COMPOSITION FOR DIAGNOSIS OR TREATMENT OF ANTICANCER DRUG RESISTANCE

Abstract

The present invention relates to a composition for diagnosing resistance to a combination of ECF (Epirubicin, Cisplatin and 5-Fluorouracil; ECF), which is used for the treatment of gastric cancer, and a diagnostic kit comprising the same. In order to solve a problem that cancer recurrence and drug resistance occur due to an increase in tumor initiating cells (TICs) after treatment with a drug belonging to the ECF family, NINJ2 may be inhibited by early detection of resistance appearance, thereby suppressing the recurrence of gastric cancer and treating resistance to the drugs.

Claims

1. A method for diagnosing anticancer drug resistance, comprising measuring an expression level of NINJ2 (nerve injury-induced protein 2; Ninjurin 2) protein or a gene encoding the protein.

2. The method of claim 1, wherein the measuring of the expression level of the protein is carried out using at least one selected from the group consisting of an antibody, an oligopeptide, a ligand, a peptide nucleic acid (PNA), and an aptamer, which bind specifically to the protein.

3. The method of claim 1, wherein the measuring of the expression level of the gene is carried out using at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide, which bind specifically to the gene.

4. The method of claim 1, wherein the anticancer drug comprises at least one selected from the group consisting of nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, bevacizumab, cisplatin, cetuximab, viscumalbum, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tusetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxyfluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludagabine, enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmofur, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin, daunorubicin, dactinomycin, pirarubicin, aclarubicin, peplomycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonine, exemestane, aminoglutethimide, anagrelide, navelbine, fadrazol, tamoxifen, toremifen, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, and carmustine.

5. The method of claim 4, wherein the anticancer drug comprises any one or more selected from the group consisting of epirubicin, cisplatin and 5-fluorouracil.

6. The method of claim 1, wherein the anticancer drug is for treatment of cancer which is thyroid cancer, parathyroid cancer, gastric cancer, ovarian cancer, colorectal cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma.

7-12. (canceled)

13. A method for preventing or treating cancer, comprising a step of administering an effective amount of an agent to a subject in need thereof, wherein the agent is for reducing an activity or expression level of NINJ2 (nerve injury-induced protein 2; Ninjurin 2) protein, or an agent for reducing an expression level of a gene encoding the protein.

14. The method of claim 13, wherein the agent for reducing the activity or expression level of the protein is any one or more selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products, which bind specifically to the NINJ2 protein or a portion thereof.

15. The method of claim 13, wherein the agent for reducing the expression level of the gene is any one or more selected from the group consisting of an antisense nucleotide, a short interfering RNA (siRNA), a short hairpin RNA, and ribozyme, which bind complementarily to the NINJ2 gene or a portion thereof.

16. The method of claim 13, wherein the cancer is thyroid cancer, parathyroid cancer, gastric cancer, ovarian cancer, colorectal cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma.

17. (canceled)

18. A method for treating anticancer drug resistance or enhancing anticancer drug sensitivity, comprising a step of administering an effective amount of an agent to a subject in need thereof, wherein the agent is for reducing an activity or expression level of NINJ2 (nerve injury-induced protein 2; Ninjurin 2) protein, or an agent for reducing an expression level of a gene encoding the protein.

19. The method of claim 18, wherein the agent for reducing the activity or expression level of the protein is any one or more selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products, which bind specifically to the NINJ2 protein or a portion thereof.

20. The method of claim 18, wherein the agent for reducing the expression level of the gene is any one or more selected from the group consisting of an antisense nucleotide, a short interfering RNA (siRNA), a short hairpin RNA, and ribozyme, which bind complementarily to the NINJ2 gene or a portion thereof.

21. The method of claim 18, wherein the anticancer drug comprises at least one selected from the group consisting of nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, bevacizumab, cisplatin, cetuximab, viscumalbum, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tusetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxyfluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludagabine, enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmofur, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin, daunorubicin, dactinomycin, pirarubicin, aclarubicin, peplomycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonine, exemestane, aminoglutethimide, anagrelide, navelbine, fadrazol, tamoxifen, toremifen, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, and carmustine.

22-28. (canceled)

28. A method for screening a drug for treating anticancer drug resistance, comprising: treating cancer cells expressing NINJ2 (nerve injury-induced protein 2; Ninjurin 2) protein or a gene encoding the protein, or an anticancer drug-resistant cancer organoid comprising cancer cells expressing NINJ2 (nerve injury-induced protein 2; Ninjurin 2) protein or a gene encoding the protein, with a candidate substance in vitro; and measuring an activity or expression level of the NINJ2 protein or an expression level of the gene encoding the protein in the cancer cells or the cancer organoid after treatment with the candidate substance.

29. The method of claim 28, further comprising a step of measuring an activity or expression level of at least one protein selected from among CD44 and periostin proteins, or measuring an expression level of a gene encoding at least one protein.

30. The method of claim 28, further comprising a step of determining that the candidate substance is the drug for treating anticancer drug resistance, when the measured activity or expression level of the NINJ2 protein decreases after treatment with the candidate substance, or the measured expression level of the gene encoding the protein decreases after treatment with the candidate substance.

31-36. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0194] FIG. 1A shows a process of administering ECF drugs to each cell line according to one example of the present invention.

