Novel biomarkers for detecting cancer metastasis

Abstract

The present invention relates to a preparation for measuring the expression level of a protein that is exclusively or specifically expressed in circulating tumor cells or a gene that encodes the same, or a method for preventing or treating cancer metastasis by regulating the expression thereof. The present invention provides a method for detecting cancer or cancer metastasis by identifying a protein that is exclusively or specifically expressed in circulating tumor cells or a gene that encodes the same and measuring the expression level thereof, and ultimately, can be useful for the efficient prevention or treatment of cancer metastasis by suppressing the expression of the factor.

Description

[Technical field]

[0001] The present invention relates to a method for predicting cancer onset or cancer metastasis by measuring the expression level of a factor that is specifically expressed in circulating tumor cells (CTCs) in relation to whether or not cancer cells have acquired the ability to metastasize, and a method for suppressing cancer metastasis by regulating the expression of the factor. [Background technology]

[0002] Cancer is known as the disease with the highest mortality rate worldwide. In Korea, the number of people who die from cancer is counted at an average of 160 per 100,000 people per year, which is the number one cause of death by disease in Korea (Cause of Death Statistics 2020). The majority of cancer patients die from cancer metastasis rather than from the primary tumor itself. Cancer metastasis is a phenomenon in which cancer cells leave the primary tumor tissue, invade the surrounding blood vessels and lymphatic vessels, and use these as a pathway to travel long distances to other parts of the body and form new tumors. Numerically, more than 90% of cancer patients die from metastasis from primary tumors (Nature Reviews Cancer, 2006, 6: 49-458), so in the diagnosis and treatment of primary cancer patients, early diagnosis of metastasis or even prediction of metastasis before metastasis is a very important issue in terms of improving the mortality rate of cancer patients.

[0003] In the process of cancer cells leaving the primary tumor and metastasizing through the blood, the adherent primary tumor cells must transform into floating cells called circulating tumor cells (CTCs) in order to leave the primary tumor site and travel through the blood or lymphatic vessels. If there were a clear biomarker for such CTCs, it could be a very useful tool for diagnosing and predicting cancer metastasis.

[0004] Currently, many researchers are having difficulty in the study of cancer metastasis, especially hematogenous cancer metastasis, due to the lack of a clear biomarker for CTCs. Currently, bloodstream cells that do not express CD45, a leukocyte-specific cell membrane protein, and at the same time express EpCAM, an epithelial cell-specific cell membrane protein, are defined as CTCs. In the case of EpCAM, it is the only marker currently used in research and clinical practice for CTC isolation. The role of EpCAM has not yet been clearly elucidated, but EpCAM is a protein expressed on the cell membrane of CTCs, and its expression does not disappear during the epithelial-mesenchymal transition (EMT) process. However, the expression of EpCAM in a mouse model was confirmed by single-cell transcriptome analysis, and it was found to be expressed in only 41.0% of CTCs. This result means that EpCAM is detected in less than half of CTCs, and therefore, if EpCAM is used as a marker to isolate CTCs, many CTCs that do not express EpCAM will be missed.

[0005] Therefore, in order to discover an efficient biomarker that can detect early progression of primary cancer to cancer metastasis and even predict this, the inventors have been studying the relationship between specific genes that are expressed exclusively in CTCs and the presence or absence of metastasis, and have attempted to confirm whether the exclusive expression of these specific genes in CTCs plays an important role in diagnosing or predicting the progression of cancer cells to cancer metastasis and thus has the potential to serve as a biomarker for cancer metastasis.

[0006] Numerous articles and patent documents are referenced throughout this specification and are incorporated by reference. The disclosures of the incorporated articles and patent documents are incorporated by reference in their entireties. In rare cases, the state of the art to which the present invention pertains and the contents of the present invention are more clearly explained. Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have been making intensive research efforts to develop a new diagnostic method that can detect cancer metastasis early or predict it before metastasis by discovering a new biomarker that can improve the low accuracy of conventional biomarkers for cancer metastasis, which is the cause of death in most cancer patients, and ultimately significantly reduce the mortality rate due to cancer metastasis. As a result, the inventors have discovered six proteins that are exclusively expressed on the cell membrane of circulating tumor cells (CTCs), confirmed the possibility of using the proteins as biomarkers for CTCs, and found that the possibility of progression of primary cancer cells to the metastasis stage can be diagnosed early and with high accuracy using these biomarkers, thereby completing the present invention.

[0008] Therefore, an object of the present invention is to provide a diagnostic composition capable of diagnosing the presence or absence of cancer or cancer metastasis.

[0009] Another object of the present invention is to provide a detection composition capable of detecting circulating tumor cells.

[0010] It is still another object of the present invention to provide a diagnostic kit capable of diagnosing the presence or absence of cancer or cancer metastasis.

[0011] It is yet another object of the present invention to provide a method that provides the information necessary to diagnose the presence or absence of cancer or cancer metastasis.

[0012] It is yet another object of the present invention to provide a composition for preventing or treating cancer metastasis.

[0013] It is still another object of the present invention to provide a method for screening a composition for preventing or treating cancer metastasis.

[0014] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings. [Means for solving the problem]

[0015] According to one aspect of the present invention, there is provided a composition for diagnosing cancer, comprising as an active ingredient a preparation for measuring the expression level of one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2, or genes encoding these proteins.

