Method for predicting anticancer drug resistance and prognosis in renal cancer patients, method for screening anti-renal cancer substances, and pharmaceutical composition for treating renal cancer

Abstract

The present invention provides: a method of predicting the anticancer drug resistance and / or prognosis in a kidney cancer patient, said method comprising a step for evaluating the expression of Largen in the kidney cancer tissue of a subject and a step for comparing the Largen expression level in the kidney cancer tissue of the subject with a control; and a method of screening an anti-kidney cancer substance, said method comprising (1) a step for contacting a test substance with kidney cancer cells that express Largen, (2) a step for measuring the expression amount of Largen in the kidney cancer cells, and (3) a step for comparing the thus determined expression amount of Largen with the expression amount of Largen in the kidney cancer cells that have not been contacted with the test substance and thus selecting a test substance that lowers the expression amount.

Description

[Technical Field]

[0001] The present invention relates to a method for predicting anticancer drug resistance and prognosis in renal cancer patients, a method for screening anti-renal cancer substances, and a pharmaceutical composition for treating renal cancer. [Background technology]

[0002] Renal cancer is one of the major cancers in the urological field, and the number of patients is on the rise due to the widespread use of health checkups and improvements in testing systems. Clinical characteristics of kidney cancer include the fact that symptoms such as hematuria and pain are not readily apparent, a significant number of patients have metastasis at the time of diagnosis, and approximately 20% of patients experience recurrence even after radical resection. Therefore, predicting and preventing postoperative recurrence and developing treatments for advanced cancer are essential to improving prognosis. In recent years, various factors have been reported as predictors of prognosis, including postoperative recurrence (e.g., Patent Documents 1 and 2), but most of these are based on genetic analysis or complex analytical models, and none have yet been used in clinical settings.

[0003] Furthermore, although molecularly targeted drugs and novel immunotherapeutic agents have emerged as treatments for advanced kidney cancer, and prognosis has improved, the 5-year survival rate remains unsatisfactory at approximately 25%. Furthermore, the lack of predictive factors for their antitumor efficacy remains a problem. Thus, to extend the prognosis of kidney cancer patients, it is necessary to develop clinically feasible and highly useful predictive models for postoperative recurrence and prognosis, as well as the effectiveness of molecularly targeted drugs and novel immunotherapeutic agents, and to develop new treatment strategies based on the development of novel drugs with different mechanisms of action.

[0004] The present inventors discovered a gene that regulates cell size and named the protein encoded by this gene "Largen" (Non-Patent Document 1). In Non-Patent Document 1, the present inventors reported that Largen increases mitochondria and promotes ATP production, that Largen activates protein synthesis, and that hepatocytes and cardiomyocytes overexpressing Largen actually grow larger in vivo in mice. The present inventors also discovered and reported that the onset and progression of lymphoma are delayed in mice with lymphocyte-specific overexpression of the PRR16 gene (Non-Patent Document 2). Furthermore, the present inventors analyzed changes in proliferation potential in a liver cancer cell line (HepG2 cells) in which Largen is stably expressed, and reported that cell proliferation was suppressed in cells stably expressing Largen (Non-Patent Document 3). However, the involvement of Largen in cancer cells other than these has not yet been clarified. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication WO2017 / 038983 [Patent Document 2] International Publication WO2013 / 168644 [Non-patent literature]

[0006] [Non-Patent Document 1] Yamamoto K et al., Molecular Cell 53 (6): 904-915, 2014. doi: 10.1016 / j.molcel.2014.02.028. [Non-patent document 2] Kazuo Yamamoto et al., Abstract of the 36th Annual Meeting of the Molecular Biology Society of Japan (December 3-6, 2013, Kobe Port Island), "The relationship between cell size regulation via translational control and cancer" [Non-patent document 3] Kazuo Yamamoto, "Research into hepatocellular carcinoma suppression using disruption of the Warburg effect by cell size-regulating genes," FY2017 Implementation Status Report, Grants-in-Aid for Scientific Research Database, https: / / kaken.nii.ac.jp / ja / report / KAKENHI-PROJECT-17K09431 / 17K094312017hokoku / Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to provide a method for predicting anticancer drug resistance in renal cancer patients and a method for predicting the prognosis of renal cancer patients.Another objective of the present invention is to provide a method for screening anti-renal cancer substances.Further objectives of the present invention are to provide a pharmaceutical composition for treating renal cancer, a kit for predicting the prognosis of renal cancer, and a kit for screening anti-renal cancer substances. [Means for solving the problem]

[0008] The present invention includes the following inventions to solve the above problems. [1] A method for predicting anticancer drug resistance and / or prognosis in a renal cancer patient, comprising the steps of assessing the expression of Largen in the renal cancer tissue of the subject and comparing the expression level of Largen in the renal cancer tissue of the subject with that of a control. [2] The prediction method according to [1], further comprising the steps of assessing necrosis in the subject's renal cancer tissue and comparing the level of necrosis in the subject's renal cancer tissue with that of a control. [3] A method for predicting anticancer drug resistance and / or prognosis of a renal cancer patient, comprising the steps of assessing the expression of Largen in the subject's renal cancer metastatic tissue and comparing the expression level of Largen in the subject's renal cancer metastatic tissue with that of a control. [4] The prediction method according to [3], further comprising the steps of assessing necrosis in the subject's renal cancer metastatic tissue and comparing the level of necrosis in the subject's renal cancer metastatic tissue with that of a control. [5] The prediction method described in [2] or [4] above, in which necrosis is evaluated based on the expression of receptor-interacting protein kinase 3 in the subject's renal cancer tissue or renal cancer metastasis tissue. [6] A method for predicting anticancer drug resistance and / or prognosis of a renal cancer patient, comprising the steps of detecting Largen in the body fluid of the subject and comparing the amount of Largen detected in the body fluid of the subject with that of a control. [7] The prediction method described in [6] above, wherein the body fluid is urine or blood. [8] The prediction method according to any one of [1] to [7] above, wherein the subject is a kidney cancer patient who has undergone surgery to remove cancer tissue. [9] The prediction method according to any one of [1] to [8] above, wherein the anticancer drug is a molecular targeted drug or an immunotherapeutic drug.