[0195] FIG. 1B shows representative IC.sub.50 values for non-ECF-resistant parental cells and ECF-resistant cells according to one example of the present invention.

[0196] FIG. 1C shows representative IC.sub.50 values for non-ECF-resistant parental cells and ECF-resistant cells according to one example of the present invention.

[0197] FIG. 1D shows the results of examining changes in tumor volume after injecting ECF into tumor mouse models xenografted with non-ECF-resistant parental cells and ECF-resistant cells (ECF-R), respectively, according to one example of the present invention.

[0198] FIG. 2 shows a process of selecting genes, which commonly appear in ECF-resistant cell lines, using a heatmap according to one example of the present invention.

[0199] FIG. 3A shows the results of quantitative qRT-PCR analysis of the expression levels of NINJ2 proteins in non-ECF-resistant parental cells and ECF-resistant cells (ECF-R) according to one example of the present invention.

[0200] FIG. 3B shows the results of Western blot analysis of the expression levels of NINJ2 proteins in non-ECF-resistant parental cells and ECF-resistant cells (ECF-R) according to one example of the present invention.

[0201] FIG. 4A shows the results of FACS performed to analyze the surface expression of NINJ2 on the wild-type cells and ECF-resistant cells (ECF-R) derived from the MKN-74 cell line, according to one example of the present invention.

[0202] FIG. 4B shows the results of FACS performed to analyze the surface expression of CD44 on the wild-type cells and ECF-resistant cells (ECF-R) derived from the MKN-74 cell line, according to one example of the present invention.

[0203] FIG. 4C shows the results of FACS performed to analyze CD44 expression on gate NINJ2(?) and NINJ2(+) populations in the ECF-resistant cells (ECF-R) derived from the MKN-74 cell line, according to one example of the present invention.

[0204] FIG. 4D depicts immunofluorescence images showing the expression level of each of markers (CD44 and NINJ2) in the wild-type cells and ECF-resistant cells (ECF-R) derived from the MKN-74 cell line, according to one example of the present invention.

[0205] FIG. 4E shows Western blot analysis results indicating the expression level of each of markers (CD44 and hNINJ2) in tumor spheres derived from the MKN-74 cell line, according to one example of the present invention.

[0206] FIG. 4F depicts immunofluorescence images showing the expression level of each of markers (CD44 and hNINJ2) in tumor spheres derived from the MKN-74 cell line, according to one example of the present invention.

[0207] FIG. 5A shows representative IC.sub.50 values for NINJ2 Iso-1 and Iso-3 overexpressing MKN-74 cell lines after ECF treatment according to one example of the present invention.

[0208] FIG. 5B shows the results of qRT-PCR performed to analyze the expression levels of CD44 mRNA in NINJ2 Iso-1 and Iso-3 overexpressing MKN-74 cell lines according to one example of the present invention.

[0209] FIG. 5C shows the results of flow cytometry performed to analyze the proportion of CD44-high cells in NINJ2 Iso-1 and Iso-3 overexpressing MKN-74 cell lines according to one example of the present invention.

[0210] FIG. 5D shows the results of in vitro limiting dilution analysis performed on NINJ2 Iso-1 and Iso-3 overexpressing MKN-74 cell lines according to one example of the present invention.

[0211] FIG. 5E shows the results of analyzing the number of tumor spheres formed from NINJ2 Iso-1 and Iso-3 overexpressing MKN-74 cell lines according to one example of the present invention.

[0212] FIG. 6 shows the results of flow cytometry performed to analyze changes in cell cycle in NINJ2 Iso-1 and Iso-3 overexpressing MKN-74 cell lines according to one example of the present invention.

[0213] FIG. 7A shows the results of performing immunoblotting analysis (co-immunoprecipitation; co-IP) using the HaloTag pull-down system (G6504, Promega) to examine NINJ2/periostin interaction from stable NINJ2-HaloTag MKN-74 cancer cells according to one example of the present invention.

[0214] FIG. 7B shows the results of measuring the expression level of periostin mRNA by qRT-PCR after isolating mRNA from ECF-resistant MKN-74 cancer cells, according to one example of the present invention.

[0215] FIG. 7C shows the results of Western blotting analysis of proteins highly expressed in NINJ2 Iso-1 and Iso-3 overexpressing MKN-74 cell lines, according to one example of the present invention.

[0216] FIG. 8 shows the results of analyzing cell viability by crystal violet staining and WST-1 assay after introducing human NINJ2-targeting shRNA lentiviral particles (Clone-1 and Clone-2) into the ECF-resistant MKN-74 cell line and then administering ECF to the cell line, according to one example of the present invention.

[0217] FIGS. 9A and 9B show the results of measuring changes in tumor volume and weight after transplanting wild-type or scrambled siRNA RES and siNINJ2 RES cancer cell lines from the MKN-28/74 cell line into nude mice and then administering ECF and siRNA to the nude mice when the tumor volume reached 100 mm.sup.3, according to one example of the present invention.

[0218] FIG. 10A shows the morphologies of a parent organoid and an ECF-resistant human gastric cancer organoid, and representative IC.sub.50 values after ECF treatment, according to one example of the present invention.