[0016] The present inventors have been making intensive research efforts to develop a new diagnostic method that can detect cancer metastasis early or predict it before metastasis by discovering a new biomarker that can improve the low accuracy of conventional biomarkers for cancer metastasis, which is the cause of death in most cancer patients, and ultimately significantly reduce the mortality rate due to cancer metastasis. As a result, the inventors have discovered six proteins that are exclusively expressed on the cell membrane of circulating tumor cells (CTCs), confirmed the possibility of using the proteins as biomarkers for CTCs, and found that the possibility of progression of primary cancer cells to the metastasis stage can be diagnosed early and with high accuracy using these biomarkers, thereby completing the present invention.

[0017] As used herein, the term "diagnosis" includes determining an individual's susceptibility to a particular disease, determining whether an individual currently has a particular disease, and determining the prognosis of a subject with a particular disease.

[0018] As used herein, the term "diagnostic composition" refers to an integrated mixture or device containing AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2 proteins; or means for measuring the expression levels of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2 genes, in order to determine whether or not cancer cells in a subject have acquired the ability to metastasize, or to predict the possibility of such acquisition, and thus may also be expressed as a "diagnostic kit."

[0019] As used herein, the term "AFDN" refers to a protein called Afadin, which in humans can be encoded by the AFDN gene, and may also be expressed as AF6, MLL-AF6, MLLT4, myeloid / lymphoid or mixed-lineage leukemia;translocated to, 4, afadin, adherens junction formation factor, l-afadin, etc.

[0020] As used herein, the term "CDC42EP1" refers to a protein called Cdc42 effector protein1, which in humans can be encoded by the CDC42EP1 gene, and may also be expressed as BORG5, CEP1, MSE55, CDC42 effector protein1, and the like, in addition to CDC42EP1.

[0021] As used herein, the term "EPHA2" refers to a protein called EPH receptor A2 or ephrin type-A receptor 2, which in humans can be encoded by the EPHA2 gene, and may also be expressed as Epha2, AW545284, Eck, Myk2, Sek-2, Sek2, ARCC2, CTPA, CTPP1, CTRCT6, EPH receptor A2, ECK, etc.

[0022] As used herein, the term "GPRC5A" refers to a protein called retinoic acid-induced protein 3, which in humans can be encoded by the GPRC5A gene, and may also be expressed by other names besides GPRC5A, such as GPCR5A, RAI3, RAIG1, PEIG-1, TIG1, G protein-coupled receptor class C group 5 member A, etc.

[0023] As used herein, the term "JPT2" refers to a protein called hematological and neurological expressed 1-like protein, which in humans can be encoded by the HN1L gene, and may also be expressed by other names such as C16orf34, L11, HN1L, hematological and neurological expressed 1-like, hematological and neurological expressed 1 like, Jupiter microtubule associated homolog 2, etc.

[0024] As used herein, the term "NIBAN2" refers to the protein Niban Apoptosis regulator 2, which in humans can be encoded by the NIBAN2 gene.

[0025] According to a specific embodiment of the invention, said cancer is a metastatic cancer.

[0026] In this specification, the term "metastatic cancer" refers to a new tumor formed when cancer cells detached from primary tumor tissue invade surrounding blood vessels or lymphatic vessels and use them as a pathway to migrate long distances to other parts of the body. Since more than 90% of cancer patient deaths are caused by metastasis from primary tumors (Nature Reviews Cancer, 2006, 6:449-458), inhibiting cancer metastasis in order to improve the mortality rate of cancer patients is as important as treating the primary cancer.

[0027] The mechanism by which cancer cells acquire mobility during metastasis is explained as EMT (epithelial to mesenchymal transition), in which tumor epithelial cells acquire mesenchymal cell traits through genetic mutation, and MET (mesenchymal to epithelial transition), which is the reverse process (J Clin Invest. 2009, 119: 1417-1419). In other words, epithelial cells that have acquired mesenchymal cell traits weaken their intercellular junctions, detach from their original location, and migrate to blood vessels. The cells that have migrated through the blood vessels regain their original epithelial characteristics (MET), settle in secondary sites far from the primary site, and grow the tumor.

[0028] The present inventors have discovered that genes specifically and exclusively expressed in circulating tumor cells (CTCs), which are floating in blood vessels after adherent tumor cells are converted to floating cells, can function as highly reliable biomarkers for diagnosing cancer metastasis or predicting whether or not primary cancer will progress to metastasis. Therefore, when the biomarkers discovered by the present inventors are highly expressed, it can be determined that circulating tumor cells that cause metastatic cancer have already been generated or that there is a high risk of them being generated in the future. For this reason, the term "diagnosis of metastatic cancer" is used interchangeably with "diagnosis of cancer metastasis," "prediction of epithelial-mesenchymal transition," "prediction of generation of circulating tumor cells," or "prediction of cancer prognosis."

[0029] According to a specific embodiment of the invention, the composition comprises a preparation for measuring the expression level of CDC42EP1 or NIBAN2 protein or the genes encoding them.

[0030] According to a specific embodiment of the present invention, the preparation for measuring the expression level of a gene encoding one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2 is a primer or probe that specifically binds to a nucleic acid molecule of the gene.

[0031] As used herein, the term "nucleic acid molecule" is intended to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and the basic building block of a nucleic acid molecule, a nucleotide, includes not only natural nucleotides but also analogues in which the sugar or base moiety has been modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0032] The term "primer" as used herein refers to an oligonucleotide that acts as an initiation point for synthesis under conditions that induce synthesis of a primer extension product complementary to a nucleic acid strand (template), i.e., the presence of nucleotides and a polymerizing agent such as DNA polymerase, at a suitable temperature and pH. Specifically, a primer is a single-stranded deoxyribonucleotide. Primers used in the present invention can contain naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides, and can also contain ribonucleotides.

