WO2016003231A1 - Composition for diagnosing or treating pancreatic cancer using kiaa1199 - Google Patents

Abstract

The present invention provides a method for screening a material for preventing or treating pancreatic cancer using a novel molecule marker for pancreatic cancer stem cells and pancreatic cancer. The present invention provides a method for screening a material for preventing or treating pancreatic cancer using a novel molecule marker for pancreatic cancer stem cells and pancreatic cancer, a kit for detecting pancreatic cancer stem cells, a kit for analyzing the diagnosis or prognosis of pancreatic cancer, a composition for inhibiting the formation of pancreatic cancer stem cells, and a pharmaceutical composition for treating pancreatic cancer or inhibiting pancreatic cancer metastasis. A target for treating pancreatic cancer of the present invention is very useful in screening therapeutic agent candidate materials specifically acting on pancreatic cancer stem cells. In addition, the use of the present invention makes it possible to accurately analyze the diagnosis and prognosis of pancreatic cancer.

Description

Composition for diagnosis or treatment of pancreatic cancer using KIAA1199

The present invention relates to a composition for diagnosing or treating pancreatic cancer using KIAA1199.

Cancer stem cells (cancer stem cells or tumor initiating 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.

The therapeutic effects of several anticancer drugs, including 5-fluorouracil, gemcitabine, and tarceva, which are known to be effective against pancreatic cancer to date, are extremely disappointing and the response rate to chemotherapy is only about 15%. This suggests that the development of more effective early diagnosis and treatment is urgently needed to improve the prognosis of pancreatic cancer patients. Cancer stem cells refer to cancer cells capable of self-renewal and have recently emerged as a major target for cancer treatment and conquest. In addition, cancers occurring in each organ of living body are known to have molecular patterns distinguished according to the producing organs. In the same vein, studies have reported that cancer stem cells from each organ also have molecular markers that differentiate them from other 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.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

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. 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 present inventors have found that 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.

Accordingly, it is an object of the present invention to provide a method for screening a substance for preventing or treating pancreatic cancer.

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 objects and advantages of the invention will become apparent from the following detailed description and claims.

According to one aspect of the present invention, the present invention provides a method for screening a substance for preventing or treating pancreatic cancer, comprising the following steps:

(a) contacting a sample to be analyzed with a cell or tissue comprising a protein of SEQ ID NO: 1 or a polynucleotide of SEQ ID NO: 2; And

(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.

According to the present invention, 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.

In the present invention, 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.

As used herein, the term “pancreatic cancer” refers to a cancer that originates in pancreatic cells. There are many types of pancreatic cancer. Pancreatic adenocarcinoma of the pancreatic ducts accounts for about 90% of the pancreatic cancer. In addition, cystic cancer (cystic adenocarcinoma), endocrine tumors and the like. About 5-10% of 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%. The reason for this is that most cancers are detected after progression. Surgical resection is possible within 20% at the time of discovery, and even if completely resected by the naked eye, the survival rate is not improved due to micrometastasis, and the response to chemotherapy and radiation treatment is poor. Because it is low. Thus, the most important way to improve survival is to detect and operate early when there are no symptoms or nonspecific.

The inventors have found that the expression of KIAA1199 protein or polynucleotides encoding it is significantly increased in pancreatic cancer stem cells compared to pancreatic cancer cells. Unlike pancreatic cancer 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. 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.

The 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. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994 and the like.

According to another aspect of the present invention, the present invention provides a method for screening a substance for preventing or treating pancreatic cancer, comprising the following steps:

(a) preparing a protein of SEQ ID NO: 1;

(b) contacting the test substance with the protein; And

(c) analyzing whether the 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.

According to the screening method of the present invention, 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.

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.

In the screening method of the present invention, the test substance or the protein may be labeled with a detectable label. For example, the detectable label may be a chemical label (eg biotin), an enzyme label (eg horseradish peroxidase, alkaline phosphatase, peroxidase, luciferase, β-galacto Cedase and β-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, chemiluminescent labels, fluorescence resonance energy transfer (FRET) labels or metal labels (eg gold and silver).