[0219] FIG. 10B shows the results of comparing the mRNA expression levels of human NINJ2 and CD44 in a parent organoid and an ECF-resistant human gastric cancer organoid, according to one example of the present invention.

[0220] FIG. 10C shows the results of performing NINJ2 scoring analysis through histological analysis of gastric tumor patients showing partial response (PR), stable disease (SD) and progressive disease (PD), according to one example of the present invention.

[0221] FIG. 10D shows Kaplan-Meier curves for the overall survival (OS) of gastric cancer patients, obtained through public data, according to one example of the present invention.

BEST MODE

[0222] One embodiment of the present invention is directed to a composition for diagnosing anticancer drug resistance, the composition containing an agent for measuring the expression level of NINJ2 (nerve injury-induced protein 2; Ninjurin 2) protein or a gene encoding the protein.

[0223] Another embodiment of the present invention is directed to a kit for diagnosing anticancer drug resistance, the kit comprising the composition for diagnosing anticancer drug resistance according to the present invention.

[0224] Still another embodiment of the present invention is directed to a method for providing information for diagnosing anticancer drug resistance, the method comprising a step of measuring the expression level of the NINJ2 protein or a gene encoding the protein in a biological sample isolated from a subject of interest.

[0225] Yet another embodiment of the present invention is directed to a pharmaceutical composition for treating anticancer drug resistance or enhancing anticancer drug sensitivity, the pharmaceutical composition containing, as an active ingredient, an agent for reducing the activity or expression level of NINJ2 protein or an agent for reducing the expression level of a gene encoding the protein.

[0226] Still yet another embodiment of the present invention is directed to a method for treating anticancer drug resistance or enhancing anticancer drug sensitivity, the method comprising a step of administering, to a subject in need thereof, an effective amount of either an agent for reducing the activity or expression level of NINJ2 (nerve injury-induced protein 2; Ninjurin 2) protein or an agent for reducing the expression level of a gene encoding the protein.

[0227] A further embodiment of the present invention is directed to a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition containing, as an active ingredient, an agent for reducing the activity or expression level of NINJ2 protein, or an agent for reducing the expression level of a gene encoding the protein.

[0228] Another further embodiment of the present invention is directed to a method for preventing or treating cancer, the method comprising a step of administering, to a subject in need thereof, an effective amount of either an agent for reducing the activity or expression level of NINJ2 (nerve injury-induced protein 2; Ninjurin 2) protein or an agent for reducing the expression level of a gene encoding the protein.

[0229] Still another further embodiment of the present invention is directed to a method for preventing or treating cancer, the method comprising a step of administering, to a subject in need thereof, an effective amount of any one or more selected from the group consisting of an antisense nucleotide, a short interfering RNA (siRNA), a short hairpin RNA, and ribozyme, which bind complementarily to a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 or 6.

[0230] Yet another further embodiment of the present invention is directed to a pharmaceutical composition for preventing or treating anticancer drug-resistant cancer, the pharmaceutical composition containing, as an active ingredient, an agent for reducing the activity or expression level of NINJ2 protein or an agent for reducing the expression level of a gene encoding the protein.

[0231] Still yet another further embodiment of the present invention is directed to a method for preventing or treating anticancer drug-resistant cancer, the method comprising a step of administering, to a subject in need thereof, an effective amount of either an agent for reducing the activity or expression level of NINJ2 (nerve injury-induced protein 2; Ninjurin 2) protein or an agent for reducing the expression level of a gene encoding the protein.

[0232] A still further embodiment of the present invention is directed to a method for preventing or treating anticancer drug-resistant cancer, the method comprising a step of administering, to a subject in need thereof, an effective amount of any one or more selected from the group consisting of an antisense nucleotide, a short interfering RNA (siRNA), a short hairpin RNA, and ribozyme, which bind complementarily to a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 or 6.

[0233] Yet still further embodiment of the present invention is directed to an anticancer drug-resistant cancer organoid comprising cancer cells expressing NINJ2 protein or a gene encoding the protein.

[0234] Another embodiment of the present invention is directed to a method for screening a drug for overcoming or treating anticancer drug resistance or a drug for enhancing anticancer drug sensitivity, the method comprising steps of: treating either cancer cells expressing NINJ2 protein or a gene encoding the protein, or a cancer organoid provided by the present invention, with a candidate substance in vitro; and measuring the activity or expression level of the NINJ2 protein or measuring the expression level of the gene encoding the protein, in the cancer cells or the cancer organoid after treatment with the candidate substance.

MODE FOR INVENTION

[0235] 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, and it will be apparent to those of ordinary skill in the art that the scope of the present invention according to the subject matter of the present invention is not limited by these examples.

