or on the virus surface, it is preferable to immobilize the cell or virus on a solid substrate in order to speed up or automate screening. It is also desirable to immobilize the isolated or purified form of the protein onto a solid substrate.
  • Available as substrates can be any conventionally used in the art, including, but not limited to, hydrocarbon polymers such as polystyrene and polypropylene, glass, metals and gels.
  • Solid phase substrates may be provided in the form of dipsticks, microtiter plates, particles (eg beads), affinity columns and immunoblot membranes (eg polyvinylidene fluoride membranes). See US Pat. No. 5,143,825. 5,374,530, 4,908,305 and 5,498,551). Most preferably, the solid substrate is a microtiter plate.
  • the screening methods of the present invention can be carried out in a variety of ways, in particular in a high throughput manner according to various binding assays known in the art.
  • the test substance or the protein may be labeled with a detectable label.
  • the detectable label may be a chemical label (eg biotin), an enzyme label (eg horseradish peroxidase, alkaline phosphatase, peroxidase, luciferase, ⁇ -galacto Cedase and ⁇ -glucosidase), radiolabels (eg C 14 , I 125 , P 32 and S 35 ), fluorescent labels [eg coumarin, fluorescein, fluoresein Isothiocyanate (FITC), rhodamine 6G (rhodamine) 6G), rhodamine B, 6-carboxytetramethyl-rhodamine, TAMRA, Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino-2-phenylindole), HEX, TET , Dabsyl and FAM], luminescent labels,
  • FITC fluorescein
  • the binding between the protein and the test substance can be analyzed by detecting a signal from the label.
  • a signal from the label For example, when alkaline phosphatase is used as a label, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS-B1-phosphate) Signal is detected using a chromogenic reaction substrate such as) and enhanced chemifluorescence (ECF).
  • BCIP bromochloroindolyl phosphate
  • NBT nitro blue tetrazolium
  • naphthol-AS-B1-phosphate naphthol-AS-B1-phosphate
  • ECF enhanced chemifluorescence
  • hose radish peroxidase When hose radish peroxidase is used as a label, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amplex Red Reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and substrates such as naphthol / pyronin to detect the signal.
  • Amplex Red Reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-pheny
  • binding of the test substance to the protein may be analyzed without labeling the interactants.
  • a microphysiometer can be used to analyze whether the test substance binds to QP-C.
  • Microphysiometers are analytical tools that measure the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). The change in acidification rate can be used as an indicator for binding between test substance and QP-C (McConnell et al., Science 257: 19061912 (1992)).
  • LAPS light-addressable potentiometric sensor
  • BIA bimolecular interaction analysis
  • the screening method of the present invention can be carried out according to a two-hybrid analysis or a three-hybrid analysis method (US Pat. No. 5,283,317; Zervos et al., Cell 72, 223232, 1993; Madura et al., J.). Biol. Chem. 268, 1204612054, 1993; Bartel et al., BioTechniques 14, 920924, 1993; Iwabuchi et al., Oncogene 8, 16931696, 1993; and W0 94/10300).
  • the protein may be used as a bait protein. According to this method, it is possible to screen substances, particularly proteins, which bind to the proteins.
  • Two-hybrid systems are based on the modular nature of the transcription factors composed of cleavable DNA-binding and activation domains.
  • this assay uses two DNA constructs.
  • the polynucleotide of SEQ ID NO: 2 sequence is fused to a DNA binding domain-encoding polynucleotide of a known transcription factor (eg, GAL-4).
  • a DNA sequence encoding a protein of interest (“prey” or “sample” is fused to a polynucleotide encoding the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factors are contiguous, which triggers transcription of the reporter gene (eg, LacZ).
  • the reporter gene eg, LacZ
  • Expression of the reporter gene can be detected, which indicates that the protein of analysis can bind to the protein, and consequently, it can be used as a material for preventing or treating cancer.
  • the sample used in the present invention is a peptide, antibody, peptide aptamer, AdNectin, affibody (US Pat. No. 5,831,012), Avimer (Avimer, Silverman, J. et al, Nature Biotechnology 23 (12): 1556 (2005)) or Kunitz domain (Kunitz domain, Arnoux B et al., Acta Crystallogr. D Biol. Crystallogr. 58 (Pt 7): 12524 (2002)) , And Nixon, AE, Current opinion in drug discovery & development 9 (2): 2618 (2006).
  • the present invention provides a pancreatic cancer stem cell detection kit comprising a binding agent that specifically binds to a protein of SEQ ID NO: 1 or a primer or probe that binds to a polynucleotide of SEQ ID NO: 2 To provide.
  • a binding agent that specifically binds to a protein may be, for example, an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a PNA (Peptide nucleic acid) or an aptamer. to be.
  • the pancreatic cancer stem cell detection kit of the present invention measures the expression level of each of the marker proteins or polynucleotides encoding the protein from human pancreatic cell samples, and the expression level of the protein or nucleotide of the normal control sample is measured. It can carry out by the method of comparing with.
  • the normal control sample is a sample already confirmed that does not include pancreatic cells, cancer-free pancreatic cells or cancer stem cells obtained from a human who does not have cancer, for example, cancer stem cells such as not forming a sphere under non-adhesive culture conditions.
  • the expression level of the protein or polynucleotide encoding the same in the normal control sample can also be measured using the same method as described above.
  • the expression level of the protein or the polynucleotide encoding the same in the normal control group and the expression level of the protein or the polynucleotide encoding the same in the pancreatic cancer patient to be detected can be compared, and a significant change in the expression level By determining whether the cancer stem cells in the pancreatic cancer patient sample can be diagnosed.