In the case of using a protein or test substance labeled with a detectable label, the binding between the protein and the test substance can be analyzed by detecting 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). 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.

Alternatively, binding of the test substance to the protein may be analyzed without labeling the interactants.

For example, 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)).

The ability of the test substance to bind to proteins can be analyzed using real-time bimolecular interaction analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63: 23382345 (1991), and Szabo et al., Curr. Opin. Struct. Biol. 5: 699705 (1995)). BIA is a technique for analyzing specific interactions in real time and can be performed without labeling of the interactions (eg, BIAcore ™). Changes in surface plasmon resonance (SPR) can be used as indicators for real-time reactions between molecules.

In addition, 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). In this case, 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. For simplicity, this assay uses two DNA constructs. For example, in one construct, 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). In another construct, 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. If bait and prey interact in in vivo to form a complex, the DNA-binding and activation domains of the transcription factors are contiguous, which triggers transcription of 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).

According to another aspect of the present invention, 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.

In the present invention, 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. Pancreatic cancer cell line samples identified as not containing, or cell samples other than cancer of pancreatic cancer patients identified as not malignant, and the like, but are not necessarily limited thereto. 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.

Through the detection methods, 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.

Specifically, 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.

According to another aspect of the present invention, 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.

In particular, the kit for pancreatic cancer diagnosis or prognosis analysis of the present invention is a kit derived from pancreatic cancer stem cells.

As used herein, 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".

As used herein, the term “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.

In the present invention, 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.

As used herein, "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies.

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.

In addition, the antibody of the present invention is not only a complete form having two full length light chains and two full length heavy chains,

Functional fragments of antibody molecules. 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).

According to a preferred embodiment of the present invention, 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.

In the present invention, the antibody is preferably an antibody conjugated with microparticles.

In addition, the microparticles are preferably colored latex or colloidal gold particles. In the present invention, 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. .

Examples of the types of kits in the present invention 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.

In addition, 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 μl) 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. On the one hand, 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. In addition, 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. Thus, 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 μl.

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.

Through the above analysis methods, 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. Thus, it is possible to diagnose whether the pancreatic cancer suspected patient actually develops pancreatic cancer.

In the present invention, the term 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. When enzymes are used as detection labels, available enzymes include β-glucuronidase, β-D-glucosidase, β-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, β-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. More preferably, 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.

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. By checking the amount of the generated antigen-antibody complexes using labeled antibodies, the amount of protein produced by the expression of genes can be confirmed to determine whether pancreatic cancer is developed. 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 μg / ml) or relative (eg relative intensity of signals) differences of the marker proteins described above.

Also preferably, immunohistostaining is performed using at least one antibody against the pancreatic cancer marker. After collecting and fixing normal tissue and suspected pancreatic cancer, 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.

Also, preferably, 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. In the method of analyzing a sample using a protein chip, 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.

The expression “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.

Preferably, 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.

By “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.). ), Chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. Nucleic acid sequences of the invention can also be modified using a label that can provide a detectable signal directly or indirectly. Examples of labels include radioisotopes, fluorescent molecules, biotin, and the like.

As used herein, the term “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.

If a probe is used, the probe is hybridized with the cDNA molecule. In the present invention, 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. N.Y. (1999). For example, among the stringent conditions, 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 ℃. Alternatively, 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.

According to another aspect of the present invention, 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 Provided is a composition for inhibiting formation.

According to another aspect of the present invention, 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.

When the composition of the present invention is made into a pharmaceutical composition, 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. In addition to the above components, 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. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

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.

The pharmaceutical 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.

As used herein, 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.