[0033] The primer of the present invention is annealed to a target nucleic acid and reacts with a template-dependent nucleic acid polymerase. The primer may be an extension primer that forms a complementary sequence with the target nucleic acid by a sequence that extends to the position where the immobilized probe is annealed and occupies the site where the probe is annealed.

[0034] The extension primer used in the present invention comprises a hybridizing nucleotide sequence complementary to a specific base sequence of a target nucleic acid, such as AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 or NIBAN2 gene. The term "complementary" means that the primer or probe is sufficiently complementary to selectively hybridize to the target nucleic acid sequence under a specific annealing or hybridization condition, and includes both substantially complementary and perfectly complementary, specifically, perfectly complementary. In this specification, the term "substantially complementary sequence" means not only a completely matching sequence, but also a sequence that is partially mismatched with the sequence to be compared within the range of annealing to a specific sequence and playing the role of a primer.

[0035] The primer must be long enough to prime the synthesis of an extension product in the presence of a polymerization agent. The suitable length of the primer is determined by many factors, such as temperature, pH, and the source of the primer, but is typically 15-30 nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template. Such primers can be easily designed by those skilled in the art with reference to the target nucleotide sequence, for example, using a primer design program (e.g., PRIMER3 program).

[0036] As used herein, the term "probe" refers to a linear oligomer having natural or modified monomers or bonds, including deoxyribonucleotides and ribonucleotides, that can hybridize to a specific nucleotide sequence. Specifically, the probe is single-stranded for maximum efficiency in hybridization, and more specifically, is a deoxyribonucleotide. As the probe used in the present invention, a sequence that is perfectly complementary to a specific nucleotide sequence of the AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2, or NIBAN2 gene may be used, but a substantially complementary sequence may also be used within a range that does not interfere with specific hybridization. In general, since the stability of a duplex formed by hybridization tends to be determined by the match of the sequences at the ends, it is preferable to use a probe that is complementary to the 3'-end or 5'-end of the target sequence.

[0037] Conditions suitable for hybridization can be determined by referring to the disclosures in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).

[0038] According to a specific embodiment of the present invention, the formulation for measuring one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2 is an antibody or antigen-binding fragment thereof that specifically binds to one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2; or an aptamer that specifically binds to one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2.

[0039] As used herein, the term "antibody" refers to a substance that specifically binds to an antigen to cause an antigen-antibody reaction. For the purposes of the present invention, antibody refers to an antibody that specifically binds to a polypeptide referred to in the present invention.

[0040] According to the present invention, the AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 or NIBAN2 protein of the present invention is detected by an immunoassay method using an antigen-antibody reaction to analyze whether or not cancer cells have acquired metastatic potential. Such an immunoassay can be performed by various immunoassay or immunostaining protocols that have been developed in the past.

[0041] For example, when the method of the present invention is carried out by a radioimmunoassay method, a radioisotope (e.g., C 14 , I 125 , P 32 and S 35 In the present invention, the antibody that specifically recognizes the AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 or NIBAN2 protein is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.

[0042] The antibodies of the present invention can be produced by methods commonly practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), recombinant DNA methods (U.S. Pat. No. 4,816,567) or phage antibody library methods (Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:58, 1-597 (1991)). A general process for the production of antibodies is described in Harlow, E. and Lane, D., Using Immunology, 1997, 1998, 1999, 10:111-115 (1998). Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; and Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984.

[0043] By analyzing the final signal intensity from the above-mentioned immunoassay process, the presence or absence of tumor metastasis or the possibility of tumor metastasis can be predicted. That is, if the signal for AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 or NIBAN2 protein is stronger from an individual's sample than from a normal sample, it is determined that the individual's tumor metastasis has progressed or is likely to progress in the future.

[0044] As used herein, the term "antigen binding fragment" refers to a portion of a polypeptide in the overall immunoglobulin structure to which an antigen can bind, including, but not limited to, F(ab')2, Fab', Fab, Fv, and scFv.

[0045] As used herein, the term "specifically binding" has the same meaning as "specifically recognizing" and means that an antigen and an antibody (or a fragment thereof) specifically interact with each other through an immunological reaction.

[0046] Instead of antibodies, the present invention may use aptamers that specifically bind to AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 or NIBAN2 proteins. As used herein, the term "aptamer" refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity. The general content of aptamers is disclosed in detail in Hoppe-Seyler F, Butz K, "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):426-30(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24):14272-7(1998).

[0047] According to a specific embodiment of the invention, said cancer is breast cancer, ovarian cancer or colon cancer.

[0048] According to yet another aspect of the present invention, there is provided a composition for detecting circulating tumor cells, comprising, as an active ingredient, a preparation for measuring the expression level of one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2, and NIBAN2, or genes encoding the same.

[0049] As used herein, the term "detection" refers to determining whether a target substance is present in a confined space, where "confined space" refers to, but is not limited to, a target organism, a sample, a reaction mixture, a molecular complex, or a substrate, etc. In particular, the term "detection" refers to any act that allows a person of ordinary skill in the art to determine the chemical or biological properties of a target substance by its ability to interact with, bind to, or activate other compounds, or other additional properties.

[0050] Detection may be quantitative or qualitative, where "quantitative" detection means measuring the amount of a target substance or signal, and measurement methods include any method that measures the amount of a target substance or the intensity of a signal. "Qualitative" detection means detecting the presence or absence of a target substance that is not quantified based on its abundance relative to other substances.