[10] The prediction method according to any one of [1] to [9] above, wherein the prognosis of a renal cancer patient is one or more selected from the group consisting of survival time, cancer progression, cancer recurrence, and event-free interval.

[11] A method for screening an anti-renal cancer substance, comprising: (1) contacting a test substance with Largen-expressing renal cancer cells in vitro; (2) measuring the expression level of Largen in the renal cancer cells; and (3) A step of comparing the obtained Largen expression level with the Largen expression level of the renal cancer cells not contacted with the test substance, and selecting a test substance that reduces the expression level. A screening method comprising the steps of:

[12] A pharmaceutical composition for treating renal cancer, the active ingredient of which is a substance that inhibits the expression of Largen in renal cancer cells.

[13] The pharmaceutical composition according to

[12] , wherein the active ingredient is a nucleic acid that inhibits the expression of Largen in renal cancer cells.

[14] A kit for predicting anticancer drug resistance and / or prognosis in kidney cancer patients, comprising an anti-Largen antibody or a primer or probe for detecting Largen mRNA.

[15] A screening kit for anti-renal cancer substances, comprising an anti-Largen antibody or a primer or probe for detecting Largen mRNA. [Effects of the Invention]

[0009] The present invention makes it possible to predict anticancer drug resistance in renal cancer patients. The present invention also makes it possible to predict the prognosis of renal cancer patients. Furthermore, the screening method of the present invention makes it possible to provide a pharmaceutical composition for treating renal cancer with a new mechanism of action. Furthermore, the present invention makes it possible to provide a kit for predicting the prognosis of renal cancer and a kit for screening anti-renal cancer substances. [Brief explanation of the drawings]

[0010] [Figure 1] Representative images of tissue sections of renal cancer tissue removed from a renal cancer patient, immunostained with anti-human Largen antibody, and observed under a microscope. (A) is an image of a normal kidney, and (B) is an image of a renal cancer area. [Figure 2] This figure shows the results of comparing the cleaved-Cas3-positive cell rate between a group of patients with high Largen expression (88 patients) and a group of patients with low Largen expression (120 patients). [Figure 3] FIG. 1 shows the results of comparing postoperative overall survival between a group of patients with high Largen expression (88 patients) and a group of patients with low Largen expression (120 patients). [Figure 4] FIG. 1 shows the results of comparing progression-free survival between a group of patients with high Largen expression (88 patients) and a group of patients with low Largen expression (120 patients). [Figure 5] This figure shows the results of comparing disease-free survival between a group of patients with high Largen expression (61 patients) and a group of patients with low Largen expression (113 patients) who underwent curative surgery without any findings suggestive of metastasis before surgery. [Figure 6] FIG. 1 shows the results of measuring serum and urinary Largen concentrations in kidney cancer patients and healthy individuals, where (A) shows serum Largen concentrations and (B) shows urinary Largen concentrations. DETAILED DESCRIPTION OF THE INVENTION

[0011] [Methods for predicting anticancer drug resistance and prognosis in kidney cancer patients] The present invention provides a method for predicting anticancer drug resistance and / or prognosis in renal cancer patients (hereinafter referred to as the "prediction method of the present invention"). A first embodiment of the prediction method of the present invention may include the steps of assessing the expression of Largen in renal cancer tissue of a subject and comparing the expression level of Largen in the renal cancer tissue of the subject with that of a control.

[0012] A second embodiment of the prediction method of the present invention may include the steps of evaluating the expression of Largen in renal cancer metastasis tissue of a subject and comparing the expression level of Largen in the renal cancer metastasis tissue of the subject with that of a control. A third embodiment of the prediction method of the present invention may include the steps of detecting Largen in the body fluid of a subject and comparing the detected amount of Largen in the body fluid of the subject with that of a control.

[0013] The subject in the prediction method of the present invention may be any subject suspected of having renal cancer. The subject may be a renal cancer patient who has undergone partial nephrectomy or a renal cancer patient who has undergone radical nephrectomy. The prediction method of the present invention is useful in that it can predict the prognosis of renal cancer patients who have undergone radical nephrectomy.

[0014] The first embodiment of the prediction method of the present invention can be applied to subjects who can provide a renal tissue sample suspected of having renal cancer. The second embodiment of the prediction method of the present invention can be applied to subjects who can provide a tissue sample suspected of having renal cancer metastasis. The third embodiment of the prediction method of the present invention can be applied to all subjects suspected of having renal cancer. The renal tissue sample or the tissue sample suspected of having renal cancer metastasis may be obtained by biopsy or surgery.

[0015] Tissues suspected of renal cancer metastasis include, but are not limited to, the ovary, uterus, breast, thyroid, brain, esophagus, tongue, lung, pancreas, stomach, small intestine, duodenum, large intestine, bladder, liver, prostate, gallbladder, pharynx, muscle, bone, skin, etc. The tissue suspected of renal cancer metastasis may be the lung, brain, or bone.

[0016] The body fluid is not particularly limited, and examples thereof include blood, lymph, interstitial fluid, body cavity fluid, digestive fluid, nasal mucus, and urine. Blood or urine is preferred. Blood may be serum, plasma, or whole blood. The body fluid may be the body fluid itself collected from a subject, or may be a body fluid that has been subjected to a commonly performed treatment such as dilution or concentration of the collected body fluid.

[0017] In the prediction method of the present invention, the anticancer drug to be predicted for resistance is not particularly limited, but may be a molecularly targeted drug or an immunotherapeutic drug. Examples of molecularly targeted drugs include tyrosine kinase inhibitors such as sorafenib, sunitinib, pazopanib, axitinib, and cabozantinib, and mTOR (mammalian target of rapamycin) inhibitors such as everolimus and temsillonimbus. Examples of immunotherapeutic drugs include immune checkpoint inhibitors, such as anti-PD-1 antibodies such as nivolumab and pembrolizumab, and anti-PD-L1 antibodies such as avelumab, atezolizumab, and durvalumab.

[0018] In the prediction method of the present invention, the prognosis is not particularly limited, but examples include survival time, cancer progression, cancer recurrence, and event-free interval. Cancer recurrence includes cancer recurrence at the resection site (progression-free survival) and cancer recurrence after a cure (disease-free survival). Event-free intervals include the period until new metastases appear in addition to existing metastases, the period until residual cancer grows, and the period until cancer-related fractures occur. In the prediction method of the present invention, if the subject is a renal cancer patient who has undergone surgery to remove cancer tissue, postoperative survival time, postoperative cancer progression, postoperative cancer recurrence, and postoperative event-free interval can be predicted.