As used herein, the term “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. In addition, 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. In addition, 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. Cholesterol moieties, cholesteryl moieties, cholic acid, thioethers, thiocholesterols, fatty chains, phospholipids, polyamines, polyethylene glycol chains, adamantane acetic acid, palmityl moieties, octadecylamine, hexylamino-carbonyl-oxy Fat-soluble moieties such as a cholesterol ester moiety, and the like. Oligonucleotides comprising fat-soluble moieties and methods of preparation are already well known in the art (US Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid can increase stability to nucleases and increase the binding affinity of the antisense nucleic acid with the target mRNA.

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.

The design of 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).

According to a specific embodiment of the present invention, the nucleic acid molecule for inhibiting expression of KIAA1199 is an siRNA or shRNA comprising a sequence complementary to the KIAA1199 gene.

The term "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.

The 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.

According to a specific embodiment of the present invention, 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 sequence complementary to the 3664 th -3688 nucleotides of the KIAA1199 gene of the sequence, more specifically the siRNA described in SEQ ID NO: 4.

According to another aspect of the invention, the present invention is directed to 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.

According to another aspect of the invention, the present invention is 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 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.

In summary, the features and advantages of the present invention are as follows:

(a) 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.

(b) 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 The composition and pharmaceutical composition for treating or inhibiting metastasis of pancreatic cancer are provided.

(c) The pancreatic cancer therapeutic target of the present invention is very useful for screening therapeutic agent candidates that specifically act on pancreatic cancer stem cells.

(d) In addition, the present invention can accurately analyze the diagnosis and prognosis of pancreatic cancer.

1 is a diagram illustrating a microarray list for selecting a protein candidate group. In 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 showing the mRNA level of KIAA1199 in the cell.

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 naïve 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.

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 β-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 β-catenin and KIAA1199 in the protoplast cells of Capan-1 and HPAC cell line by immunoprecipitation method using β-catenin antibody.

Figure 8d is a diagram showing the results of measuring the interaction of β-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 β-catenin and KIAA1199 act as a transcription factor for OCT4 and Nanog promoter.

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.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Experimental Materials and Experimental Methods

1. Tumor sphere culture

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.

2. RT-PCR and qRT-PCR

RNA was extracted from pancreatic cancer cells or pancreatic cancer cells derived from tumor progenitor cells using the RNeasy Mini Kit (Quiagen). CDNA was synthesized from the RNA obtained using the Super Script II System (Invitrogen). RT-PCR and qRT-PCR were performed with primers specific to the target using Taq polymerase (Takara) and SYBR mast mix (ABI), respectively.

3. Cell Lines, Antibodies, and siRNAs

Human pancreatic cancer cell lines AsPC-1, Capan-1 and HPAC were used in this experiment. KIAA1199 (abcam), nanog (CST), OCT4 (CST), Snail (CST), Slug (CST), pAKT (CST), pmTOR (CST), pGSK3β (for western blotting or other assays) CST), E-cadherin, N-cadherin, β-catenin (Santa cruz), IGFR1α (Santa cruz), IGFR1β (Santa cruz), SUMO-1 (Santa cruz) and GAPDH (Santa) cruz) specific antibody was used. SiRNA specific for KIAA1199 gene was purchased from Invitrogen (Stealth).

4. Plasmid and Stable Cell Line Construction

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.

5. Migration, invasiveness and wound healing assays

To 2x10 ⁵ or 3x10 ⁵ cells / mL of the cells it was dispensed in a trans-well (3422, corning). After 3 days, the cells were fixed for 10 minutes with 5% glutaaldehyde and then washed three times with distilled water. Cells were stained for 20-30 minutes using crystal violet. The cells were washed several times with distilled water, then dried and mounted on slides.

For infiltration assays, 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.

For wound healing assays, 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.

6. Sumoylation assay

Each sample (naive) using 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. Cells, E6 cells, K10 cells and cells treated with siRNA). Lysates were reacted with appropriate antibodies overnight at 4 ° C. and treated with ultra biolink support resin for 2 hours to aid binding. The resin was collected by centrifugation and washed three times with a wash buffer containing 20 mM Tris-HCL, 500 mM Nacl, 1 mM EDTA and 0.5% NP40. SDS-PAGE was performed using the sample, followed by Western blotting analysis.