  • pancreatic cancer stem cells when the expression level of the protein or each polynucleotide encoding the same in the patient sample is 150% or more of the expression level of the protein or the polynucleotide encoding the normal control sample, it is determined to include pancreatic cancer stem cells.
  • the present invention is for pancreatic cancer diagnosis or prognostic analysis comprising a binding agent that specifically binds to a protein of SEQ ID NO: 1 or a primer or probe that binds to a polynucleotide of SEQ ID NO: 2 Provide the kit.
  • the kit for pancreatic cancer diagnosis or prognosis analysis of the present invention is a kit derived from pancreatic cancer stem cells.
  • the expression “kit for diagnosing or prognosticting pancreatic cancer” refers to a kit including a composition for diagnosing or prognosticing pancreatic cancer. Therefore, the expression “kit for diagnosis or prognosis of pancreatic cancer” can be used interchangeably or mixed with "composition for diagnosis or prognosis of pancreatic cancer”.
  • diagnosis refers to determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a particular disease or condition, or as long as a person has a particular disease or condition. Determining the prognosis of the subject (eg, identifying a metastatic or metastatic cancer state, determining the stage of the cancer, or determining the responsiveness of the cancer to treatment), or therametrics (eg, for treatment efficacy Monitoring the state of an object to provide information).
  • Expression protein expression analysis used in diagnosing pancreatic cancer in the present invention is a process of confirming the presence and degree of expression of the protein expressed from the gene in a biological sample, preferably, an antibody that specifically binds to the protein Means to check the amount of protein.
  • Analytical methods for this purpose include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket. Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc.
  • the method of analysis of the invention is not limited.
  • a binding agent that specifically binds to a protein may be, for example, an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a PNA (Peptide nucleic acid) or an aptamer. to be.
  • antibody refers to a specific protein molecule directed against an antigenic site.
  • an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies.
  • pancreatic cancer marker proteins Since new pancreatic cancer marker proteins have been identified as described above, the production of antibodies using them can be readily prepared using techniques well known in the art.
  • Polyclonal antibodies can be produced by methods well known in the art for injecting pancreatic cancer marker protein antigens described above into an animal and collecting blood from the animal to obtain serum comprising the antibody.
  • Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog.
  • Monoclonal antibodies are well known in the art by the hybridoma method (see Kohler and Milstein (1976) European Journal of Immunology 6: 511-519), or phage antibody libraries (Clackson et al, Nature, 352: 624-628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991).
  • Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.
  • the antibody of the present invention is not only a complete form having two full length light chains and two full length heavy chains,
  • a functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 and Fv.
  • the aptamer binding to the active form of the enzyme in the present invention is an oligonucleic acid or peptide molecule, the general content of aptamers are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine”. J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. 95 (24): 142727 (1998).
  • the present invention provides oligopeptides, monoclonal antibodies, polyclonal antibodies, chimerics that specifically bind to the proteins for diagnosing pancreatic cancer and / or pancreatic cancer stem cells.
  • Antibodies, ligands, peptide nucleic acids (PNAs) or aptamers more preferably oligopeptides, monoclonal antibodies, polyclonal antibodies or chimeric antibodies, even more preferably monoclonal Antibodies or polyclonal antibodies, most preferably monoclonal antibodies.
  • the antibody is preferably an antibody conjugated with microparticles.
  • the microparticles are preferably colored latex or colloidal gold particles.
  • the antibody may be any antibody capable of measuring the expression level of a protein encoded by a known mRNA gene for the marker described above, but preferably, the kit is for immunoassay. Kit, most preferably the kit is a Luminex assay kit, a protein microarray kit or an ELISA kit.
  • the Luminex Assay Kit, Protein Microarray Kit, and Eliza Kit include polyclonal and monoclonal antibodies directed against the protein, and secondary antibodies against the polyclonal and monoclonal antibodies bound to a label. .
  • kits in the present invention examples include immunochromatography strip kits, luminex assay kits, protein microarray kits, eliza kits, or immunological dot kits. kind is not limited.
  • the kit may further include the necessary elements necessary to perform the ELISA.
  • ELISA kits include antibodies specific for the marker protein. Antibodies are antibodies that have high specificity and affinity for marker proteins and have little cross-reactivity to other proteins and are monoclonal, polyclonal, or recombinant antibodies.
  • the ELISA kit can also include antibodies specific for the control protein.
  • Other ELISA kits can bind reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg conjugated with the antibody) and substrates or antibodies thereof. Other materials and the like.
  • the kit may additionally include the necessary elements necessary for performing protein microarrays to simultaneously analyze the complex markers.
  • the microarray kit includes antibodies specific for the marker protein bound to the solid phase. Antibodies are antibodies that have high specificity and affinity for marker proteins and have little cross-reactivity to other proteins and are monoclonal, polyclonal, or recombinant antibodies.
  • the protein microarray kit can also include antibodies specific for the control protein.
  • Other protein microarray kits include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (such as fused with antibodies), and substrates thereof or other materials that can bind to antibodies. And the like.
  • the method of analyzing a sample using a protein microarray is to diagnose a pancreatic cancer by separating the protein from the sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, and confirming the presence or expression level of the protein by reading the protein. You can provide the necessary information.
  • Luminex Assay is a high-throughput quantitative method that can simultaneously measure up to 100 different analytes without pretreatment of small (10-20 ⁇ l) patient samples As a result, it has good sensitivity (pg unit) and can be quantified in a short time (3-4 hours), and it is an analysis method that can replace ELISA or ELISPOT.
  • the Luminex Assay is a multiplexed fluorescent microplate assay that can simultaneously analyze more than 100 biological samples in each well of a 96-well plate. By using the laser detector of the real-time signal transmission to distinguish the polystyrene bead (polystyrene bead) of more than 100 different color groups.