[0019] Evaluation of Largen expression in a subject's renal cancer tissue or renal cancer metastasis tissue can be performed, for example, by preparing tissue sections of the subject's renal cancer tissue or renal cancer metastasis tissue and immunostaining them with an anti-Largen antibody. Evaluation of Largen expression in a subject's renal cancer tissue or renal cancer metastasis tissue can also be performed, for example, by measuring the amount of Largen protein or Largen mRNA in the renal cancer tissue or renal cancer metastasis tissue. Immunostaining is preferred from the standpoints of simplicity, widespread use, and ability to exclude Largen expression in cells other than cancer cells. Information on the amino acid sequence of human Largen and the nucleotide sequence of the gene encoding it can be obtained from publicly known databases such as NCBI. Examples include, but are not limited to, the amino acid and nucleotide sequences listed in Table 1 below. The gene encoding Largen is known as the PRR16 (proline rich 16) gene (hereinafter referred to as the "PRR16 gene").

[0020] [Table 1]

[0021] The immunostaining method for tissue specimens is not particularly limited, and can be appropriately selected from known immunostaining methods. Commercially available anti-Largen antibodies (anti-PRR16 antibodies) can be used. When measuring protein levels, proteins can be extracted from renal cancer tissues or renal cancer metastasis tissues using known methods and quantified using known protein measurement methods. Known protein measurement methods include, for example, Western blotting, EIA, ELISA, RIA, and methods using protein measurement reagents. When measuring mRNA levels, RNA can be extracted from renal cancer tissues or renal cancer metastasis tissues using known methods and quantified using known mRNA measurement methods. Known mRNA measurement methods include Northern blotting, RT-PCR, quantitative RT-PCR, and RNase protection assay.

[0022] When evaluating Largen expression in a subject's renal cancer tissue by immunostaining, normal kidney tissue can be used as a control, preferably, but not limited to, normal kidney tissue collected at the same time. For example, renal cells collected from a healthy subject can be used as a control, or an organ or tissue other than the kidney known to have a low level of Largen expression can be used as a control. When evaluating Largen expression in a subject's renal cancer metastasis tissue by immunostaining, normal tissue corresponding to the metastatic tissue can be used as a control, preferably, but not limited to, normal tissue collected at the same time. For example, corresponding normal tissue collected from a healthy subject can be used as a control, or an organ or tissue other than the corresponding tissue known to have a low level of Largen expression can be used as a control.

[0023] Information on Largen expression in various organs and tissues can be found in publicly known databases. Largen expression levels can be expressed, for example, by classifying Largen expression intensity into four levels (0: no expression, mild; 1: moderate; 2: high; 3) and expression rate into four levels (less than 10%; 0: 10% to less than 25%; 1: 25% to less than 50%; 2: 50% or more; 3), and then adding these two levels together. The expression level of the control and the expression level of the subject's renal cancer tissue are compared to determine whether the subject's expression level is high or not.

[0024] When assessing Largen expression in a subject's renal cancer tissue by protein or mRNA level, normal kidney tissue can be used as a control, preferably a normal kidney tissue collected at the same time. When assessing Largen expression in a subject's renal cancer metastasis tissue by protein or mRNA level, corresponding normal tissue can be used as a control, preferably a corresponding normal tissue collected at the same time. Alternatively, a cutoff value based on accumulated data from normal tissue can be used as the measured value of Largen protein or mRNA in the control. The measured values ​​of Largen protein or mRNA in the subject's renal cancer tissue or renal cancer metastasis tissue are compared with the measured values ​​in the control to determine whether the expression level in the subject is high or not. For example, multiple measurements can be performed on each sample and the presence or absence of a statistically significant difference can be determined.

[0025] In the prediction method of the present invention, if the expression level of Largen in a subject's renal cancer tissue or renal cancer metastasis tissue is determined to be higher than that of a control, the subject can be predicted to be resistant to anticancer drugs (anticancer drugs will be less effective) and have a poor prognosis (short survival time, rapid cancer progression, high possibility of recurrence, short event-free interval). On the other hand, if the expression level of Largen in a subject's renal cancer tissue or renal cancer metastasis tissue is determined to be no different from that of a control, the subject can be predicted to be sensitive to anticancer drugs (anticancer drugs will be more effective) and have a good prognosis (long survival time, slow cancer progression, low possibility of recurrence, long event-free interval). Therefore, the prediction method of the present invention can be used to assist in determining a subject's treatment plan and selecting an anticancer drug to administer.

[0026] The prediction methods of the present invention preferably combine evaluation of Largen expression in a subject's renal cancer tissue or renal cancer metastasis tissue with evaluation of necrosis in the subject's renal cancer tissue or renal cancer metastasis tissue. That is, the prediction methods of the present invention may include, in addition to the steps of evaluating Largen expression in the subject's renal cancer tissue or renal cancer metastasis tissue and comparing the Largen expression level in the subject's renal cancer tissue or renal cancer metastasis tissue with a control, the steps of evaluating necrosis in the subject's renal cancer tissue or renal cancer metastasis tissue and comparing the necrosis level in the subject's renal cancer tissue or renal cancer metastasis tissue with a control. Combining these two evaluations can improve the accuracy of anticancer drug resistance and prognosis predictions for renal cancer patients.

[0027] Necrosis in a subject's renal cancer tissue or renal cancer metastasis tissue can be evaluated, for example, by hematoxylin-eosin staining (HE staining) tissue sections of the subject's renal cancer tissue or renal cancer metastasis tissue and examining them with a pathologist. Alternatively, tissue sections of the subject's renal cancer tissue can be prepared and evaluated for the expression of receptor-interacting protein kinase 3 (RIP3), a representative marker of tissue necrosis.