7. Self-renewal and colony formation assay

Specific for pancreatic cancer cells, naïve cells, scrabble siRNA transfected cells and KIAA1199 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. For colony formation, 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.

8. Chip (chromatin immunoprecipitation) assay

Cells transfected with E6, K10, AsPC-1 scramble, cells transfected with AsPC-1 siKIAA1199, adhesion and protoplast cells derived from capan-1, adhesion and protoplast cells derived from HPAC were used for the ChIP assay. This experiment was performed according to the EZ ChIP kit (Up state) protocol. The antibody used for IP is the same one used for western blotting.

9. In vivo  Xenograft

Five Balb / C nude mice were used for each experimental group. 5x10 E6 and K10 cells⁵ The cells were injected at the ratio of cells / mouse and mice were sacrificed after 7 days.

10. TMA (Tissue Microarray) and Immunohistochemical Staining (IHC)

Immunohistochemical staining (IHC) 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. After tapping for 10 minutes, the tap water was washed three times with PBS and treated with 10% normal donkey serum at room temperature for 1 hour. After washing three times with PBS, it was reacted with an antibody specific for KIAA1199 protein (Sigma-Aldrich) for 24 hours at 4 ℃ and washed with PBS. After processing DAKO Envision kit solution, it was left in the chamber for 20 minutes. After washing three times with PBS and distilled water, and then treated with hematoxylin for 1 minute, washed three times with distilled water and dried, the permount was raised and covered with a cover slide and observed under a microscope. Immune responsiveness of cancer tissues were divided into not expressed according to staining intensity, weakly expressed, moderately expressed and strongly expressed. To score for each, at least 50% of the cells under the low-power field (100 ×) 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.

11.MTT Assay

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.

12. Statistical Analysis

All statistical analyzes were performed using SPSS of Windows version 18.0 (SPSS, Inc., Chicago, IL, USA). Survival curves were generated using the Kaplan-Meier method, and the differences between the curves were evaluated using a log rank test. P-value <0.05 was considered statistically significant.

Experiment result

1. Selection of Protein Candidates by Microarray

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.

1 shows fold change values (FC_HS and FC_CS) in tumor progenitor cells relative to adherent cells. Of the candidates in the list described in FIG. 1, KIAA1199 was selected as the study target.

2. Validation of the expression level of KIAA1199 protein in cell lines

To culture the tumor cells of each cell line through tumor protoplast culture in human pancreatic cancer cell lines AsPC-1, Capan-1 and HPAC and Miapaca-2, and to determine the mRNA level of KIAA1199 using control cells as adherent cells, qRT-PCR Was performed. As a result, as shown in Figure 2a, it was confirmed that the mRNA of KIAA1199 is increased in the tumor protoplast form of each cell line than the adherent cells.

In addition, 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).

Western blotting was performed to confirm the protein levels of KIAA1199 in the adherent cells and tumor progenitor cells of human pancreatic cancer cell lines, Capan-1 and HPAC. It was confirmed (Fig. 2c).

As a result of immunohistostaining using antibodies specific to cancer tissues, normal tissues and KIAA1199 protein of pancreatic cancer patients, it was confirmed that KIAA1199 protein was increased in cancer tissues than normal tissues of pancreatic cancer patients (FIG. 2D).

3. Establishment and Characterization of Stable Cell Line Overexpressed with Flag-KIAA1199

Flag-tagged KIAA1199-cDNA vectors (K8, K9 and K10 cells) and empty vectors (E6 and E9 cells) were transfected into the Capan-1 cell line and selected with the selective drug G418. 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.