Example 1: Preparation of Cells Resistant to Anticancer Drugs (Epirubicin, Cisplatin and 5-Fluorouracil; ECF)

[0236] To obtain gastric cancer cells resistant to an ECF combination comprising Epirubicin, Cisplatin and 5-Fluorouracil (ECF), primary human gastric cancer cell lines (SNU-488 and SNU-520) and metastatic human gastric cancer cell lines (MKN-28/74, MKN-74, MKN-45, and SNU-668) were first prepared. Thereafter, as shown in FIGS. 1A-ID, each of the gastric cancer cell lines was treated sequentially with ECF IC.sub.50, ECF IC.sub.70 and ECF IC.sub.50, and then given a schedule of drug-on (3 days) and drug-off (1 to 3 weeks) for a long period of more than 2 months. To determine whether ECF-resistant gastric cancer cell lines were created, drug responsiveness was evaluated in vitro and in xenograft animal models. As a result, it could be confirmed that the IC.sub.50 values for ECF-resistant gastric cancer cell lines significantly increased in the primary and metastatic gastric cancer cell lines compared to the parental cells (FIGS. 1B and 1C). In the case of the xenograft animal models, it could be confirmed that the volume of the tumor derived from the ECF-resistant gastric cancer cell line increased gradually, but the tumor volume decreased in the control group (FIG. 1D).

Example 2: Selection of Gene Resistant to Anticancer Drugs (Epirubicin, Cisplatin and 5-Fluorouracil; ECF) and Validation of the Potential of NINJ2 Biomarker to Diagnose ECF Resistance

2.1. Selection of NINJ2 (Nerve Injury-Induced Protein 2; Ninjurin 2)

[0237] ECF-resistant gastric cancer cells were obtained from Example 1, and a heatmap was used to select genes commonly appearing in ECF-resistant cell lines compared to wild-type cells (see FIG. 2). For a novel gene involved in ECF drug resistance, mRNA was extracted by a protocol performed in a conventional procedure, and then mRNA expression was measured using the affymetrix HG-U133A, HG-U133 Plus 2.0 and HG-U133A 2.0 platforms. The measured gene expression was set to one gene expression value, and then a correlation analysis procedure was further performed and the results were assessed by transcriptome analysis using RNA sequencing. The present inventors could finally select a biomarker of NINJ2 (nerve injury-induced protein 2; Ninjurin 2) by focusing on changed genes in RES and WT (wild type) related to integral component of plasma membrane and cell adhesion protein. Referring to the heatmap results, it was confirmed that the gene of NINJ2 was highly expressed in the RES cell line, like CD44 which is an already known stem cell marker, and showed the same trend as CD44 (see FIG. 2). By using the marker, it is possible to select only the RES cell line, that is, cells resistant to the drugs.

2.2. Validation of the Potential of NINJ2 Marker to Diagnose Anticancer Drug (ECF) Resistance

[0238] In order to verify whether the NINJ2 marker among the selected markers can diagnose anticancer drug (ECF) resistance, additional experiments were conducted at the cellular level and tissue level. First, the expression levels of NINJ2 mRNA and protein in ECF-resistant primary gastric cancer cell lines (SNU-488 and SNU-520) and metastatic gastric cancer cell lines (MKN-28/74, MKN-74, MKN-45, and SNU-668) were determined by qRT-PCR and Western blot analysis. As a result, as shown in FIGS. 3A and 3B, it could be confirmed that the expression levels of NINJ2 mRNA and protein were significantly higher in the ECF-resistant gastric cancer cell lines than in the parental cells.

Example 3: Correlation Between NINJ2 and CD44 Markers in ECF-Resistant Gastric Cancer Cell Lines

[0239] In gastric cancer initiating cells, CD133, CD44, aldehyde dehydrogenase 1 (ALDH1), and ATP-binding cassette subfamily G member 2 (ABCG2) were expressed. It is well known that, in the case of cancer stem cells, the effect of anticancer therapy is lower and the risk of recurrence is also higher than in the case of conventional cancer cells. CD44 is also one of the markers expressed in cancer stem cells, and it was confirmed that the expression level of the CD44 marker was high in the ECF-resistant cell lines in which NINJ2 was highly expressed (see FIGS. 4B and 4C). Also, among ECF-resistant MKN-74 cells, the proportion of surface NINJ2-positive cells was 9.6%+1.3%, but the proportion of parental cells was 0.8%?0.09%, and among ECF-resistant MKN-74 cells, the proportion of surface NINJ2 protein-expressing cells was 13.4%+2.7%, but the proportion of parental cells was only 1.4%+0.7% (FIGS. 4A and 4B). To analyze the correlation between NINJ2 and CD44, the expression level of CD44 in NINJ2 (+) or (?) cells among ECF-resistant cells was analyzed. As a result, as shown in FIGS. 4C and 4D, it could be confirmed that, among ECF-resistant cells, the NINJ2(+) cell population was composed mostly of CD44 high-expressing cells, whereas the NINJ2(?) cell population was composed mostly of CD44(?) cells. Thereby, it could be confirmed that the NINJ2(+)CD44hi gastric cancer-initiating cells significantly increased in the ECF-resistant gastric cancer cells.