[0051] The term "circulating tumor cells" as used herein, also known as CTCs (Circulating Tumor Cells), refers to tumor cells that detach from a primary tumor or a metastatic tumor from a primary tumor, invade blood vessels, and then circulate with the bloodstream. Circulating tumor cells can transform back into adherent cells at sites far from the primary tumor site, causing new cancer metastasis.

[0052] According to the present invention, the biomarkers of the present invention can be used to detect CTCs from biological samples such as blood, serum, plasma or body fluids, and such detection of CTCs may be performed by non-invasive "liquid biopsy". Compared with traditional biopsies, liquid biopsies have the advantages of being applicable to most patients and easy to collect (Front Oncol. 11: 652253., 2021).

[0053] Examples of diagnosing cancer or cancer metastasis by detecting CTCs as described above include colorectal cancer (Biomarker Research volume 9, Article number: 85, 2021), gastric cancer (J Cancer Metastasis Treat; 4: 32., 2018) and breast cancer (Front Oncol. 11: 652253., 2021). Considering the mechanism of action of cancer metastasis through the generation of circulating tumor cells, this method is applicable to all cancers in addition to the carcinomas listed above.

[0054] CTCs are known to be statistically significantly correlated with the stage of a cancer patient's disease, tumor size, the invasiveness of cancer cells, or the presence or absence of metastasis to lymph nodes (Cancer Med. 2020 Mar; 9 (5): 1638-1647). Therefore, detection of the CTCs can be used to diagnose not only cancer or cancer metastasis, but also the stage of the disease, tumor size, and the presence or absence of invasiveness.

[0055] Therefore, in the present invention, the term "detection of circulating tumor cells" is a concept that includes all of the diagnosis of cancer or cancer metastasis, prediction of the presence or absence of metastasis, diagnosis of the disease stage, diagnosis of tumor size, and diagnosis of the presence or absence of invasiveness of cancer cells.

[0056] According to still another aspect of the present invention, there is provided a kit for diagnosing cancer, comprising the composition according to any one of claims 1 to 7.

[0057] In the present invention, the kit may be, but is not limited to, an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0058] The cancer metastasis diagnosis kit of the present invention may further comprise one or more other component compositions, solutions or devices suitable for an analytical method.

[0059] For example, in the present invention, the cancer diagnostic kit may further include essential elements required for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes a primer pair specific to a gene encoding a marker protein. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. In addition, a primer specific to the nucleic acid sequence of a control gene may be included. In addition, the reverse transcription polymerase reaction kit may include a test tube or other suitable container, a reaction buffer (varies in pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

[0060] The diagnostic kit of the present invention can also include essential elements required for performing DNA chips. The DNA chip kit can include a substrate to which cDNA or oligonucleotides corresponding to genes or fragments thereof are attached, and reagents, preparations, enzymes, and the like for producing fluorescently labeled probes. The substrate can also include cDNA or oligonucleotides corresponding to control genes or fragments thereof.

[0061] Furthermore, the diagnostic kit of the present invention may include essential elements required for performing ELISA. The ELISA kit includes an antibody specific to the protein. The antibody has high specificity and affinity for the marker protein and little cross-reactivity with other proteins, and may be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. The ELISA kit may also include an antibody specific to a control protein. In addition, the ELISA kit may include reagents capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e.g., conjugated with the antibody) and its substrate or other substances capable of binding to the antibody.

[0062] In the diagnostic kit of the present invention, the immobilization medium for the antigen-antibody binding reaction is a nitrocellulose membrane, a PVDF membrane, a polyvinyl resin, or a polystyrene membrane. A well plate made of polystyrene resin, a glass slide, etc. can be used, but is not limited to these.

[0063] In the diagnostic kit of the present invention, the label of the secondary antibody is preferably a common coloring agent that undergoes a color-developing reaction, and labels such as HRP (horseradish peroxidase), alkaline phosphatase, colloid gold, fluorescent substances such as FITC (poly-L-lysine-fluorescein isothiocyanate) and RITC (rhodamine-B-isothiocyanate) and dyes can be used, but are not limited to these.

[0064] In addition, in the diagnostic kit of the present invention, the chromogenic substrate for inducing color development is preferably used according to the label that undergoes the color development reaction, and TMB (3,3',5,5'-tetramethylbenzidine), ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-phenylenediamine), etc. can be used. In this case, it is more preferable that the chromogenic substrate is provided in a state of being dissolved in a buffer solution (0.1M NaAc, pH 5.5). The chromogenic substrate such as TMB is decomposed by HRP used as the label of the secondary antibody conjugate to generate a chromogenic immersion body, and the presence or absence of the marker protein is detected by visually checking the degree of immersion of the chromogenic immersion body.

[0065] In the diagnostic kit of the present invention, the washing solution preferably contains phosphate buffer solution, NaCl, and Tween 20, and more preferably a buffer solution (PBST) consisting of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20. After the antigen-antibody binding reaction, the secondary antibody is reacted with the antigen-antibody complex, and then an appropriate amount of the washing solution is added to the immobilized body to wash 3 to 6 times. As the reaction stop solution, a sulfuric acid solution (H2SO4) can be preferably used.

[0066] According to a specific embodiment of the invention, said cancer is a metastatic cancer.

[0067] The metastatic cancer of the present invention has already been described above, so it will be omitted to avoid excessive repetition.

[0068] According to yet another aspect of the present invention, there is provided a method for providing information necessary for diagnosing cancer, comprising the step of measuring the expression level of one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2, or genes encoding same, in a biological sample isolated from an individual.