  • the 100 beads are configured to be distinguished in the following manner.
  • the red fluorescence bead is divided into ten or more steps, and on the other, the orange fluorescence bead is divided into ten steps, showing the difference in intensity, and the beads therebetween.
  • the red and orange ratios are mixed in different proportions, making up a total of 100 color-coded bead sets.
  • each bead is attached to the antibody of the protein to be analyzed, it is possible to quantify the protein by an immune antibody reaction using the same.
  • the sample is analyzed using two lasers, one of which detects the beads to determine the bead identification number, and the other laser reacts with the antibody attached to the beads.
  • the protein in the sample is detected.
  • 100 in vivo proteins can be analyzed simultaneously in one well. This analysis has the advantage of being able to detect samples as small as 15 ⁇ l.
  • Luminex kits capable of performing the Luminex assay of the present invention include antibodies specific for the marker protein.
  • Antibodies are antibodies that have high specificity and affinity for marker proteins and have little cross-reactivity to other proteins and are monoclonal, polyclonal, or recombinant antibodies.
  • Luminex kits can also include antibodies specific for control proteins.
  • Other Luminex kits include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with antibodies) and their substrates or other substances that can bind to antibodies, and the like. It may include.
  • the antibody may be a conjugated antibody to microparticles, and the microparticles may be colored latex or colloidal gold particles.
  • the pancreatic cancer diagnostic kit comprising the immunochromatography strip for pancreatic cancer diagnosis in the pancreatic cancer diagnosis or prognostic analysis kit of the present invention includes an essential element necessary for performing a rapid test in which the analysis result can be known within 5 minutes. It may be a diagnostic kit characterized.
  • the immunochromatography strip may include (a) a sample pad into which a sample is absorbed; (b) a conjugate pad that binds to the protein of the gene in the sample; (c) a test membrane in which a test line and a control line including a monoclonal antibody against the protein of the gene are treated; (d) an absorption pad on which the remaining sample is absorbed; And (e) a support.
  • Rapid test kits which also include immunochromatographic strips, include antibodies specific for the marker protein.
  • the antibody is a monoclonal antibody, polyclonal antibody or recombinant antibody having high specificity and affinity for a marker protein and having little cross-reactivity to other proteins.
  • Rapid test kits may also include antibodies specific for the control protein.
  • Other rapid test kits include reagents that can detect bound antibodies, such as nitrocellulose membranes to which specific and secondary antibodies are immobilized, membranes bound to beads to which antibodies are bound, absorbent pads and sample pads, and the like. Other substances necessary for diagnosis, and the like.
  • Determination of protein expression levels by immunological dot assay in the present invention comprises the steps of (a) dotting a biological sample on the membrane; (b) reacting the antibody specific for the protein of the gene to the dipped membrane; And (c) adding and developing a secondary antibody conjugated with a marker to the reacted membrane, wherein the ELISA assay comprises (a) a protein of a gene having a nucleotide sequence for the marker.
  • Adsorbing specific antibody 1 to solid phase (b) contacting the antibody 1 adsorbed to the solid body with a biological sample of a suspected cancer patient to form an antigen-antibody complex; (c) treating the antibody 2 specific for the protein encoded by the gene having a nucleotide sequence for the marker to which the labeling substance is bound and binding to the complex; And (d) is preferably a sandwich ELISA assay comprising the step of detecting the concentration of the protein by detecting the label, the protein microarray assay is (a) polyclonal specific for the protein of the marker gene Immobilizing the antibody on the chip; (b) contacting the immobilized polyclonal antibody 1 with a biological sample of a suspected cancer patient to form an antigen-antibody complex; (c) treating a monoclonal antibody specific for a protein encoded by a gene having a nucleotide sequence for the marker to which the labeling agent is bound and binding to the complex; And (d) detecting the label and measuring the concentration of the protein.
  • the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in suspected pancreatic cancer patients can be compared, and the significant expression level of the pancreatic cancer marker gene to the protein can be determined.
  • the pancreatic cancer marker gene to the protein can be determined.
  • antigen-antibody complex means a combination of a pancreatic cancer marker protein and an antibody specific thereto, and the amount of antigen-antibody complex formed can be quantitatively measured through the size of a signal of a detection label.
  • Such a detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not necessarily limited thereto.
  • enzymes include ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese Therapies, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate deca Carboxylase, ⁇ -latamase, and the like, but are not limited thereto.
  • Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like.
  • Ligands include, but are not limited to, biotin derivatives.
  • Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like.
  • Microparticles include, but are not limited to, colloidal gold, colored latex, and the like.
  • Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W (CN) 8, [Os (bpy) 3] 2+, [RU (bpy) 3] 2+, [MO (CN) 8] 4- and the like.
  • Radioisotopes include, but are not limited to, 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re, and the like. .
  • Protein expression level measurement is preferably by using an ELISA method.
  • ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of an antibody that recognizes an antigen attached to a solid support, an attached to a solid support
  • Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the antibody-antigen complex a labeled antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the complex of the antigen with the antibody attached to the solid support
  • Various ELISA methods include indirect sandwich ELISA using secondary antibodies.
  • the antibody is enzymatically developed by attaching the antibody to the solid support, reacting the sample, and then attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex, or to an antibody that recognizes the antigen of the antigen-antibody complex. It is detected by the sandwich ELISA method which attaches a labeled secondary antibody and enzymatically develops. Pancreatic cancer marker protein and antibody can be confirmed by determining the degree of complex formation of pancreatic cancer.
  • Western blot using at least one antibody against the pancreatic cancer marker is also preferably, Western blot using at least one antibody against the pancreatic cancer marker.