[0240] Next, in order to evaluate NINJ2 expression in cancer stem cells known as an anticancer drug-resistant population, cancer spheroids were prepared by culturing the MKN-74 cell line in a serum-free culture medium supplemented with growth factors. As a result of measuring CD44 and NINJ2 mRNA expression levels in the MKN-74-derived tumor spheres by qRT-PCR and analyzing the expression level of NINJ2 protein by Western blot analysis, as shown in FIG. 4E, it was confirmed that, in the MKN-74-derived tumor spheres, CD44 and NINJ2 mRNA expression levels and NINJ2 protein expression levels significantly increased. In addition, the cultured spheroids were placed on a slide, and the cells were fixed with 1% (w/v) paraformaldehyde (PFA), incubated for 30 minutes, and washed three times with PBS. The cells were incubated with a blocking buffer (BSA 1%, Triton X-100 0.05%) for 30 minutes. The cells were treated with NINJ2 antibody (R&D Systems) and incubated at 4? C. for 16 hours. The cells were washed three times with PBS. Alexa-488-tagged secondary antibody (Thermo Fisher Scientific) was added to the cells, followed by incubation for 1 hour. The cells were washed three times with PBS, treated with CD44 antibody, incubated at 4? C. for 16 hours, and then washed three times with PBS for 20 minutes. Alexa-555-tagged secondary antibody (Thermo Fisher Scientific) was added to the cells, followed by incubation for 1 hour. The cells were washed three times with PBS for 20 minutes and stained with DAPI, and then immunofluorescence images thereof were observed using a confocal microscope. As a result of examining the immunofluorescence image, as shown in FIG. 4F, it was confirmed that NINJ2 and CD44 were co-localized in the spheroid and that NINJ2(+)CD44hi gastric cancer-initiating cells were located on the outside of the spheroid. Thereby, it could be seen that the NINJ2(+) cell population among ECF-resistant gastric cancer cells mainly corresponded to CD44 high-expressing gastric cancer initiating cells.

Example 4: Increase in Cancer Stem Cells (Cancer Initiating Cells) by NINJ2 Overexpression

[0241] In order to overexpress NINJ2 in the MKN-74 cell line, NINJ2 isoform-1 (Iso-1) (NP 057617.3) and isoform-3 (Iso-3) (NP_001281275.1) were each cloned into the pHTC HaloTag? CMV-neo vectors (Promega, G7711). Thereafter, the NINJ2 Iso-1 and Iso-3 vectors were transfected into MKN-74 cells using ViaFect? transfection reagent. Cells transfected with a growth medium containing G-418 (Promega) were selected. As a result of measuring cell viability after ECF treatment of the NINJ2 Iso-1 and Iso-3 overexpressing gastric cancer cell lines, as shown in FIG. 5A, it was confirmed that the viabilities of the NINJ2 Iso-1 and Iso-3 overexpressing gastric cancer cell lines were significantly higher than that of parental cells. In addition, it could be confirmed that the CD44 mRNA expression level and the number of CD44-expressing cells significantly increased in the NINJ2 Iso-1 and Iso-3 overexpressing gastric cancer cell lines (FIGS. 5B and 5C).

[0242] Next, in order to examine the frequency of gastric cancer initiating cells inducing tumorigenic properties, the sphere-forming ability was evaluated by in-vitro limiting dilution analysis. More specifically, NINJ2 Iso-1 and Iso-3 overexpressing cell lines were each diluted 2-fold and dispensed in 96-well plates at a density of 1,000 to 8 cells/well. The cells were cultured in DMEM-F12 supplemented with 20 ng/ml rhEGF, 20 ng/ml rhbFGF and 5 ?g/ml insulin, and quantified by extreme limiting dilution assay (ELDA). As a result, as shown in FIG. 5D, it was confirmed that gastric cancer initiating cells were abundant in the NINJ2 Iso-1 and Iso-3 overexpressing gastric cancer cells.

[0243] 1,000 NINJ2 Iso-1 or Iso-3 overexpressing cells were dispensed in 24-well plates, and the cells were cultured in DMEM-F12 supplemented with 20 ng/ml rhEGF, 20 ng/ml rhbFGF, and 5 ?g/ml insulin in the same manner as described above. After 10 days, the number of tumor spheres was counted. In this case, only spheres measured to have a size of 5,000 ?m.sup.2 or more were counted. As a result, it could be confirmed that the number of tumor spheres was increased by overexpression of NINJ2 Iso-1 and Iso-3 (see FIG. 5E).

Example 5: Increase in Cell Cycle Arrest by NINJ2 Overexpression

[0244] Cancer cells in quiescence are known as the major factor of resistance to many anticancer drugs. Accordingly, the present inventors examined an NINJ2-induced change in cell cycle. Bromodeoxyuridine (BrdU) was added to the cells overexpressing NINJ2 Iso-1 and Iso-3 from the gastric cancer cell line MKN-74, and the cells were cultured for an additional hour. After staining with anti-bromodeoxyuridine and bis-benzamide (Hoeschest 33342), the cell cycle was analyzed using a flow cytometer. As a result, it was confirmed that cell cycle arrest was significantly increased by inhibiting the progression from the G0/G1 phase to the S phase inducing anti-proliferative activity, in the NINJ2 Iso-1 and Iso-3 overexpressing gastric cancer cells (see FIG. 6).