[0069] The AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2 proteins of the present invention, the genes encoding them and the cancers that can be diagnosed using them have already been described above, so they will not be described here to avoid excessive repetition.

[0070] The present inventors first found that the expression level of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 or NIBAN2 protein is positively correlated with the amount of circulating tumor cells in the blood or the acquisition of metastatic ability of cancer cells. Therefore, when AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 or NIBAN2 protein or the gene encoding it is highly expressed in an individual, the individual is judged to have or may have cancer cells with metastatic ability in the future.

[0071] As used herein, the term "high expression" refers to a case where the expression level of the protein or gene is significantly higher than that of a control group in which metastasis does not occur or is unlikely to occur. means that the expression level is 130% or more of that of the control group, more specifically, 150% or more, and most specifically, 170% or more.

[0072] In the present specification, the term "individual" refers to an individual who provides a sample for measuring the expression level of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 or NIBAN2 protein or the gene encoding it, and who ultimately becomes the subject of analysis of the acquisition of metastatic ability of cancer cells. Individuals include, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys, and specifically, humans. Since the composition of the present invention provides information not only on the current acquisition of metastatic ability of cancer cells, but also on the genetic risk of cancer cells acquiring metastatic ability in the future, the individual of the present invention may be a patient with advanced cancer metastasis or a primary cancer patient who has not yet acquired metastatic ability.

[0073] According to a specific embodiment of the invention, said cancer is a metastatic cancer.

[0074] The metastatic cancer of the present invention has already been described above, so it will be omitted to avoid excessive repetition.

[0075] According to a specific embodiment of the invention, said biological sample is selected from the group consisting of whole blood, plasma and serum.

[0076] As used herein, the term "whole blood" refers to blood that is generally composed of non-clotting plasma and cellular components. Plasma may make up approximately 50-60% of the whole blood volume, and cellular components (e.g., red blood cells, white blood cells, or platelets) may make up approximately 40-50%.

[0077] As used herein, the term "plasma" refers to the liquid component of blood, which functions as a transport medium in providing nutrients to the cells and organs of the body.

[0078] The term "serum" used in this specification refers to a pale yellow liquid collected from blood. Specifically, when blood is collected and left to stand, the fluidity of the blood decreases and red coagulation occurs. The term "serum" refers to the pale yellow body fluid component that remains when the red coagulation is removed.

[0079] According to the present invention, cancer or cancer metastasis can be diagnosed using the biomarkers of the present invention, and the diagnosis may be performed by liquid biopsy using a patient-derived body fluid such as blood. Such liquid biopsy methods are non-invasive compared to conventional tissue biopsy procedures, and therefore have the advantage of minimizing patient pain and providing information about cancer more quickly.

[0080] According to a specific embodiment of the invention, said cancer is breast cancer, ovarian cancer or colon cancer.

[0081] According to yet another aspect of the present invention, there is provided a composition for preventing or treating cancer metastasis, comprising as an active ingredient an inhibitor of one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2 or genes encoding the same.

[0082] As used herein, the term "inhibitor" refers to an agent that induces a decrease in the activity or expression of a target gene. This refers to a substance that reduces the activity or expression of a target gene not only when its activity or expression is undetectable or present at insignificant levels, but also when it reduces the activity or expression to such an extent that the biological function of the target gene is significantly reduced.

[0083] Examples of target gene inhibitors include shRNA, siRNA, miRNA, ribozymes, peptide nucleic acids (PNA), antisense oligonucleotides, or CRISPR systems containing guide RNAs that recognize target genes, which inhibit the expression of the gene at the gene level and whose sequences are already known in the art, and antibodies or aptamers that inhibit the expression at the protein level, as well as compounds, peptides, and natural products that inhibit the activity of these. However, all gene and protein level inhibition means known in the art can be used.

[0084] As used herein, the term "shRNA (small hairpin RNA)" refers to an RNA sequence that is a single strand consisting of 50-70 nucleotides that forms a stem-loop structure in vivo and forms a tight hairpin structure to suppress the expression of a target gene by RNA interference. In general, a long RNA of 19-29 nucleotides is base-paired complementary to both sides of a loop region of 5-10 nucleotides to form a double-stranded stem, which is transduced into cells via a vector containing a U6 promoter for constant expression, and is generally transmitted to daughter cells so that suppression of the expression of the target gene is inherited.

[0085] As used herein, the term "siRNA" refers to a short double-stranded RNA capable of inducing the RNAi (RNA interference) phenomenon by cleavage of a specific mRNA. It is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases, and may have a blunt end or a cohesive end as long as it can suppress the expression of the target gene by the RNAi effect. The cohesive end structure may be a 3'-end overhanging structure or a 5'-end overhanging structure.

[0086] As used herein, the term "miRNA (microRNA)" refers to an oligonucleotide that is not expressed in cells, a single-stranded RNA molecule that has a short stem-loop structure and suppresses the expression of a target gene by complementary binding to the mRNA of the target gene.

[0087] As used herein, the term "ribozyme" refers to a type of RNA molecule that has an enzyme-like function of recognizing a specific RNA base sequence and cleaving it by itself. Ribozymes are composed of a region that specifically binds to a complementary base sequence of a target mRNA strand and a region that cleaves the target RNA.

[0088] In this specification, the term "PNA (peptide nucleic acid)" refers to a molecule that has all the properties of nucleic acids and proteins and can bind to DNA or RNA in a complementary manner. PNA is not found in nature but is artificially synthesized by chemical methods, and forms a double strand by hybridization with natural nucleic acids of complementary base sequences to regulate the expression of target genes.