[0028] Evaluation of RIP3 expression in a subject's renal cancer tissue or renal cancer metastasis tissue can be performed, for example, by preparing tissue sections of the subject's renal cancer tissue or renal cancer metastasis tissue and immunostaining them with an anti-RIP3 antibody. Commercially available anti-RIP3 antibodies can be used. Evaluation of RIP3 expression in a subject's renal cancer tissue or renal cancer metastasis tissue can also be performed, for example, by measuring the amount of RIP3 protein or RIP3 mRNA in the renal cancer tissue or renal cancer metastasis tissue. Information on the amino acid sequence of human RIP3 protein and the nucleotide sequence of the gene encoding it can be obtained from publicly known databases such as NCBI. The RefSeq ID for the amino acid sequence of human RIP3 protein is NP_006862.2, and the RefSeq ID for the nucleotide sequence of the gene encoding it is NM_006871.4. The immunostaining method, protein amount measurement method, and mRNA amount measurement method can be performed using the same methods as those used to evaluate the expression of Largen described above.

[0029] When evaluating necrosis in a subject's renal cancer tissue or renal cancer metastasis tissue using HE-stained specimens, the same controls as those used to evaluate Largen expression by immunostaining can be used. The level of necrosis in HE-stained specimens can be determined by a pathologist through microscopic observation based on the presence or absence of disrupted cell morphology, cytoplasmic oxidation, and condensed or lost nuclei. The necrosis level of the control and the necrosis level of the subject's renal cancer tissue or renal cancer metastasis tissue are compared based on standard criteria by a pathologist to determine whether the subject's expression level is high or not.

[0030] When evaluating RIP3 expression in a subject's renal cancer tissue or renal cancer metastasis tissue by immunostaining, the same control as used to evaluate Largen expression by immunostaining can be used as the control. For example, the RIP3 expression level can be expressed as a sum of the four levels of RIP3 expression intensity (0: no expression, mild; 1: moderate; 2: severe; 3) and the four levels of expression percentage (less than 10%; 0: 10% to less than 25%; 1: 25% to less than 50%; 2: 50% or more; 3). The expression level of the control and the expression level of the subject's renal cancer tissue or renal cancer metastasis tissue are compared to determine whether the subject's expression level is high or not.

[0031] In the prediction method of the present invention, if it is determined that the expression level of Largen in a subject's renal cancer tissue or renal cancer metastasis tissue is higher than that of a control and the level of necrosis is no different from that of the control, the subject can be predicted with high accuracy to be resistant to anticancer drugs (anticancer drugs will be less effective) and to have a poor prognosis (short survival time, rapid cancer progression, high possibility of recurrence, short event-free interval).On the other hand, if it is determined that the expression level of Largen in a subject's renal cancer tissue or renal cancer metastasis tissue is no different from that of the control and the level of necrosis is higher than that of the control, the subject can be predicted with high accuracy to be anticancer drug sensitive (anticancer drugs will be more effective) and to have a good prognosis (long survival time, slow cancer progression, low possibility of recurrence, long event-free interval).

[0032] Methods for detecting Largen in body fluids are not particularly limited and may be immunoreaction or non-immunoreaction methods. Examples include EIA, ELISA, RIA, Western blotting, immunoprecipitation, latex agglutination, methods using protein measurement reagents, high-performance liquid chromatography, gas chromatography, and mass spectrometry. A method that allows simultaneous detection and quantification is preferred. For example, in ELISA, a sandwich method can be used to simultaneously detect and quantitate Largen. Specifically, an anti-Largen antibody is immobilized on a solid support, and an appropriately pretreated body fluid is added and reacted. Then, an enzyme-labeled anti-Largen antibody that recognizes a different epitope is added and reacted. After washing, the antibody reacts with an enzyme substrate, develops color, and the absorbance is measured, allowing Largen to be detected and quantified simultaneously. Alternatively, a method may be used in which an anti-Largen antibody immobilized on a solid support reacts with Largen in a biological sample, followed by the addition of an unlabeled Largen antibody (primary antibody), and then an enzyme-labeled antibody (secondary antibody) against the unlabeled antibody is added. The ELISA method is not limited to the sandwich method, and may be a direct method, a competitive method, etc. In these cases, a detection system may be prepared using a commercially available ELISA preparation kit (manufactured by MyBioSource, etc.).

[0033] Largen in body fluids can be detected and quantified by detecting and quantifying the amount of mRNA in the body fluid. mRNA can be simultaneously detected and quantified using Northern blot, microarray, quantitative PCR, real-time PCR, digital PCR, next-generation sequencing, and other methods. mRNA in body fluids may be contained in exosomes. In this case, exosomes can be isolated from the body fluid using ultracentrifugation, commercially available kits (e.g., Hitachi Chemical), filters, or columns, and then mRNA can be extracted and quantified.

[0034] When the prediction method of the present invention is carried out to evaluate the amount of Largen detected in the body fluid of a subject (third embodiment), the corresponding body fluid of a healthy subject can be used as a control. It is preferable to detect Largen using the corresponding body fluid of a healthy subject using the same method as the body fluid of the subject. Alternatively, a cutoff value based on accumulated data on the amount of Largen detected in the body fluid of a healthy subject may be used as the amount of Largen detected in the control body fluid.

[0035] In the prediction method of the present invention, if it is determined that the amount of Largen detected in the body fluid of a subject is higher than that of a control, the subject can be predicted to be resistant to anticancer drugs (anticancer drugs will be less effective) and have a poor prognosis (short survival time, rapid cancer progression, high possibility of recurrence, short event-free interval).On the other hand, if it is determined that the amount of Largen detected in the body fluid of a subject is no different from that of a control, the subject can be predicted to be sensitive to anticancer drugs (anticancer drugs will be more effective) and have a good prognosis (long survival time, slow cancer progression, low possibility of recurrence, long event-free interval).

[0036] [Screening method] The present invention provides a method for screening for an anti-renal cancer substance. The screening method of the present invention may include the following steps (1) to (3): (1) contacting a test substance with Largen-expressing renal cancer cells in vitro; (2) measuring the expression level of Largen in the renal cancer cells; and (3) A step of comparing the obtained Largen expression level with the Largen expression level of the renal cancer cells not contacted with the test substance, and selecting a test substance that reduces the expression level.

[0037] Preferred test substances include, for example, nucleic acids, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, cell culture supernatants, plant extracts, mammalian tissue extracts, and plasma. However, the test substances are not limited to these. The test substances may be novel or known substances. These test substances may form salts. Salts of test substances with physiologically acceptable acids or bases are used.