Example 6: Identification of ECF Drug Resistance-Inducing Mechanism

[0245] To investigate the mechanism by which NINJ2 induces drug resistance, the present inventors selected candidate proteins that interact with NINJ2. To this end, the NINJ2 complex was pulled-down using a HaloTag pull-down system (G6504, Promega) according to the manufacturer's instructions, and then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As NINJ2-interacting proteins, periostin, PTPRk (Protein Tyrosine Phosphatase Receptor Type K), RNA-binding protein 28, and fibrinogen gamma chain were identified. Among the candidate proteins, periostin and PTPRk are known to be involved in drug resistance, and only periostin is involved in induction of ECF drug resistance. It could be confirmed through immunoblotting analysis after pull-down that NINJ2 interacted with periostin to induce ECF resistance (see FIG. 7A). In addition, the expression level of periostin in the ECF-resistant MKN74 cell line was additionally measured. As a result, it could be confirmed that the expression level of periostin in the ECF-resistant cell line was as high as that of NINJ2 (see FIG. 7B). Meanwhile, as a result of performing phospho-antibody array using the NINJ2-overexpressing gastric cancer cell line, the four phosphorylated proteins VE-Cadherin (Phospho-Tyr731), VAV2 (Phospho-Tyr142), JunD (Phospho-Ser255) and ATF2 (Phospho-Ser112/94), which were up-regulated by 1.5 times or more, were identified. Consistent results were identified by Western blot analysis (see FIG. 7C). From the above results, it can be seen that NINJ2 induces ECF drug resistance in the VAV2, JunD and ATF2 pathways through VE-cadherin activation. Taking the above results together, it is suggested that not only the NINJ2 marker may be used to diagnosis ECF resistance, but also the periostin marker interacting with NINJ2 may also be additionally used.

Example 7: Evaluation of Resistance Treatment Potential of shNINJ2 Using ECF-Resistant Gastric Cancer Cell Line

[0246] To evaluate whether NINJ2 is involved in drug resistance, a stable NINJ2 knockdown (K/D) ECF-resistant cancer cell line was prepared using shRNA lentiviral particles and puromycin. More specifically, for stable NINJ2 knockdown in ECF-resistant MKN-74 cells, two shRNAs (TRCN0000063773 (Clone-1) and TRCN0000063775 (Clone-2)) targeting different partial regions of NINJ2 genes (Isoform-1, Isoform-2 and Isoform-3) as shown in Table 1 below were used, and a non-targeting pLKO.1-puro shRNA control (SHC002) was used as a negative control. The shRNA clones (TRCN0000063773, TRCN0000063775 and SHC002) containing the pMDLg/pRRE, pRSV-Rev and pMD2.G plasmids, respectively, were transfected into 293T cells using Fugene HD (Promega) according to the manufacturer's instructions. After 48 hours, each supernatant was collected and filtered, and ECF-resistant MKN-74 cells were transduced with lentiviral particles and cultured in a growth medium containing puromycin. After NINJ2 knockdown in the lentivirus-transduced MKN-74 cells was confirmed using qRT-PCR, the cells were treated with ECF, and an examination was made as to whether cancer cells in the negative control (Mock) and the shNINJ2 groups (clone 1 and clone 2) re-grew.

TABLE-US-00001 TABLE1 NINJ2-targeting Gene Clone regionsequence Hairpinsequence NINJ2 TRCN0000063773 CGTGGTCATTGCA 5-CCGG- CGGCTGAA(SEQ CGTGGTCATTGCACGGCTGAA- IDNO:5) CTCGAG- TTCAGCCGTGCAATGACCACG- TTTTTG-3(SEQIDNO:9) NINJ2 TRCN0000063775 CTGAACCTGAATG 5-CCGG- AGGTAGAA(SEQ CTGAACCTGAATGAGGTAGAA- IDNO:6) CTCGAG- TTCTACCTCATTCAGGTTCAG- TTTTTG-3(SEQIDNO:10) Control SHC002 5- CCGGCAACAAGATGAAGAGCACCAA CTCGAGTTGGTGCTCTTCATCTTGTTG TTTTT-3

[0247] As shown in FIG. 8, as a result of performing crystal violet staining and WST-1 analysis 3 weeks after ECF treatment, it could be confirmed that, in the case of the NINJ2 knockdown (KID) resistant cancer cells (clone-1 and clone-2), the recurrence and regrowth of cancer cells were significantly inhibited compared to those in the negative control (Mock). This suggests that resistance to ECF drugs is overcome upon knockdown of NINJ2 at the cellular level.

Example 8: Evaluation of Resistance Treatment Potential of siNINJ2 Using Animal Model Transplanted with ECF-Resistant Gastric Cancer Cell Line

[0248] 8.1. Preparation of Animal Model Transplanted with ECF-Resistant Gastric Cancer Cell Line

[0249] A mouse model was prepared by xenografting Balb/c nude mice with the spheroids prepared from the MKN-28/74 cell line by the method of Example 3. Specifically, 10.sup.7 ECF-resistant cancer cells (ECF-R cancer cells) and parental cancer cells were subcutaneously injected into Balb/c nude mice, respectively, and an examination was made as to whether the animal model xenografted with the ECF-resistant cancer cells (RES) established in vitro had acquired ECF drug resistance. Thereafter, to evaluate the resistance treatment effect of NINJ2 inhibition using the prepared ECF-resistant animal model, expression of NINJ2 was inhibited using primers for siNINJ2 (represented by SEQ ID NOs: 11 and 12) and primers for a control as shown in Table 2 below, and then an additional drug administration experiment was performed. To this end, 5.7 mg/kg of epirubicin, 6.67 mg/kg of cisplatin and 22 mg/kg of 5-FU were administered intratumorally to xenograft model mice having a tumor size of 100 mm.sup.3 once a week for 15 days, and the tumor weight and the tumor volume were measured every 3 days using digital calipers. As described above, the ECF administration experiment was conducted on each of a control group, an NINJ2-knockout RES cell line group and an RES cell line group highly expressing NINJ2 for about one month.