  • the whole protein is isolated from the sample, electrophoresed to separate the protein according to size, and then transferred to the nitrocellulose membrane to react with the antibody.
  • the detection method consists of examining the expression level of the marker gene in the control group and the expression level of the marker gene in the cancer-causing cells.
  • mRNA or protein levels can be expressed as absolute (eg ⁇ g / ml) or relative (eg relative intensity of signals) differences of the marker proteins described above.
  • immunohistostaining is performed using at least one antibody against the pancreatic cancer marker.
  • paraffin embedding blocks are prepared by methods well known in the art. These are sliced to a thickness of several micrometers and attached to glass slides, and then reacted with one of the above antibodies by a known method. The unreacted antibody is then washed and labeled with one of the above-mentioned detection labels to read whether or not the antibody is labeled on a microscope.
  • At least one antibody against the pancreatic cancer marker is arranged at a predetermined position on the substrate, and the protein chip is immobilized at a high density.
  • the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex, which is then read and checked for the presence or expression of the protein to determine cancer. You can check whether you have the disease.
  • Biological sample in the present invention means tissue, cells, blood, serum, plasma, saliva, cerebrospinal fluid or urine, preferably means tissue or cells, most preferably cells.
  • polynucleotide measurement used for diagnosing pancreatic cancer in the present invention refers to measuring the amount of polynucleotide by checking the presence and degree of expression of the polynucleotide encoding the protein marker of the present invention in a biological sample.
  • Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real-time reverse transcriptase (Real-time RT-PCR), RNase protection assay (RPA). assays, Northern blotting, DNA chips, and the like.
  • the base sequence encoding the protein used as a marker in the present invention may include a base sequence having homology with this sequence.
  • polynucleotide in the present invention means a fragment of DNA or mRNA.
  • the probe or primer used in the diagnostic kit of the present invention has a sequence complementary to the polynucleotide sequence and the polynucleotide sequence encoding the protein.
  • primer of the present invention is meant a nucleic acid sequence having a short free 3-terminal hydroxyl group which can form complementary templates and base pairs and which serves as a starting point for template strand copying.
  • Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.
  • Primers of the invention are sense and antisense nucleic acids with 7 to 50 nucleotide sequences as primers specific for each marker gene.
  • Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis.
  • Primers of the invention can be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art.
  • Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, phosphoroami Date, carbamate, etc.) or charged linkages such as phosphorothioate, phosphorodithioate and the like.
  • Nucleic acids may be selected from one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine, psoralene, etc.).
  • Nucleic acid sequences of the invention can also be modified using a label that can provide a detectable signal directly or indirectly.
  • labels include radioisotopes, fluorescent molecules, biotin, and the like.
  • probe refers to a linear oligomer of natural or modified monomers or linkages, includes deoxyribonucleotides and ribonucleotides, and can specifically hybridize to a target nucleotide sequence, naturally Present or artificially synthesized. Probes of the invention are preferably single chain and oligodioxyribonucleotides.
  • suitable hybridization conditions can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and wash times, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); And M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc.
  • the high stringency conditions were hybridized to 65 ° C. in 0.5 M NaHPO 4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA, and 68 at 0.1 ⁇ standard saline citrate / 0.1% SDS. It means washing under the condition of °C.
  • high stringency conditions mean washing at 48 ° C. in 6 ⁇ SSC / 0.05% sodium pyrophosphate.
  • Low stringency means washing at 42 ° C. conditions, for example, at 0.2 ⁇ SSC / 0.1% SDS.
  • the present invention provides a pancreatic cancer stem cell comprising a nucleic acid molecule for inhibiting the expression of an antibody or a polynucleotide of SEQ ID NO: 2, which specifically binds to a protein of SEQ ID NO: 1
  • a composition for inhibiting formation is provided.
  • the present invention is for treating pancreatic cancer comprising a nucleic acid molecule for inhibiting the expression of an antibody or a polynucleotide of SEQ ID NO: 2 sequence specifically binding to a protein of SEQ ID NO: 1 sequence Or it provides a pharmaceutical composition for inhibiting metastasis.
  • the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen.
  • the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.
  • a lubricant e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, a kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mann
  • the pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, mucosal administration, and eyedrop administration.
  • Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.0001-10000 / kg on an adult basis.
  • compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container.
  • the formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
  • the antibody against the KIAA1199 protein or the nucleic acid molecule for inhibiting the expression of KIAA1199 reduces spear and colony formation, and decreases the proliferation rate and migration capacity of cancer stem cells, resulting in the recognition of cancer stem cells as early stages of cancer development. Proliferation can be suppressed.
  • the nucleic acid molecule for inhibiting KIAA1199 expression used in the present invention is an antisense oligonucleotide or siRNA oligonucleotide or shRNA oligonucleotide that specifically binds to the KIAA1199 gene.
  • antisense oligonucleotide refers to DNA or RNA or derivatives thereof that contain a nucleic acid sequence complementary to a sequence of a particular mRNA, and binds to a complementary sequence within the mRNA to inhibit translation of the mRNA into a protein. It works. Antisense sequence means a DNA or RNA sequence that is complementary to KIAA1199 mRNA and capable of binding to KIAA1199 mRNA, and that is essential for translation, translation into the cytoplasm, maturation or any other overall biological function of KIAA1199 mRNA. May inhibit.
  • the antisense nucleic acid has a length of 6 to 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases.
  • the antisense nucleic acid can be modified at one or more base, sugar or backbone positions to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol., 5 (3): 343-55 (1995) ).
  • the nucleic acid backbone can be modified with phosphorothioate, phosphoroester, methyl phosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic intersaccharide linkages and the like.
  • antisense nucleic acids may comprise one or more substituted sugar moieties.