[0038] Renal cancer cells expressing Largen can be primary cultured cells obtained from cancer tissues of renal cancer patients, renal cancer cell lines expressing Largen, renal cancer cell lines transfected with a Largen expression vector, etc. Renal cancer cell lines are preferably human renal cancer cell lines.

[0039] In step (1), the method for contacting the test substance with Largen-expressing renal cancer cells in vitro is not particularly limited, and examples include adding the test substance to the medium in which the renal cancer cells are cultured. It is preferable to provide a control group that is not contacted with the test substance.

[0040] In step (2), the expression level of Largen in renal cancer cells contacted with the test substance is measured. The expression level of Largen may be measured by measuring the amount of Largen protein or the amount of Largen mRNA. When measuring the amount of protein, protein can be extracted from renal cancer cells using a known method and quantified using a known method for measuring protein amount. Known methods for measuring protein amount include, for example, Western blotting, EIA, ELISA, RIA, and methods using protein measurement reagents. When measuring the amount of mRNA, RNA can be extracted from renal cancer cells using a known method and quantified using a known method for measuring mRNA amount. Known methods for measuring mRNA amount include Northern blotting, RT-PCR, quantitative RT-PCR, and RNase protection assay.

[0041] In step (3), the Largen expression level measured in step (2) is compared with that of the renal cancer cells not contacted with the test substance, and a test substance that reduces the expression level is selected. The criteria for expression reduction are not particularly limited, but it is preferable to select a test substance that reduces the expression level to a level that is significant in statistical analysis. Because the selected test substance reduces the Largen expression level in renal cancer cells, renal cancer patients administered with the test substance are expected to have a better prognosis. Furthermore, renal cancer patients administered with the test substance are expected to become more sensitive to anticancer drugs. Therefore, the test substance selected by the screening method of the present invention is expected to be an active ingredient in a renal cancer therapeutic agent that, when used in combination with other anticancer drugs, can enhance the anticancer effect of the combined anticancer drugs.

[0042] [Pharmaceutical composition for treating kidney cancer] The present invention provides a pharmaceutical composition for treating renal cancer. The pharmaceutical composition of the present invention may contain, as an active ingredient, a substance that inhibits the expression of Largen in renal cancer cells. A test substance selected by the above-described screening method of the present invention can be suitably used as the active ingredient of the pharmaceutical composition of the present invention.

[0043] The pharmaceutical compositions of the present invention contain a substance that inhibits Largen expression in renal cancer cells as an active ingredient and can be formulated according to conventional methods. For example, formulations for oral administration include solid or liquid dosage forms, such as tablets (including sugar-coated and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, and suspensions. These formulations are produced by known methods and contain carriers, diluents, or excipients commonly used in the pharmaceutical field. For example, carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate. Formulations for parenteral administration include injections and suppositories, including intravenous, subcutaneous, intradermal, intramuscular, infusion, and intraarticular injections. These injections are prepared according to known methods, for example, by dissolving, suspending, or emulsifying the active ingredient in a sterile aqueous or oily liquid commonly used for injections. Aqueous solutions for injection include, for example, physiological saline, isotonic solutions containing glucose or other adjuvants, and may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50). Oily solutions include, for example, sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. Suppositories for rectal administration are prepared by mixing the active ingredient with a standard suppository base. The formulations obtained in this manner are safe and low-toxic, and can be administered orally or parenterally to humans and mammals (e.g., rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.).

[0044] The active ingredient of the pharmaceutical composition of the present invention may be a nucleic acid that inhibits the expression of Largen in renal cancer cells. Examples of nucleic acids that inhibit Largen expression include short interfering RNA (siRNA), short hairpin RNA (shRNA), and antisense oligonucleotides of the PRR16 gene. Nucleic acids that inhibit Largen expression can be designed by known methods based on the nucleotide sequence of the PRR16 gene (see RefSeq ID in Table 1).

[0045] siRNA is a double-stranded RNA of approximately 20 bases (e.g., approximately 21-23 bases) or less in length. Expression of such siRNA in cells can suppress the expression of the gene targeted by the siRNA (in the present invention, the PRR16 gene). shRNA refers to a single-stranded RNA molecule of approximately 20 base pairs or more that contains a partially palindromic base sequence, forms a double-stranded structure within the molecule, and consists of a short hairpin structure with an overhang at the 3' end. After being introduced into cells, such shRNA is degraded to lengths of approximately 20 bases (typically, e.g., 21, 22, or 23 bases) within the cell, and can suppress the expression of the target gene (in the present invention, the PRR16 gene) in the same way as siRNA. siRNA and shRNA may be in any form as long as they can suppress the expression of Largen. siRNA or shRNA can be designed by known methods based on the base sequence of the target gene. siRNA or shRNA can also be artificially chemically synthesized. Alternatively, antisense and sense RNAs can be synthesized in vitro from template DNA using, for example, T7 RNA polymerase and a T7 promoter. Antisense oligonucleotides can be either DNA or RNA, as long as they are complementary to or hybridize with a sequence of 5 to 100 consecutive bases in the DNA sequence of the PRR16 gene. Modifications may also be used as long as they do not interfere with function. Antisense oligonucleotides can be synthesized by conventional methods, for example, easily using a commercially available DNA synthesizer.

[0046] When the active ingredient of the pharmaceutical composition of the present invention is a nucleic acid that inhibits Largen expression, it can be administered in the form of a non-viral or viral vector. When administered in the form of a non-viral vector, methods for introducing nucleic acid molecules using liposomes (such as the liposome method, HVJ-liposome method, cationic liposome method, lipofection method, and lipofectamine method), microinjection, and methods for transferring nucleic acid molecules into cells together with carriers (metal particles) using a gene gun can be used. When siRNA or shRNA is administered to the body using a viral vector, DNA expressing the siRNA or shRNA can be introduced into a DNA or RNA virus such as a detoxified retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, or SV40, and the gene can be introduced into cells or tissues by infecting the cells or tissues with this recombinant virus. It is preferable that the DNA expressing the siRNA or shRNA has a renal cell-specific promoter sequence.

[0047] While it is desirable that the siRNA and the target sequence are identical, they do not need to be completely identical as long as they are able to induce RNA interference. Specifically, as long as the antisense strand sequence of the siRNA and the target sequence hybridize, one to several (e.g., two, three, or four) mismatches are acceptable. That is, the siRNA may have one to several base substitutions, additions, or deletions relative to the target sequence and be able to induce RNA interference. Alternatively, the siRNA may have 85% or more, 90% or more, 95% or more, or 98% or more sequence identity with the target sequence and be able to induce RNA interference.