As a result of observing the cell shapes of the E6 cell line and the K10 cell line by fluorescence microscopy, it was confirmed that the shape of the K10 cell line changed to the branched cell shape unlike the E6 (FIG. 3B).

As a result of the migration assay and invasion assay for the E6 and K10 cell lines, there were a large number of cells with high migration or invasiveness in the K10 cell line (FIG. 3C).

In addition, after dispensing and maintaining E6 and K10 cells in a 24-well plate dish, scratching with a tip and observing the recovery pattern after 24 hours confirmed that the degree of recovery in the K10 cell line was fast (FIG. 3D).

On the other hand, Western blotting confirmed the difference in molecular patterns of E6 and K10 cells. The major molecules of OCT4, Nanog and Wnt signaling, which are known to regulate EMT-related molecules, IGF-1 signaling-related molecules, and self-renewal, It was confirmed that the expression pattern of beta-catenin was changed (FIG. 3E).

4. Function Verification of KIAA1199

In order to examine the function of KIAA1199, 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.

Naive Capan1 cells, E6, K10 cells; Scrambled oligo group to K10 cells; And KIAA1199 expression level was confirmed in the group treated with siRNA specific to K10 cells (FIG. 4B).

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).

In addition, Western blotting was performed after treatment of scrambled or KIAA1199 specific siRNAs on AsPC-1 cells. As a result, molecular changes similar to those of K10 cell lines produced in Capan-1 cells were confirmed (FIG. 4D).

5. Verification of KIAA1199's Association with Wnt Signaling

The expression pattern of beta-catenin was changed according to the degree of KIAA1199 expression, and the association with Wnt signaling was verified.

Western blotting was used to determine the expression patterns of each molecule in E6, K10 and K10 + scramble oligos and K10 + siRNA samples, and mRNA expression patterns of KIAA1199, beta-catenin, Dvl2, Wnt3, Wnt3a and Wnt7b were measured by qRT-PCR. As a result, as shown in Figures 5a and 5b, it was confirmed that mRNA levels of beta-catenin, Dvl2, Wnt3 and Wnt3a, which are molecules related to typical Wnt signaling, change corresponding to the levels of KIAA1199. However, it was confirmed that the level of Wnt7b mRNA, an atypical Wnt signaling-related molecule, is opposite to that of KIAA1199.

QRT-PCR was performed on cells treated with scramble and siRNA, respectively, in the AsPC-1 cell line. As a result, it was confirmed that mRNA levels of Dvl2, Wnt3, and Wnt3a, which are related to typical Wnt signaling, change according to the mRNA levels of KIAA1199 (FIG. 5C).

Luciferase in an open reading frame (ORF) with a Tcf / Lef promoter sequence that binds to beta catenin after transfer to the nucleus to confirm that KIAA1199 acts on typical Wnt signaling, where beta-catenin acts as the main molecule. 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. As a result, 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).

Scrambled and siRNA-treated samples of the E6, K10, and AsPC-1 cell lines of the Capan1 cell line confirmed the mRNA levels of OCT4, Nanog, and Sox-2, which are known to be typical targets of Wnt signaling, and both cell lines showed KIAA1199 levels. It was confirmed that each of three molecular mRNA expression level changes according to (Fig. 5e).

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. As a result, beta-catenin increased binding to the target promoter as KIAA1199 protein expression level was increased (Panel a of FIG. 5G), and KIAA1199 protein expression. As level decreased, binding with the target promoter decreased (Panel e of FIG. 5G). In addition, it was confirmed that 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).

6. Verification of relationship with OCT4

As KIAA1199 increased, the strength of the band in which the OCT4 was deformed became stronger. Therefore, the relationship between KIAA1199 and OCT4 was investigated. The results are shown in FIGS. 6A and 6B.

As shown in Figure 6a, it was confirmed that 110 kDa KIAA1199 protein is related to OCT4.