TABLE-US-00002 TABLE2 Primer Direction Sequence(5.fwdarw.3) SEQIDNO siNINJ2 Sense GUAAGGCAUGUCUGUCUAAGG SEQIDNO:11 CC Antisense GGCCUUAGACAGACAUGCCUU SEQIDNO:12 AC Control Sense UUCUCCGAACGUGUCACGUTT Antisense ACGUGACACGUUCGGAGAATT
8.2. Evaluation of Resistance Treatment Effect of siNINJ2

[0250] Referring to FIG. 9A, it was confirmed that the tumor volume changed after the ECF drugs were administered to the animal model xenografted with each cell line group. FIG. 9B depicts graphs showing the results of quantifying the changes in the tumor volume and weight. It could be confirmed that the tumor volume and weight significantly decreased in the NINJ2-knockout RES cell line in which the expression of NINJ2 mRNA was inhibited, compared to the RES group. A large difference in tumor volume appeared over time, suggesting that resistance to ECF drugs was overcome.

Example 9: Identification of Increased Expression of NINJ2 in ECF-Resistant Gastric Cancer Organoid and Clinical Significance for Cancer Progression

[0251] Patient-derived human gastric tumor organoids (HCM-BROD-0115-C16, PDM-135) were purchased from the American Type Culture Collection (ATCC), subcultured according to the ATCC guidelines, and used in the experiment. Organoids were treated with ECF at an IC.sub.50 concentration, and after 72 hours, the medium was replaced with a drug-free medium, and two additional passages were performed. Next, the organoids were exposed to appropriate IC.sub.70 and IC.sub.80 concentrations, and then the above procedure was repeated. In order to prevent the organoids from reverting to the ECF drug-sensitive state, the organoids were treated with ECF at an IC.sub.80 concentration every 3 weeks, thereby constructing ECF-resistant organoids. FIG. T0A shows microscopic observation photographs of parental gastric cancer organoids and ECF-resistant gastric cancer organoids, and shows the results of measuring IC.sub.50 for ECF of each organoid. As a result of quantifying the expression levels of NINJ2 and CD44 mRNA in parental gastric cancer organoids and ECF-resistant gastric cancer organoids by qRT-PCR, as shown in FIG. 10B, it could be confirmed that the expression levels of NINJ2 and CD44 mRNA were significantly higher in the ECF-resistant gastric cancer organoids than in the parenteral gastric cancer organoids, like the results obtained in the cell lines.

[0252] Next, to examine the clinical relevance between NINJ2 expression level and drug response, two pathologists analyzed the intensity and extensive expression of NINJ2 through histological analysis of gastric cancer patients showing partial response (PR), stable disease (SD) and progressive disease (PD). As a result, as shown in FIG. 10C, it was confirmed that the extensive expression of NINJ2 was significantly higher in PD than in PR/SD. Next, in order to confirm the clinical relevance between NINJ2 expression level and survival rate, Kaplan-Meier analysis and log-rank test were performed using the hazard ratio (HR) of gastric cancer patients and using public data. As a result, it could be seen that the survival rate was very low in the NINJ2-expressing group among gastric cancer patients, and in particular, it was confirmed that the NINJ2 high-expressing group in the Her2-negative group showed an increase in overall survival compared to the Her2-positive group (see FIG. 10D).

[0253] Taking the above-described results of Examples 1 to 9 together, it can be seen that it is possible to use the NINJ2 marker and the periostin marker for diagnosis of ECF resistance, and furthermore, it is possible to overcome ECF drug resistance by inhibiting the expression of NINJ2. Accordingly, it is expected that anticancer effects can be significantly improved by treating anticancer drug resistance in patients who have developed anticancer drug resistance after ECF treatment.

[0254] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this detailed description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.

INDUSTRIAL APPLICABILITY

[0255] The composition according to the present invention can not only diagnose anticancer drug resistance, but can also treat cancer very effectively. In addition, the composition may be very effectively used to overcome anticancer drug resistance, and furthermore, to effectively prevent, ameliorate or treat anticancer drug-resistant cancer.

Sequence List Free Text

[0256]