  • Antisense nucleic acids can include modified bases.
  • Modified bases include hypoxanthine, 6-methyladenine, 5-me pyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosil HMC, 2-aminoadenine, 2 Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, etc. There is this.
  • the antisense nucleic acids of the present invention may be chemically bound to one or more moieties or conjugates that enhance the activity and cellular adsorption of the antisense nucleic acids.
  • Antisense oligonucleotides can be synthesized in vitro by conventional methods to be administered in vivo or to allow antisense oligonucleotides to be synthesized in vivo.
  • One example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I.
  • One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose origin is in the opposite direction of the recognition site (MCS). Such antisense RNA is desirable to ensure that there is a translation stop codon in the sequence so that it is not translated into the peptide sequence.
  • antisense oligonucleotides that can be used in the present invention can be readily prepared according to methods known in the art with reference to the nucleotide sequences of SEQ ID NO: 2 (Weiss, B. (ed.): Antisense Oligodeoxynucleotides and Antisense RNA: Novel Pharmacological and Therapeutic Agents, CRC Press, Boca Raton, FL, 1997; Weiss, B., et al., Antisense RNA gene therapy for studying and modulating biological processes.Cell.Mol.Life Sci., 55: 334- 358 (1999).
  • the nucleic acid molecule for inhibiting expression of KIAA1199 is an siRNA or shRNA comprising a sequence complementary to the KIAA1199 gene.
  • siRNA in the present invention means a nucleic acid molecule capable of mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409). And WO 00/44914) siRNAs are provided as efficient gene knockdown methods or gene therapy methods because they can inhibit the expression of target genes.
  • siRNAs are not limited to completely paired double-stranded RNA moieties paired with RNA, but paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. May be included.
  • the total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases.
  • the siRNA terminal structure can be either blunt or cohesive, as long as the expression of the gene can be inhibited by the RNAi effect.
  • the cohesive end structure is possible for both 3'-end protrusion structures and 5'-end protrusion structures.
  • siRNA molecules of the present invention may have a form in which a short nucleotide sequence (eg, about 5-15 nt) is inserted between a self-complementary sense and an antisense strand, in which case it is formed by expression of the nucleotide sequence.
  • siRNA molecules form a hairpin structure by intramolecular hybridization, and form a stem-and-loop structure as a whole. This stem-and-loop structure is processed in vitro or in vivo to produce an active siRNA molecule capable of mediating RNAi.
  • the siRNA is a sequence complementary to the 2137 th to 2161 nucleotides of the KIAA1199 gene of SEQ ID NO: 2, more specifically the siRNA described in SEQ ID NO: 3, SEQ ID NO: 2
  • the present invention is directed to a composition
  • a composition comprising a nucleic acid molecule for inhibiting the expression of an antibody or a polynucleotide of SEQ ID NO: 2 sequence specifically binding to the protein of SEQ ID NO: 1 target It provides a method for inhibiting pancreatic cancer stem cell formation comprising administering to a subject.
  • the present invention is a composition
  • a nucleic acid molecule for inhibiting the expression of an antibody or a polynucleotide of SEQ ID NO: 2 sequence specifically binding to a protein of SEQ ID NO: 1 target Provided is a method for inhibiting metastasis or treating pancreatic cancer, the method comprising administering to a subject.
  • the present invention provides a method for screening a pancreatic cancer preventing or treating substance using pancreatic cancer stem cells and novel molecular markers for pancreatic cancer.
  • the present invention is a method for screening pancreatic cancer prevention or treatment using pancreatic cancer stem cells and novel molecular markers for pancreatic cancer, pancreatic cancer stem cell detection kit, pancreatic cancer diagnosis or prognostic kit, pancreatic cancer stem cell formation inhibition
  • pancreatic cancer stem cell detection kit pancreatic cancer stem cell detection kit
  • pancreatic cancer diagnosis or prognostic kit pancreatic cancer stem cell formation inhibition
  • the composition and pharmaceutical composition for treating or inhibiting metastasis of pancreatic cancer are provided.
  • pancreatic cancer therapeutic target of the present invention is very useful for screening therapeutic agent candidates that specifically act on pancreatic cancer stem cells.
  • the present invention can accurately analyze the diagnosis and prognosis of pancreatic cancer.
  • FIG. 1 is a diagram illustrating a microarray list for selecting a protein candidate group.
  • Capan-1 and HPAC cell lines adherent and prototypical cell types were formed, and the difference in the genetic patterns between the two was confirmed by microarray, and some of the results of the microarray were listed.
  • Figure 2a shows adherent cells and protozoa in AsPC-1, Capan-1, HPAC and Miapaca-2
  • Figure 2b is a diagram showing the mRNA level of KIAA1199 in capan-1, HPAC adherent cells and spheroidal cells.
  • Figure 2c is a diagram showing the protein level of KIAA1199 in capan-1, HPAC adherent cells and tumor progenitor cells.
  • Figure 2d is a diagram showing the results of immunohistostaining experiments of cancer tissue, normal tissue and KIAA1199 in pancreatic cancer patients.
  • Figure 3a is a diagram showing the results of Western blotting experiments of KIAA1199 cDNA vectors (K8, K9 and K10) and empty vectors (E6 and E9) tagged with a Capan-1 cell line.
  • Figure 3b is a diagram showing a photograph of the observation of the cell shape of the E6 cell line and K10 cell line with a microscope.
  • Figure 3c is a diagram showing the migration assay and invasion assay using E6 and K10 cell line.
  • Figure 3d is a diagram showing the recovery of E6 and K10 cells.
  • Figure 3e is a diagram showing the difference in the molecular pattern of E6 and K10 cells.