[0048] As long as the siRNA can induce RNA interference, it may be one in which all nucleotides in either the sense strand or the antisense strand are converted to DNA (hybrid type), or one in which some nucleotides in the sense strand and / or the antisense strand are converted to DNA (chimera type). Examples of hybrid types include those in which nucleotides in the sense strand are converted to DNA. Examples of chimera types include those in which some nucleotides on the downstream side (the 3'-end of the sense strand, the 5'-end of the antisense strand) are converted to DNA. Specifically, examples include those in which both the 3'-end of the sense strand and the 5'-end of the antisense strand are converted to DNA, and those in which either the 3'-end of the sense strand or the 5'-end of the antisense strand is converted to DNA. Furthermore, the nucleotide length to be converted may be any length up to half the nucleotides of the RNA molecule.

[0049] As long as siRNA can induce RNA interference, its nucleotide (ribonucleotide, deoxyribonucleotide) can be a nucleotide analogue whose sugar, base and / or phosphate are chemically modified.The nucleotide analogue whose base is modified includes, for example, 5-position modified uridine or cytidine (for example, 5-propynyluridine, 5-propynylcytidine, 5-methylcytidine, 5-methyluridine, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 5-methyloxyuridine, etc.); 8-position modified adenosine or guanosine (for example, 8-bromoguanosine, etc.); deazanucleotide (for example, 7-deaza-adenosine, etc.); O- and N-alkylated nucleotide (for example, N6-methyladenosine, etc.). Examples of sugar-modified nucleotide analogs include 2'-sugar modifications in which the 2'-OH of a ribonucleotide is replaced with H, OR, R, a halogen atom, SH, SR, NH, NHR, NR, or CN (where R represents an alkyl, alkenyl, or alkynyl group having 1 to 6 carbon atoms), and 5'-terminal phosphorylation modifications in which the 5' terminus is monophosphorylated. Examples of phosphate-modified nucleotide analogs include those in which the phosphoester group linking adjacent ribonucleotides is replaced with a phosphothioate group.

[0050] The pharmaceutical composition of the present invention can improve the prognosis of renal cancer patients to whom it is administered. Furthermore, the pharmaceutical composition of the present invention can enhance the sensitivity of renal cancer patients to anticancer drugs to whom it is administered. Therefore, the pharmaceutical composition of the present invention is preferably used in combination with other anticancer drugs.

[0051] 〔kit〕 The present invention provides a kit for predicting anticancer drug resistance and / or prognosis in renal cancer patients. The present invention also provides a kit for screening for anti-renal cancer substances. The kit of the present invention may contain an anti-Largen antibody or a primer or probe for detecting Largen mRNA. The anti-Largen antibody can be used for immunostaining, Western blotting, ELISA, etc. Primers for detecting Largen mRNA can be used for RT-PCR and quantitative RT-PCR. The primers may be a primer set for RT-PCR or quantitative RT-PCR. A probe for detecting Largen mRNA can be used for Northern blotting.

[0052] The components of the kit of the present invention other than the anti-Largen antibody or the primers or probes for detecting Largen mRNA are not particularly limited, and depending on the intended use, the kit may include equipment such as reaction tubes or plates, necessary reagents, instruction manuals, etc. By using the kit of the present invention, the prediction method of the present invention and the screening method of the present invention can be carried out simply and quickly. [Example]

[0053] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0054] [Experimental materials] All clinical specimens and clinical information used in the following examples were collected and used with permission from the Nagasaki University Hospital Clinical Research Ethics Committee (Nos. 12052899 and 19081920). Specifically, for the study of Largen expression in renal cancer tissue (Example 1), the relationship between Largen expression and cleaved caspase 3 (cleaved-Cas3) expression (Example 2), the relationship between Largen expression and the occurrence of histological necrosis or RIP3 (receptor-interacting protein kinase 3) expression (Example 3), the relationship between Largen expression and postoperative overall survival (Example 4-1), and the relationship between Largen expression and progression-free survival (Example 4-2), 208 renal cancer patients who underwent surgical resection of renal cancer tissue at Nagasaki University Hospital between 1993 and 2008 and did not receive preoperative treatment were studied. For the study of the relationship between Largen expression and disease-free survival (Example 4-3), 174 of the 208 renal cancer patients who did not have findings suggestive of metastasis on preoperative imaging were studied. In addition, regarding the relationship between Largen and the therapeutic effects of anticancer drugs (Example 5), 41 renal cancer patients who underwent surgical resection of renal cancer tissue at Nagasaki University Hospital between 2017 and 2020 and received molecular targeted drugs and immunotherapy after surgery were studied.

[0055] [Statistical analysis] For statistical analysis, JMP software manufactured by SAS was used.

[0056] Example 1: Evaluation of Largen expression in cancer tissues of renal cancer patients Using tissue specimens from the above 208 renal cancer patients, the expression of Largen in the renal cancer tissue specimens was evaluated by immunostaining.

[0057] (1) Immunostaining method Renal cancer tissue was fixed in 10% formalin, embedded in paraffin, and then cut into 5-μm-thick sections. After antigen retrieval in the sections using Target Retrieval Solution (DAKO), the sections were incubated at 4°C for 12 hours with a 60-fold diluted mouse polyclonal antibody against human Largen (Abnova) as the primary antibody. The sections were then stained using the EnVision detection system (DAKO), which combines a secondary antibody and peroxidase with a dextran polymer.

[0058] (2) Expression determination method Immunostained specimens were evaluated by scoring the expression intensity and expression rate. Specifically, expression intensity was classified into four levels (0: no expression, mild; 1: moderate; 2: severe; 3: high expression), and expression rate was classified into four levels (less than 10%; 0: 10% to less than 25%; 1: 25% to less than 50%; 2: 50% or more; 3: high expression). The scores were then added together. Ten or more fields were observed at 200x magnification, and the field with the highest expression was used for evaluation. Because the highest score for normal kidney tissue was 3, renal cancer tissues with a score of 4 or higher were classified as patients with high Largen expression, and those with a score of 3 or lower were classified as patients with low Largen expression.