[0089] As used herein, the term "antisense oligonucleotide" refers to a nucleotide sequence that is complementary to a specific mRNA sequence and binds to a complementary sequence in the target mRNA to inhibit its translation into protein, translocation into the cytoplasm, maturation, or any other activity essential to its overall biological function. Antisense oligonucleotides refer to nucleic acid molecules that inhibit the activity of a target molecule. Antisense oligonucleotides can be modified at one or more base, sugar, or backbone positions to enhance efficacy. Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55, 1995. The oligonucleotide backbones can be modified to phosphorothioates, phosphotriesters, methylphosphonates, short chain alkyls, cycloalkyls, short chain heteroatomic, heterocyclic sugar sulfonates, and the like.

[0090] As used herein, the term "guideRNA (gRNA)" refers to an RNA molecule used in a gene editing system that recognizes a target gene and induces a nuclease to specifically cleave the recognized site. A representative example of such a gene editing system is the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system.

[0091] According to the present invention, the expression inhibitor of the present invention may be a specific antibody that inhibits the activity of the protein encoded by the gene. The antibody that specifically recognizes the target protein is a polyclonal or monoclonal antibody, and is preferably a monoclonal antibody.

[0092] The antibodies and aptamers used in the present invention have already been described above, so their description will be omitted to avoid excessive duplication.

[0093] As used herein, the term "prevention" refers to inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition, but is susceptible to such disease or condition.

[0094] As used herein, the term "treatment" means (a) suppressing the development of a disease, illness, or symptom; (b) alleviating a disease, illness, or symptom; or (c) eliminating a disease, illness, or symptom. When the composition of the present invention is administered to a subject, the expression of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2, NIBAN2 protein or the gene encoding the same is suppressed, and the production of circulating tumor cells is inhibited, thereby suppressing the development of symptoms caused by tumors, specifically metastatic tumors, or eliminating or alleviating the symptoms. Therefore, the composition of the present invention may be a composition for treating these diseases by itself, or may be administered together with other pharmacological ingredients and applied as a therapeutic adjuvant for the above diseases. Therefore, as used herein, the term "treatment" or "therapeutic agent" includes the meaning of "therapeutic adjuvant" or "therapeutic adjuvant".

[0095] According to yet another aspect of the present invention, there is provided a method for screening a composition for preventing or treating cancer metastasis, comprising the steps of: (a) contacting a test substance with a biological sample containing one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2, genes encoding same, or cells expressing same; (b) measuring the expression level of one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2 or genes encoding same in said biological sample; If the expression level of the protein or the gene in the biological sample is decreased, the test substance is determined to be a composition for preventing or treating cancer metastasis.

[0096] The factors that are specifically and exclusively expressed in circulating tumor cells used in the present invention and the types of cancer that can be prevented or treated by regulating their expression have already been described above, so a description thereof will be omitted to avoid excessive duplication.

[0097] In the present invention, the term "biological sample" refers to any sample containing cells expressing the above-mentioned genes obtained from a mammal, including a human, including, but not limited to, tissues, organs, cells, or cell cultures. More specifically, the biological sample may be cancer tissues, cancer cells, or cultures thereof.

[0098] The term "test substance" used in reference to the screening method of the present invention refers to an unknown substance that is added to a sample containing cells expressing the genes of the present invention and used in screening to examine whether it affects the activity or expression level of these genes. The test substance includes, but is not limited to, chemical compounds, nucleotides, peptides, and natural extracts. The step of measuring the expression level or activity of the genes in the biological sample treated with the test substance may be performed by various expression and activity measuring methods known in the art.

[0099] According to a specific embodiment of the invention, said biological sample comprises cancer tissue or cancer cells.

[0100] According to yet another aspect of the present invention, there is provided a method for diagnosing cancer, comprising the step of administering to a subject a composition containing as an active ingredient a preparation for measuring the expression level of one or more proteins selected from the group consisting of FDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2, or genes encoding these proteins.

[0101] According to yet another aspect of the present invention, there is provided a method for preventing or treating cancer metastasis, comprising administering to a subject a composition containing as an active ingredient an inhibitor of one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2 or genes encoding the same.

[0102] The preparations for measuring the expression levels of one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2 of the present invention or genes encoding the same, their inhibitors, and carcinomas that can be diagnosed, or whose metastasis can be prevented or treated by inhibiting the above genes have already been described above, so they will be omitted here to avoid excessive repetition. Effect of the Invention

[0103] The features and advantages of the present invention can be summarized as follows: (a) The present invention provides a method for preventing or treating cancer metastasis by measuring the expression levels of proteins that are exclusively or specifically expressed in circulating tumor cells or genes encoding such proteins, or by regulating their expression. (b) The present invention provides a method for detecting cancer or cancer metastasis by discovering a protein or a gene encoding the protein that is exclusively and specifically expressed in circulating tumor cells and measuring the expression level of the protein or the gene encoding the protein, and ultimately, the method can be useful for the efficient prevention or treatment of cancer metastasis by suppressing the expression of the factor. [Brief description of the drawings]