  • Figure 4a is a diagram showing the expression of KIAA1199 after treatment of KIAA1199-specific siRNA variants # 1 and # 2 to K10 cell line (overexpressing cell line of KIAA1199), respectively.
  • 4B is a diagram showing KIAA1199 expression levels after na ⁇ ve Capan1 cells, samples treated with scrambled oligos in E6, K10 and K10 cells, and siRNA specific to K10 cells.
  • 5A is a diagram showing the expression pattern of each molecule in E6, K10 and K10 + scramble oligos and K10 + siRNA samples by Western blotting.
  • Figure 5b is a diagram showing the mRNA expression of KIAA1199, beta-catenin, Dvl2, Wnt3, Wnt3a and Wnt7b by qRT-PCR.
  • Figure 5c is a diagram showing the results of the experiment performed qRT-PCR with cells treated with scramble and siRNA, respectively, in the AsPC-1 cell line.
  • FIG. 5D shows a TOP plasmid tagged luciferase in an open reading frame (ORF) with a cf / Lef promoter sequence and a FOP plasmid tagged with a luciferase in an ORF with a muted Tcf / Lef promoter sequence.
  • Figure shows the results of the luciferase assay (luciferase assay).
  • 5E shows mRNA levels of OCT4, Nanog and Sox-2 in four samples treated with scrambled siRNA and E6, K10 and AsPC-1 cell lines.
  • FIG. 5F shows the results of Western blotting experiments on the protein expression patterns of KIAA1199 in fractions of cytoplasm, membrane, nucleus and cytoskeleton by performing subcellular fractionation of K10 cells.
  • Figure 5g is a diagram showing the results of ChIP assay experiments to determine whether KIAA1199 acts as a transcription factor in the nucleus.
  • Figure 5h is a diagram showing the results of Western blotting secretion Wnt3.
  • Figure 6a is a diagram showing the interaction of KIAA1199 and OCT4.
  • Figure 6b is a diagram showing the results of the experiment to perform the sumolation assay (imoylation assay) after immunoprecipitation with OCT4 and SUMO-1, respectively.
  • FIG. 7 is a diagram showing the results of experiments verifying the tumorigenic ability of in vivo KIAA1199.
  • Figure 8a is a diagram showing the results of measuring the mRNA levels of Oct4, Nanog, Dvl2, Wnt3, Wnt3a in adherent cells and protoplast cells of Capan-1 and HPAC cell lines.
  • Figure 8b is a diagram showing the results of measuring the protein expression level of ⁇ -catenin and Dvl2 in adherent cells and protoplast cells of Capan-1 and HPAC cell lines.
  • Figure 8c is a diagram showing the results of measuring the interaction of ⁇ -catenin and KIAA1199 in the protoplast cells of Capan-1 and HPAC cell line by immunoprecipitation method using ⁇ -catenin antibody.
  • Figure 8d is a diagram showing the results of measuring the interaction of ⁇ -catenin and KIAA1199 in the adherent cells and protoplast cells of Capan-1 and HPAC cell line by immunoprecipitation method using KIAA1199 antibody.
  • Figure 8e is a diagram showing the results measured by chromatin immunoprecipitation assay whether ⁇ -catenin and KIAA1199 act as a transcription factor for OCT4 and Nanog promoter.
  • FIG. 9 is a diagram showing the results of performing IHC using KIAA1199 on TMA tissue of pancreatic cancer patients and the average survival rate of the patients.
  • 10a-f show IC50 values of irinotecan, Carboplatin, 5-FU, Gemcitabine, Etoposide, Paclitaxel in E6 and K10 cells, respectively. It is a figure which shows the result of a measurement.
  • Figure 10g is a diagram showing the results of measuring the endpoint of the survival rate for gemcitabine after treatment with scrambled siRNA or siKIAA1199 to E6 and K10 cells, respectively.
  • Cultures were incubated for 5 or 7 days to maintain a concentration of 1,000 cells / mL in culture medium containing 0.5% BSA, 0.5% FBS, 1xITS, bFGF 10 ng / mL and EGF 10 ng / mL.
  • KIAA1199 (abcam), nanog (CST), OCT4 (CST), Snail (CST), Slug (CST), pAKT (CST), pmTOR (CST), pGSK3 ⁇ (for western blotting or other assays) CST), E-cadherin, N-cadherin, ⁇ -catenin (Santa cruz), IGFR1 ⁇ (Santa cruz), IGFR1 ⁇ (Santa cruz), SUMO-1 (Santa cruz) and GAPDH (Santa) cruz) specific antibody was used.
  • SiRNA specific for KIAA1199 gene was purchased from Invitrogen (Stealth).
  • Human KIAA1199 ORF (NM_018689) was purchased from GenScript and cloned into the pcDNA3.1 (+) plasmid tagged with Flag at the c-terminus.
  • the pcDNA3.1 (+) plasmid and pcDNA3.1 plasmid were transfected into Capan-1 cell line using GENEIN transfection reagent (Gliostem). Stabilizing cell lines K10 overexpressing stabilizing cell lines E6 and KIAA1199 with control vectors were constructed.
  • the upper side was coated with agarose and then the same number of cells were dispensed into the inserts of the transwells. The same steps were followed as with the migration assay.
  • culture plates of E6 and K10 cells having a density of at least 90% were scratched with a tip and incubated in growth medium for 24 hours.
  • IP buffer containing 50 mM Tris-HCl (pH 6.7) buffer, 50 mM Tris-HCl (pH 7.4), 120 mM NaCl, 0.5% NP40 and 1 ⁇ protease inhibitor cocktail.
  • Tumor protoplasts of cells transfected with siRNA were isolated into single cells.