[0059] (3) Results Figure 1 shows representative immunostained images of renal cancer tissue removed from a renal cancer patient. (A) is an observation image of a normal kidney section (magnification 200x), and (B) is an observation image of a renal cancer section (top row: magnification 400x, bottom row: magnification 200x). Largen was barely stained in the normal kidney section in (A), but it can be seen that Largen is deeply stained in the renal cancer section in the top row of (B). In the bottom row of (B), the renal cancer cells in the upper right are deeply stained, while those in the lower left are lightly stained, indicating low Largen expression. In other words, this shows that there are areas of high Largen expression and areas of low Largen expression in renal cancer cells in the same field of view. Of the 208 renal cancer patients studied, 88 were determined to be patients with high Largen expression and 120 were determined to be patients with low Largen expression.

[0060] Example 2: Evaluation of cleaved-Cas3 expression in cancer tissues from renal cancer patients The expression of cleaved-Cas3 in renal cancer tissue specimens from 208 renal cancer patients in Example 1 was evaluated by immunohistochemistry, and the expression of cleaved-Cas3 in the group of patients with high Largen expression (88 patients) and the group of patients with low Largen expression (120 patients) was compared.

[0061] (1) Immunostaining and positive rate calculation method Paraffin-embedded sections with a thickness of 5 μm were prepared as in Example 1. Antigen retrieval was performed using 0.01 M (pH 6.0) citrate buffer, and the expression of cleaved-Cas3 (Asp175) was evaluated. Immunostaining was performed in the same manner as in Example 1, using a mouse monoclonal antibody against human cleaved-Cas3 (R&D Systems) as the primary antibody. The cleaved-Cas3-positive cell rate was calculated using the following formula: Cleaved-Cas3 positive cell rate (%) = [number of cleaved-Cas3 expressing cells / total number of cells] × 100

[0062] (2) Results The results are shown in Figure 2. The expression of cleaved-Cas3 was significantly lower in patients with high Largen expression. This result suggests that Largen may suppress the induction of apoptosis.

[0063] Example 3: Evaluation of histological necrosis in cancer tissues of renal cancer patients Histological necrosis in renal cancer tissue specimens from the 208 renal cancer patients in Example 1 was assessed by hematoxylin-eosin (HE) staining and immunostaining for the tissue necrosis marker RIP3, and the occurrence of histological necrosis was compared between the group of patients with high Largen expression (88 patients) and the group of patients with low Largen expression (120 patients).

[0064] (1) Evaluation by HE staining Paraffin-embedded sections of each patient's cancer tissue were prepared and HE stained according to standard methods. All observations were performed by a pathologist, who examined at least 10 fields at 200x magnification. Necrosis was determined when cellular morphology was disrupted, cytoplasmic oxidation was increased, or nuclei were condensed or lost. The results were evaluated by logistic regression analysis.

[0065] (2) Evaluation by RIP3 immunostaining RIP3 is a representative marker of tissue necrosis. Immunostaining was performed using a rabbit polyclonal antibody against human RIP3 (Abcam) as the primary antibody, as described in Example 1. RIP3 expression intensity was classified into four levels (0: no expression, mild, 1: moderate, 2: severe, 3: high), and expression rate was classified into four levels (less than 10%, 0: 10% to less than 25%, 1: 25% to less than 50%, 2: 50% or more, 3: high), and the scores were added together. Ten or more fields were observed at 200x magnification, and the field with the highest expression was evaluated. A combined score of 4 or higher was considered to indicate necrosis.

[0066] (3) Results Evaluation was performed using logistic regression analysis. The results are shown in Table 2. In both HE staining and RIP3 immunostaining, the proportion of cancer tissues from patients with high Largen expression that were determined to have undergone necrosis immunohistochemically was significantly lower (0.492 times) than in patients with low Largen expression. Furthermore, the same results were confirmed in multivariate analysis including pathological characteristics. These results suggest that cancer cells with high Largen expression have acquired resistance to necrosis.

[0067] [Table 2]

[0068] Example 4: Evaluation of postoperative survival time in renal cancer patients 4-1 Postoperative overall survival Postoperative overall survival was compared between the group of patients with high Largen expression (88 patients) and the group with low Largen expression (120 patients). Cumulative survival rates were calculated using the Kaplan-Meier method, and P values ​​were calculated using the log-rank test.

[0069] The results are shown in Figure 3. Patients with high Largen expression had a significantly shorter survival time than those with low Largen expression (5-year survival time: 91.1% vs. 54.5%; P < 0.001).

[0070] 4-2 Progression-free survival period Progression-free survival (FS) is the period after surgery during which the cancer remains stable without recurrence or progression. Patients were divided into a high Largen expression group (88 patients) and a low Largen expression group (120 patients). All patients were followed up, regardless of their Largen expression level, with necessary imaging tests and treatment. Cumulative survival rates were calculated using the Kaplan-Meier method, and P values ​​were calculated using the log-rank test.

[0071] The results are shown in Figure 4. Patients with high Largen expression had a significantly shorter progression-free survival time than patients with low Largen expression (5-year progression-free survival time, 93.4% vs. 55.4%; P <0.001).

[0072] 4-3 Disease-free survival period Disease-free survival (DFS) refers to the period of time during which patients who underwent curative surgery without any preoperative findings suggesting metastasis survive without cancer recurrence. Of the 174 kidney cancer patients studied, 61 had high Largen expression and 113 had low Largen expression. Regardless of Largen expression level, all patients were followed up while undergoing necessary imaging tests and treatment. Cumulative survival rates were calculated using the Kaplan-Meier method, and P values ​​were calculated using the log-rank test.

[0073] The results are shown in Figure 5. Patients with high Largen expression had a significantly shorter disease-free survival period than patients with low Largen expression (5-year disease-free period: 97.6% vs. 76.2%; P < 0.001). These results suggest that patients with high Largen expression have a shorter disease-free period after radical surgery, i.e., a higher recurrence rate.

[0074] Example 5: Therapeutic effect of anticancer drugs on renal cancer patients Of the 41 renal cancer patients studied, 17 had high Largen expression and 24 had low Largen expression. The anticancer drugs used were the tyrosine kinase inhibitors sorafenib and pazopanib as molecular targeted drugs, and the immune checkpoint inhibitor nivolumab as immunotherapy. Regarding antitumor efficacy, patients who achieved a complete response (CR), partial response (PR), or stable disease (SD) were considered to have an "antitumor effect" according to the Response Evaluation Criteria in Solid Tumors (RECIST).