[0104] [Figure 1] The graph shows that cancer mortality has been the number one cause of death since 1990, with 160.1 people per 100,000 dying from cancer in 2020. [Diagram 2] FIG. 1 is a schematic diagram of the single cell transcriptome analysis process for primary cancer cells and circulating tumor cells, using a mouse model xenografted with cancer cell lines and a high density micropore chip (HDM Chip). [Diagram 3] Immunofluorescence was used to identify EpCAM+ / CD45- circulating tumor cells and EpCAM- / CD45+ leukocytes in blood vessels. [Figure 4]FIG. 1 shows UMAP maps for primary cancer cells and circulating tumor cells, as well as feature and violin plots showing the expression levels of EpCAM and CD45 transcripts in primary cancer cells and circulating tumor cells. [Diagram 5] 1 is a table showing the number of EpCAM+ / CD45- circulating tumor cells as a result of single cell transcriptome analysis. [Figure 6] This presents a group of eight biomarker candidates that are exclusively expressed in metastatic cancers and not expressed in leukocytes. The selection criteria were proteins that are expressed in more than 50% of circulating tumor cells and less than 1% of leukocytes. Among these, MET and BCAR1, which are widely known as markers of breast cancer, were excluded from the candidate group. [Figure 7] FIG. 1 shows UMAP maps of tumor cells from ovarian cancer patients, colon cancer patients and healthy controls. [Figure 8] FIG. 1 shows a Violin Plot of six novel biomarker candidates and EpCAM vs. CD45 (PTPRC), demonstrating that the six novel biomarkers are more exclusively expressed in circulating tumor cells than the conventional CTC biomarker, EpCAM. [Figure 9] FIG. 1 shows feature plots of six novel biomarker candidates and EpCAM and CD45 (PTPRC). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0105] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention in more detail, and that the scope of the present invention is not limited to these examples according to the gist of the present invention.

[0106] Working Example Experimental and analytical methods CDX models and animal studies LM3, a breast cancer cell line, was kindly provided by HW Park, Department of Bioengineering, College of Life Systems, Yonsei University, Korea. Orthotopic implantation of cultured LM3-GFP was performed after removal of the mammary fat pads of immunocompromised 3-4 week-old NOD / SCID or NGS mice by established procedures. Tumor growth and metastasis were monitored weekly. When the tumors reached 2.0-2.5 cm in length or thickness, the animals were euthanized, and tissues and blood were collected.

[0107] Tissue harvesting and isolation The terminally ill experimental animals were euthanized by CO2 asphyxiation and cervical dislocation, and blood samples were extracted from the heart using a syringe. After the blood sample preparation was completed, the mice were treated with 10 mM EDTA (D-PBS solvent). The primary tumors of the mice were mechanically dissected for flow cytometry, and then incubated at 37°C for 45 minutes in a solvent (RPMI-F12 medium with 5% FBS, 5 μg ml -1 The cells were treated with collagenase IV (Sigma-Aldrich cat. no. C5138-1G) in insulin and 1% penicillin / streptomycin solution. The cell suspension was diluted with 2 μg ml -1 After washing with DNAse I (Worthington Biochemical, cat. no. LS002139) for 5 min, cells were additionally dissociated with 0.05% trypsin for 10 min. After washing with Hanks balanced salt solution with 2% PBS, cells were passed through a 70 μm filter. Primary tumor cells and blood cells were treated with 1× RBC lysis buffer to remove red blood cells by lysis, and then resuspended in 2% FBS in PBS for immunofluorescence assay and cell separation.

[0108] Immunofluorescence assay and imaging To measure epithelial (EpCAM) and leukocyte (CD45) markers on the membrane surface of circulating tumor cells, cells were attached to slides by cytocentrifugation. For immunofluorescence, slides were incubated overnight at 4°C with antibodies against EpCAM (Abcam, ab213500, 1:100 dilution) and CD45 (Abcam, ab33923, 1:100 dilution), followed by incubation with a fluorescent-conjugated anti-IgG secondary antibody (antibody Info, cat.num.) and DAPI. Immunolabeled cells were mounted for imaging, and confocal images were taken with a Zeiss LSM780 inverted microscope.

[0109] Generation of scRNA-seq data derived from circulating tumor cells To sort LM3-GFP cells from mouse breast tissue and blood, single cells were sorted onto HDM chips (Smart Biopsy™ Cell Isolator-CIS030) and single-cell RNA sequencing libraries were generated using the Chromium Single Cell 3' Library, Gel Bead & Multiplex kit and chip kit (10X Genomics) targeting 10,000 cells per library according to the manufacturer's conditions. Such a droplet-based system used barcodes (one for each cell) and unique molecular identifiers (Unique Molecular Identifiers, UMIs, one for each unique transcript) to obtain unique 3'-mRNA gene expression profiles from every captured cell. All samples were sequenced using an Illumina HiSeq4000 and mapped to the human reference genome (GRCh38) using Cell Ranger (10X Genomics).

[0110] scRNA-seq bioinformatics for CDX models Illumina output from 10X Genomics Chromium sequencing was processed using Cell Ranger 6.0.0. A minimum of 500 Genewise reads were used, and data was exported from Cell Ranger in Matrix Market format and read in R using Seurat's Read 10X function. An average of >135 million reads and 13,000 cells were obtained for each sample. Each 10X library was individually quality checked, and cells were filtered to ensure good gene coverage, consistent read range, and low mitochondrial read counts. In general, at least 500 genes were required to be detected per cell, although the lower limit was reduced to 200 or 300 for some libraries. For mitochondrial read counts, only 10% or less per cell was accepted, but the upper limit increased to a maximum of 65% and 92% for cells derived from primary tumor and blood samples, respectively. Cells with an unusually large number of reads or detected genes were filtered to minimize the generation of doublets (libraries generated from two cells). Cells with more than 20% unmapped reads were also filtered to prevent mouse-derived cells from contaminating the results. After quality filtering, cells from 3,006 primary tumor samples and 5,511 blood samples were prepared for analysis. Statistical analysis of the 10X data was performed using the Seurat (v4.1.0, Seurat et al., 2019) software package for R.