In addition, a sumoylation assay was performed to identify modified bands of OCT4. After treatment with scrambled and KIAA1199-specific siRNA on the K10 cell line and AsPC-1 cell line of Capan1, immunoprecipitation with OCT4 and SUMO-1 was performed, followed by the hydromolecular assay, and according to the amount of protein expression of KIAA1199 It was confirmed that the degree of hydromolation was changed (Fig. 6b).

7. in vivo  Tumor Formation of KIAA1199

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.

8. Reaffirm the association of KIAA1199 with basic WNT signaling in adherent cells and tumor protoplasts

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 '; Reverse Nanog, 5'-CGT CAC ACC ATT GCT ATT CTT CG-3 '; Dvl2 forward, 5′-TCC ATA CGG ACA TGG CAT CGG T-3 ′; Dvl2 reverse, 5'-CGT GAT GGT AGA GCC AGT CAA C-3 '. Protein levels were determined using antibodies to β-catenin and Dvl2 (Santa cruz) in both cell lines. As a result, both cell lines increased their respective mRNA and protein levels in protoplast cells (FIGS. 8A-B).

The interaction of β-catenin with KIAA1199 in protoplast cells of the two cell lines was confirmed by immunoprecipitation method, and the transcriptional activities of OCT4 and Nanog genes of β-catenin and KIAA1199 were confirmed by chromatin immunoprecipitation assay ( 8C-D). In addition, it was confirmed that KIAA1199 also acts as a transcription factor to OCT4 and Nanog promoters using β-catenin known as OCT4 and Nanog transcription factors using the EZ Chip Kit of Upstate (Fig. 8E). These results confirmed that KIAA1199 acts as a transcription factor for OCT4 and Nanog promoters in cell protoplasts compared to adherent cells.

9. Survival Analysis of KIAA1199 and Pancreatic Cancer Patients Using TMA (Tissue Microarray) Analysis

30 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.

10. On anticancer drugs  Sensitivity test

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). On the other hand, the opposite result was found for Paclitaxel (FIG. 10F). In addition, after measuring scrambled siRNA or siKIAA1199 to E6 and K10 cells, respectively, the end point of survival rate for gemcitabine was measured, and the IC50 value was decreased in the KIAA1199 knocked-down sample (FIG. 10G).

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (11)

A method for screening a substance for preventing or treating pancreatic cancer, comprising the following steps: (a) contacting a sample to be analyzed with a cell or tissue comprising a protein of SEQ ID NO: 1 or a polynucleotide of SEQ ID NO: 2; And (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. A method for screening a substance for preventing or treating pancreatic cancer, comprising the following steps: (a) preparing a protein of SEQ ID NO: 1; (b) contacting the test substance with the protein; And (c) analyzing whether the 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. 3. The method of claim 2, wherein the protein is on the cell surface or the virus surface or in isolated form. A kit for pancreatic cancer stem cell detection 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. A kit for diagnosing or prognostic pancreatic cancer 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. The kit according to claim 4 or 5, wherein the kit is a kit for immunoassay. The kit according to claim 4 or 5, wherein the kit is a kit for gene amplification or microarray. Composition for inhibiting pancreatic cancer stem cell formation 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. A pharmaceutical composition for treating or inhibiting pancreatic cancer comprising a nucleic acid molecule for inhibiting expression of an antibody or a polynucleotide of SEQ ID NO: 2 as an active ingredient that specifically binds to a protein of SEQ ID NO: 1. A pancreatic cancer stem comprising administering to a subject a composition comprising an antibody that specifically binds to a protein of SEQ ID NO: 1 or a nucleic acid molecule for inhibiting expression of a polynucleotide of SEQ ID NO: 2 as an active ingredient Method of inhibiting cell formation. A composition comprising an antibody that specifically binds to a protein of SEQ ID NO: 1 or a nucleic acid molecule for inhibiting expression of a polynucleotide of SEQ ID NO: 2 as an active ingredient comprises administering to a subject a pancreatic cancer Methods of inhibiting treatment or metastasis.

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