  • Cells were cultured in stem cell medium and second and third generation tumor protoplasts were obtained. Floating tumor protoplasts and total cell numbers were counted under light microscopy.
  • isolated single cells were dispensed into 24 well low attached dishes (Corning) at a density of 100 cells per well in 0.3% soft agar with growth medium. Plates were then incubated for 14 days at 5% CO 2 , 37 ° C. until colonies were visible. Colonies were stained using 0.01% crystal violet and colony counts were counted under an inverted microscope.
  • Chip chromatin immunoprecipitation
  • mice Five Balb / C nude mice were used for each experimental group. 5x10 E6 and K10 cells 5 The cells were injected at the ratio of cells / mouse and mice were sacrificed after 7 days.
  • TMA tissue Microarray
  • IHC Immunohistochemical Staining
  • Immunohistochemical staining was performed using KIAA1199 antibody (Sigma-Aldrich) on TMA including cancerous and adjacent normal tissues of pancreatic cancer patients. Immunohistochemical staining was performed by the following methods: The tissues on the slides were incubated at 60 ° C. for 30 minutes, and then immersed in diluted alcohol, such as 70%, 50%, 30%, starting with 100% ethanol, from the tissues. Paraffin was removed. Soon washed three times with PBS, soaked in peroxidase quenching solution (100% methanol and 30% hydrogen peroxide mixture) for 20 minutes, washed three times with PBS and then simmered in boiled 10 mM sodium citrate solution (pH 6.0) Soak for 3 minutes.
  • peroxidase quenching solution 100% methanol and 30% hydrogen peroxide mixture
  • the cells under the low-power field should indicate the extent to which they correspond to immunoreactivity.
  • the OS was defined as the interval between the date of diagnosis and the date of death.
  • Recurrence-free survival (RFS) was defined as the interval between death and relapse or last visit.
  • E6 and K10 cells were seeded at 2,500 cells / well in 96 well plates. After 24 hours, 5-FU, Gemcitabine (Gemcitabine), irinotecan (Irinotecan), etoposide (Etoposide), carboplatin (Carboplatin) and paclitaxel (Paclitaxel) were treated in each plate by concentration. After incubation for 72 hours, cells were washed with PBS and treated with MTT solution (Amresco) diluted in growth medium for 3 hours. Then, fluorescence was measured at 570 nm wavelength using an ELISA instrument.
  • Tumor spheroid cells of each cell line are cultured through tumor stem culture, which is a method of culturing cancer stem cells, in human pancreatic cancer cell lines CAPAN-1 and HPAC, and microarrays using control cells as adherent cells. Analysis by chip was performed. The results are shown in FIG.
  • RT-PCR was performed to confirm protein levels of KIAA1199 in human pancreatic cancer cell lines, Capan-1 and HPAC, and tumor protoplast cells. It confirmed that it was (FIG. 2B).
  • Flag-tagged KIAA1199-cDNA vectors K8, K9 and K10 cells
  • empty vectors E6 and E9 cells
  • the expression pattern of Flag-KIAA1199 protein of each clone was verified by Western blotting using an antibody specific for Flag. The results are shown in Figure 3a.
  • siRNA variants # 1 and # 2 specific to KIAA1199 were treated with K10 cell lines (overexpressing cell lines of KIAA1199), respectively, and their expression patterns were confirmed. As shown in FIG. 4A, it was confirmed that # 2 more efficiently reduced the expression of KIAA1199 than the variant # 1. In addition, it was confirmed that molecules such as OCT4 and IGF1R, which were increased in K10, also decreased.
  • Tumor protoplast formation and colony formation experiments were performed in E6, K10 cells, K10 cells treated with scrambled oligo, and K10 cells treated with KIAA1199-specific siRNA, and formed in cells treated with scrambled oligos in K10 and K10 cells compared to E6.
  • the number of tumor protoplasts and colonies was increased, and cells treated with KIAA1199-specific siRNA at K10 were found to have poor tumor protoplasts and colony forming ability (FIG. 4C).
  • the expression pattern of beta-catenin was changed according to the degree of KIAA1199 expression, and the association with Wnt signaling was verified.
  • Luciferase assay was performed using an FOP plasmid tagged with luciferase in an ORF with a Tcf / Lef promoter sequence mutated with a T tagged TOP plasmid.
  • the TOP / FOP ratio was increased in K10 cells overexpressing KIAA1199, and the TOP / FOP ratio was decreased in the samples in which KIAA1199 expression was reduced by siRNA in the remaining cell lines (FIG. 5D).
  • K10 cells were subjected to subcellular fractionation to obtain fractions of cytoplasm, membrane, nucleus and cytoskeleton, and the protein expression pattern of KIAA1199 in each fraction was confirmed by western blotting. As a result, it was confirmed that KIAA1199 was expressed in the nucleus (FIG. 5F).
  • ChIP assay was performed to confirm that KIAA1199 acts as a transcription factor in the nucleus.
  • beta-catenin increased binding to the target promoter as KIAA1199 protein expression level was increased (Panel a of FIG. 5G), and KIAA1199 protein expression.
  • binding with the target promoter decreased (Panel e of FIG. 5G).
  • KIAA1199 binds to the OCT4 and Nanog promoters (Panels b, c and d of FIG. 5G).
  • the medium of E6 and K10 cells was discarded, precipitated with acetone, and the secreted Wnt3 was confirmed by Western blotting. As a result, the secreted Wnt3 was increased in K10 cells (FIG. 5H).
  • mice were sacrificed 7 weeks after 5 ⁇ 10 5 E6 and K10 cells were injected into the subcutaneous fat on the right leg of 5 nude Balb / C mice each. As a result, as shown in Fig. 7, it was confirmed that no mice developed tumors among the five mice injected with E6. However, it was confirmed that tumors developed in 4 out of 5 mice injected with K10 cells.