[0075] The results are shown in Table 3. Patients with high Largen expression showed a lower response to anticancer drugs compared to patients with low Largen expression. These results suggest that cancer cells with high Largen expression have acquired resistance to anticancer drugs. Furthermore, when combined with RIP3 expression, renal cancer patients with high Largen expression and low RIP3 expression (low necrosis rate) in renal cancer tissue were found to have particularly low sensitivity to anticancer drugs. Therefore, it can be inferred that renal cancer patients with high Largen expression / low RIP3 expression have a poor prognosis.

[0076] [Table 3]

[0077] Example 6: Detection and quantification of Largen in the blood and urine of renal cancer patients Blood and urine samples were collected from healthy volunteers and renal cancer patients, and the amounts of Largen protein and mRNA were quantified and compared. The study subjects were 11 renal cancer patients who underwent surgical resection of renal cancer tissue at Nagasaki University Hospital, and blood and urine samples were collected preoperatively. Blood and urine samples were also collected from three healthy volunteers without any pathological conditions, including malignant tumors and renal dysfunction. Largen protein was quantified using a sandwich ELISA method. A quantitative sandwich ELISA kit from MyBioSource was used for the ELISA. Largen mRNA was quantified by RT-PCR.

[0078] The results of measuring Largen protein are shown in Figure 6. (A) shows serum Largen concentrations, and (B) shows urinary Largen concentrations. In both serum and urine, Largen concentrations were significantly higher in renal cancer patients (n=11) compared to healthy subjects (n=3). Statistical analysis was performed using the Mann-Whitney U test.

[0079] Example 7: Relationship between antitumor effect of anticancer drugs and Largen expression Among patients treated at Nagasaki University Hospital with the molecular targeted therapy sunitinib or the immune checkpoint inhibitor nivolumab, for whom Largen expression in tissues before treatment could be analyzed, the relationship between the tumor shrinkage effect obtained with each treatment and Largen expression was analyzed. Statistical analysis was performed using the chi-square test.

[0080] The results are shown in Tables 4-1, 4-2, and 4-3. Table 4-1 shows the results for all patients (n=41), Table 4-2 shows the results for patients treated with sunitinib (n=26), and Table 4-3 shows the results for patients treated with nivolumab (n=15). In the tables, CR stands for Complete Response, indicating a state in which the tumor has completely or almost disappeared (strong therapeutic effect), PR stands for Partial Response, indicating a state in which the tumor size has decreased (therapeutic effect), SD stands for Stable Disease, indicating a state in which the tumor size has not changed, and PD stands for Progressive Disease, indicating a state in which the tumor has increased / new lesions have appeared (no therapeutic effect). For both sunitinib and nivolumab treatments overall (Table 4-1), sunitinib treatment (Table 4-2), and nivolumab treatment (Table 4-3), the number of patients who did not respond to treatment (PD) was significantly higher in Largen-positive patients. These results indicate that renal cancers expressing Largen are resistant to molecular targeted therapeutic drugs and immune checkpoint inhibitors, and are less likely to respond to them.

[0081] [Table 4-1]

[0082] [Table 4-2]

[0083] [Table 4-3]

[0084] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

1. A method for predicting anticancer drug resistance and / or prognosis of a renal cancer patient, comprising the steps of assessing the expression of Largen in the renal cancer tissue of the subject and comparing the expression level of Largen in the renal cancer tissue of the subject with that of a control.

2. The method for prediction according to claim 1, further comprising the steps of assessing necrosis in the subject's renal cancer tissue and comparing the level of necrosis in the subject's renal cancer tissue with a control.

3. A method for predicting anticancer drug resistance and / or prognosis of a renal cancer patient, comprising the steps of assessing the expression of Largen in the subject's renal cancer metastatic tissue and comparing the expression level of Largen in the subject's renal cancer metastatic tissue with that of a control.

4. The method for prediction according to claim 3, further comprising the steps of assessing necrosis in the subject's renal cancer metastatic tissue and comparing the level of necrosis in the subject's renal cancer metastatic tissue with that of a control.

5. The method for prediction according to claim 2 or 4, wherein necrosis is assessed based on the expression of receptor-interacting protein kinase 3 in the subject's renal cancer tissue or renal cancer metastasis tissue.

6. A method for predicting anticancer drug resistance and / or prognosis of a renal cancer patient, comprising the steps of detecting Largen in the body fluid of the subject and comparing the amount of Largen detected in the body fluid of the subject with that of a control.

7. The method for prediction according to claim 6, wherein the body fluid is urine or blood.

8. The method for prediction according to any one of claims 1 to 7, wherein the subject is a kidney cancer patient who has undergone surgery to remove cancer tissue.

9. The method for prediction according to any one of claims 1 to 8, wherein the anticancer drug is a molecular targeted drug or an immunotherapeutic drug.

10. The method for prediction according to any one of claims 1 to 9, wherein the prognosis of a renal cancer patient is one or more selected from the group consisting of survival time, cancer progression, cancer recurrence, and event-free interval.

11. A method for screening an anti-renal cancer substance, comprising: (1) contacting a test substance with renal cancer cells expressing Largen in vitro; (2) measuring the expression level of Largen in the renal cancer cells; and (3) comparing the obtained Largen expression level with the Largen expression level of the renal cancer cells not contacted with the test substance, and selecting a test substance that reduces the expression level. A screening method comprising the steps of:

12. A pharmaceutical composition for treating renal cancer, comprising as an active ingredient a substance that inhibits the expression of Largen in renal cancer cells, wherein the active ingredient is siRNA (short interfering RNA), shRNA (short hairpin RNA), or antisense oligonucleotide of the PRR16 (proline rich 16) gene.

13. A kit for predicting anticancer drug resistance and / or prognosis in renal cancer patients, comprising an anti-Largen antibody and reagent for measuring the amount of Largen protein in the subject's renal cancer tissue or renal cancer metastasis tissue, or a primer or probe and reagent for detecting Largen mRNA in the subject's renal cancer tissue or renal cancer metastasis tissue, and an instruction manual.

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