[0111] Samples were combined using merging in Seurat, and cell clusters were identified using the basic Louvain clustering algorithm in Seurat. The basic Seurat function settings were used, with a principal component dimension of 1:10 for all dimension reduction and integration steps. Cluster resolution values ​​were set to 1.0 unless otherwise specified. RunUMAP Random Seed was set to 1,000 to ensure reproducibility. To detect genes not expressed in hemocytes, marker genes for cell clusters were identified using the FindAllMarkers function in Seurat with basic settings, except for the proportion of cells expressing a particular gene.

[0112] scRNA-seq bioinformatics for validation scRNA-seq data for ascites from ovarian cancer patients were searched in DUOS (Data Using Oversight System, Dataset ID: DUOS-000118). scRNA-seq data for ascites from colorectal cancer patients was provided by HS Kim, Department of Internal Medicine, Yonsei University College of Medicine. scRNA-seq data for healthy PBMCs was provided by HY Gee, Department of Pharmacology, Yonsei University College of Medicine. All data were processed with the datat processing workflow of the CDX model, and circulating tumor cells were selected based on epithelial cell marker expression scores using the celldex (v1.4.0, Liu et al., 2020) software package for R.

[0113] Experimental Results Limitation analysis of the existing biomarker EpCAM The expression of EpCAM in a mouse model was confirmed by single-cell transcriptome analysis, and only 41.0% of CTCs expressed EpCAM and did not have CD45 (Figures 4 and 5). This result means that EpCAM is only detected in half of CTCs, and therefore, if EpCAM is used as a marker to separate CTCs, many CTCs that do not express EpCAM will be missed. To overcome these limitations of EpCAM, the present inventors searched for a new biomarker for CTCs.

[0114] Selection of candidate biomarkers When a comparison and analysis was performed between the isolated CTCs and single-cell transcriptomes from blood, it was confirmed that the RNA of eight of the genes that code for proteins located in the cell membrane (AFDN, BCAR1, CDC42EP1, EPHA2, GPRC5A, JPT2, MET, and NIBAN2) was exclusively expressed in CTCs (Figure 6). Of the eight genes mentioned, six cell membrane proteins, excluding MET and BCAR1, which are well known to be associated with the progression of breast cancer, were selected as candidate CTC biomarkers.

[0115] The nucleotide sequences of the AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2 genes are attached herein as Sequence Listings 1, 2, 3, 4, 5 and 6, respectively, and the sequences of the proteins encoded by the genes are attached herein as Sequence Listings 7, 8, 9, 10, 11 and 12, respectively.

[0116] Circulating tumor cells in patients with ovarian and colorectal cancer We confirmed that CTCs isolated from ascites of patients with breast cancer, ovarian cancer, and colorectal cancer expressed six biomarkers, including AFDN, exclusively in comparison with blood.

[0117] Potential as a biomarker Based on the above results, it was confirmed that the RNA expression levels of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2, MET, or NIBAN2 in cells present in the bloodstream could be used as biomarkers for various cancer metastasis.

[0118] Although certain parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific descriptions are merely preferred embodiments and therefore do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for diagnosing cancer, comprising as an active ingredient a preparation for measuring the expression level of one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2, or genes encoding these proteins.

2. The composition of claim 1, wherein the cancer is a metastatic cancer.

3. The composition according to claim 1, characterized in that the composition comprises a preparation for measuring the expression level of CDC42EP1 or NIBAN2 protein or the gene encoding same.

4. The composition described in claim 1, characterized in that the preparation for measuring the expression level of a gene encoding one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2 is a primer or probe that specifically binds to the nucleic acid molecule of the gene.

5. The composition of claim 1, characterized in that the formulation for measuring one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2 is an antibody or an antigen-binding fragment thereof that specifically binds to one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2; or an aptamer that specifically binds to one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2.

6. The composition of claim 1, wherein the cancer is breast cancer, ovarian cancer or colon cancer.

7. A composition for detecting circulating tumor cells, comprising as an active ingredient a preparation for measuring the expression level of one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2, or genes encoding these proteins.

8. A cancer diagnostic kit comprising the composition according to any one of claims 1 to 7.

9. The kit according to claim 8, wherein the cancer is a metastatic cancer.

10. A method for providing information necessary for diagnosing cancer, comprising the step of measuring the expression level of one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2, or genes encoding these, in a biological sample isolated from an individual.

11. The method for providing information according to claim 10, characterized in that the cancer is a metastatic cancer.

12. The method for providing information according to claim 10, characterized in that the biological sample is selected from the group consisting of whole blood, plasma and serum.

13. The method for providing information according to claim 10, characterized in that the cancer is breast cancer, ovarian cancer or colon cancer.

14. A composition for preventing or treating cancer metastasis, comprising as an active ingredient an inhibitor of one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2, or genes encoding the same.

15. A method for screening for a composition for preventing or treating cancer metastasis, comprising the steps of: (a) contacting a test substance with a biological sample containing one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2, genes encoding same, or cells expressing same; (b) measuring the expression level of one or more proteins selected from the group consisting of AFDN, CDC42EP1, EPHA2, GPRC5A, JPT2 and NIBAN2, or genes encoding same, in the biological sample; If the expression level of the protein or the gene in the biological sample is decreased, the test substance is determined to be a composition for preventing or treating cancer metastasis.

16. 16. The method of claim 15, wherein the biological sample comprises cancer tissue or cancer cells.

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