  • Capan-1 and HPAC cell lines were used to generate adherent and protoplast cells, where the mRNA levels of Oct4, Nanog, Dvl2, Wnt3, and Wnt3a were identified.
  • Primer sequences used to confirm mRNA levels are as follows: Wnt3 forward, 5′-GCG TGT TAG TGT CCA GGG AGT T-3 ′; Wnt3 reverse, 5'-TGA GGT GCA TGT GGT CCA GGA T-3 '; Wnt3a forward, 5'-ATG AAC CGC CAC AAC AAC GAG G-3 '; Wnt3a forward, 5'-GTC CTT GAG GAA GTC ACC GAT G-3 '; Oct4 forward, 5'-CCT GAA GCA GAA GAG GAT CAC C-3 ', Oct4 reverse, 5'-AAA GCG GCA GAT GGT CGT TTG G-3'; Nanog forward, 5'-CTC CAA CAT CCT GAA CCT CAG C-3
  • KIAA1199 acts as a transcription factor for OCT4 and Nanog promoters in cell protoplasts compared to adherent cells.
  • pancreatic cancer TMA tissues were subjected to IHC with an antibody specific for KIAA1199 (Sigma-Aldrich). As a result, 40% of the cancer tissue showed little or weak staining, and 60% of the cancer tissue showed medium or strong staining (FIG. 9). The average total survival of patients with little or no staining was 1,847 days, whereas the average total survival of patients with moderate or strong staining was 226 days. This analysis was carried out on Windows version 18.0 with SPSS program, survival curve was obtained by Kaplan-Meier method, and p-value is 0.018. As a result, it was confirmed that the stronger the expression of KIAA1199, the shorter the average survival days of patients.
  • MTT assays were performed by diluting each anticancer agent by one-tenth at the maximum concentration on the graph x-axis and measuring the endpoint of survival after three days of culture.
  • 5-FU, Gemcitabine, Irinotecan, Etoposide, Carboplatin which are used as anti-cancer agents for pancreatic cancer in K10 cells expressing KIAA1199 more than E6 cells expressing less KIAA1199
  • the IC50 value for was shown to increase (FIGS. 10A-E).
  • the opposite result was found for Paclitaxel (FIG. 10F).

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Abstract

La présente invention concerne un procédé de criblage de produits destinés à la prévention ou au traitement du cancer du pancréas, utilisant une nouvelle molécule marqueur des cellules souches du cancer du pancréas et du cancer du pancréas. La présente invention concerne un procédé de criblage de produits destinés à la prévention ou au traitement du cancer du pancréas, utilisant une nouvelle molécule marqueur des cellules souches du cancer du pancréas et du cancer du pancréas, un kit pour détecter les cellules souches du cancer du pancréas, un kit pour évaluer le diagnostic ou le pronostic du cancer du pancréas, une composition pour inhiber la formation de cellules souches du cancer du pancréas, et une composition pharmaceutique pour le traitement du cancer du pancréas ou l'inhibition de la métastase du cancer du pancréas. Une cible pour le traitement du cancer du pancréas de la présente invention est particulièrement utile pour le criblage de produits candidats au rôle d'agent thérapeutique à action spécifique contre les cellules souches du cancer du pancréas. En outre, l'utilisation de la présente invention permet d'évaluer avec précision le diagnostic et le pronostic du cancer du pancréas.
PCT/KR2015/006855 2014-07-04 2015-07-03 Composition pour le diagnostic ou le traitement du cancer du pancréas utilisant kiaa1199 WO2016003231A1 (fr)

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KR102122452B1 (ko) * 2018-02-27 2020-06-12 연세대학교 산학협력단 암의 진단용 조성물

Citations (5)

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US20030180747A1 (en) * 2001-10-11 2003-09-25 Hruban Ralph H. Pancreatic cancer diagnosis and therapies
KR20090040391A (ko) * 2006-08-18 2009-04-23 온코세라피 사이언스 가부시키가이샤 Ｒｅｇ４ 또는 ｋｉａａ０１０１을 과발현하는 암의 예방 및 치료 기술
JP2009276153A (ja) * 2008-05-13 2009-11-26 Sumitomo Bakelite Co Ltd 胃癌の判定方法
WO2010064702A1 (fr) * 2008-12-05 2010-06-10 国立大学法人 東京大学 Biomarqueur pour prédire un pronostic de cancer
KR20140057361A (ko) * 2011-08-31 2014-05-12 온코사이트 코포레이션 암의 치료 및 진단을 위한 방법 및 조성물

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* Cited by examiner, † Cited by third party
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EP3603671A3 (fr) * 2011-10-28 2020-07-29 Chugai Seiyaku Kabushiki Kaisha Molécule spécifique de cellules souches du cancer

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030180747A1 (en) * 2001-10-11 2003-09-25 Hruban Ralph H. Pancreatic cancer diagnosis and therapies
KR20090040391A (ko) * 2006-08-18 2009-04-23 온코세라피 사이언스 가부시키가이샤 Ｒｅｇ４ 또는 ｋｉａａ０１０１을 과발현하는 암의 예방 및 치료 기술
JP2009276153A (ja) * 2008-05-13 2009-11-26 Sumitomo Bakelite Co Ltd 胃癌の判定方法
WO2010064702A1 (fr) * 2008-12-05 2010-06-10 国立大学法人 東京大学 Biomarqueur pour prédire un pronostic de cancer
KR20140057361A (ko) * 2011-08-31 2014-05-12 온코사이트 코포레이션 암의 치료 및 진단을 위한 방법 및 조성물

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