TABLE-US-00003 SEQIDNO1: mesarenidlqpgssdprsqpinlnhyatkksvaesmldvalfmsnamrl kavleqgpsshyyttlvtlislslllqvvigvllvviarlnlnevekqwrInqlnnaatilvfftvvinvfitafgahkt gflaarasrnpl SEQIDNO2: mldvalfmsnamrlkavleqgpsshyyttlvtlislslllqvvigvllvviarlnlneve kqwrlnglnnaatilvfftvvinvfitafgahktgflaarasrnpl SEQIDNO3: agagactcagacggcggagcctggaggageccacgcagtctgttcccggc acccggtgcgtgtgaagggacttgagggcagcgagatggaatcagcaagagaaaacategaccttcaacc tggaagctccgaccccaggagccagcccatcaacctgaaccattacgccaccaagaagagcgtggcggag agcatgctggacgtggccctgttcatgtccaacgccatgcggctgaaggcggtgctggagcagggaccatcctctcacta ctacaccaccctggtcaccctcatcagcctctctctgctcctgcaggtggtcatcggtgtcctgctcgtggtcattgcac ggctgaacctgaatgaggtagaaaagcagtggcgactcaaccagctcaacaacgcagccaccatcttggtcttcttcact gtggtcatcaatgttttcattacagccttcggggcacataaaacagggttcctggetgccagggcctcaaggaatcctct ctgaatgcagcctgggacccaggttctgggcctggaacttctgcctccttcctccgtgatctgccaggctcgtgggcact ttccacagcccaggagagcttctgaaaggacagtatagctgcccttgctccctacccacagcacctgagttaaaaagtga tttttatgttattggtctaagggacttccatettggtctgaagtcctgagctcagacgcaggtactgccagccatacctt cctggtagcatctgctggacctaagtaaggcatgtctgtctaaggccaagtctgcccggcttaaggatgctggttctgac tctaccccactgcttccttctgctccaggcctcaattttcccttcttgtaaaatggaatctatatctataaaggtttctt caaatcca SEQIDNO4: gttgcaaagcagccgctcggtggccgtacaacgcttcatctctccgagcctcggtttcct catctccagccctaaaatgacgacacgccccacaggtcttgggaggattaagtgaggggacatgagcctg gaagctccgaccccaggagccagcccatcaacctgaaccattacgccaccaagaagagcgtggcggagag catgctggacgtggccctgttcatgtccaacgccatgcggctgaaggcggtgctggagcagggaccatcctctcactact acaccaccctggtcaccctcatcagcctctctctgctcctgcaggtggtcatcggtgtcctgctcgtggtcattgcacgg ctgaacctgaatgaggtagaaaagcagtggcgactcaaccagctcaacaacgcagccaccatcttggtcttcttcactgt ggtcatcaatgttttcattacagccttcggggcacataaaacagggttcctggctgccagggcctcaaggaatcctctct gaatgcagcctgggacccaggttctgggcctggaacttctgcctccttcctccgtgatctgccaggctcgtgggcacttt ccacagcccaggagagcttctgaaaggacagtatagctgcccttgctccctacccacagcacctgagttaaaaagtgatt tttatgttattggtctaagggacttccatcttggtctgaagtcctgagctcagacgcaggtactgccagccataccttcc tggtagcatctgctggacctaagtaaggcatgtctgtctaaggccaagtctgcccggcttaaggatgctggttctgactc taccccactgcttccttctgctccaggcctcaattttcccttcttgtaaaatggaatctatatctataaaggtttcttcaaatcca SEQIDNO5: CGTGGTCATTGCACGGCTGAA SEQIDNO6: CTGAACCTGAATGAGGTAGAA SEQIDNO7: mipflpmfslllllivnpinannhydkilahsrirgrdqgpnvcalqqil gtkkkyfstcknwykksicgqkttvlyeccpgymrmegmkgcpavlpidhvygtlgivgatttqrysdas klreeiegkgsftyfapsneawdnldsdirrglesnvnvellnalhshminkrmltkdlkngmiipsmyn nlglfinhypngvvtvncariihgnqiatngvvhvidrvltqigtsiqdfieaeddlssfraaaitsdilealgrdghft lfaptneafeklprgvlerimgdkvasealmkyhilntlqcsesimggavfetlegntieigcdgdsitv ngikmvnkkdivtnngvihlidqvlipdsakqvielagkqqttftdlvaqlglasalrpdgeytllapvnnafsddtlsm dqrllklilqnhilkvkvglnelyngqiletiggkqlrvfvyrtavcienscmekgskqgrngaihifreiikpaekslh eklkqdkrfstflslleaadlkelltqpgdwtlfvptndafkgmtseekeilirdknalqniilyhltpgvfigkgfepg vtnilkttqgskiflkevndtllvnelkskesdimttngvihvvdkllypadtpvgndqlleilnklikyiqikfvrgst fkeipvtvykpiikkytkiidgvpveiteketreeriitgpeikytristgggeteetlkkllqeevtkvtkfieggdgh lfedeeikrllqgdtpvrklqankkvqgsrrrlregrsq SEQIDNO8: mdkfwwhaawglclvplslaqidlnitcrfagvfhvekngrysisrteaa dlckafnstlptmaqmekalsigfetcrygfieghvviprihpnsicaanntgvyiltsntsqydtycfnasappeedct svtdlpnafdgpititivnrdgtryvqkgeyrtnpediypsnptdddvssgsssersstsggyifytfstvhpipdedsp witdstdripatrhshgsqegganttsgpirtpqipewliilasllalalilavciavnsrrrcgqkkklvinsgngave drkpsglngeasksqemvhlvnkessetpdqfmtadetrnlqnvdmkigv SEQIDNO9: CCGG-CGTGGTCATTGCACGGCTGAA-CTCGAG- TTCAGCCGTGCAATGACCACG-TTTTTG SEQIDNO10: CCGG-CTGAACCTGAATGAGGTAGAA-CTCGAG- TTCTACCTCATTCAGGTTCAG-TTTTTG SEQIDNO11: GUAAGGCAUGUCUGUCUAAGGCC SEQIDNO12: GGCCUUAGACAGACAUGCCUUAC