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WO2018074770A1 - Utilisation du rnf20 pour le diagnostic et le traitement du cancer du rein ou du foie et criblage pour identifier des agents thérapeutiques contre le cancer du rein ou du foie - Google Patents

Utilisation du rnf20 pour le diagnostic et le traitement du cancer du rein ou du foie et criblage pour identifier des agents thérapeutiques contre le cancer du rein ou du foie Download PDF

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WO2018074770A1
WO2018074770A1 PCT/KR2017/011172 KR2017011172W WO2018074770A1 WO 2018074770 A1 WO2018074770 A1 WO 2018074770A1 KR 2017011172 W KR2017011172 W KR 2017011172W WO 2018074770 A1 WO2018074770 A1 WO 2018074770A1
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rnf20
expression
gene
protein
srebp1c
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김재범
이재호
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SNU R&DB Foundation
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Definitions

  • the present application is a technique related to the diagnosis, treatment and treatment drug development of cancer associated with abnormalities of RNF20.
  • Kidney cancer occurs mainly in elderly people in their 60's and 70's, and continues to increase, and Korea ranks 10th with 2.0% of cancers occurring in men and 15th with 1.2% in women.
  • Renal cell carcinoma includes clear cell RCC, papillary RCC, chromophobe RCC, medullary RCC, and nonclassifiable renal cell carcinoma. (unclassified RCC), kidney transitional cell carcinoma (TCC), and renal eosinophilic granuloma (ranal oncocytoma). Clear cell type renal cell carcinoma accounted for 66-75%, papillary renal cell carcinoma accounted for 15%, and anachromosomal renal cell carcinoma accounted for 5%.
  • Kidney cancer has little symptoms when the tumor is small, and symptoms do not appear until the tumor is large enough to push the organs. Therefore, since the diagnosis is often delayed, about 30% of the patients are already metastasized when the first diagnosis is made. Therefore, a method for early diagnosis of kidney cancer is needed.
  • kidney cancer Currently, methods used for diagnosing kidney cancer include abdominal ultrasonography, abdominal computed tomography (CT), etc., and use of expensive equipment has a disadvantage in that it is inexpensive to use for screening purposes.
  • CT computed tomography
  • Hepatocellular carcinoma is also the most common type of adult liver cancer and is the third leading cause of death from cancer (Stefaniuk P, et al., 2010, World J Gastroenterol 16: 418-424). HCC is a disease that manifests only when it progresses considerably, which often leads to a missed time for proper treatment and a very poor prognosis. In particular, surgical removal of the disease is a serious disease that dies within one year. When the diagnostic method is improved, a significant improvement in treatment is expected.
  • Ring finger protein 20 is an E3 ubiquitin ligase that plays a variety of roles in transcriptional regulation, DNA damage response, stem cell differentiation and adipose synthesis.
  • RNF20 promotes monoubiquitination of histone H2B, which regulates transcription of gene subsets and contributes to chromatin remodeling (Minsky et al., 2008, Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells. Nat Cell Biol 10 , 483-488; Shema et al., 2011).
  • US Patent Publication No. 2011-0081362 relates to the treatment of cancer, a technique for inhibiting modulators involved in a number of intracellular processes including RNF20 to selectively inhibit the growth or survival of cancer cells comprising RAS active mutations. Is disclosed.
  • US Patent Publication No. 2013-0295584 relates to the use of histone protein polyubiquitonation in cancer biomarkers, which uses monopolyquitinization of histone 2B in the identification of thyroid cancer, based on the interaction between CDC73 and RNF20. Disclosed are methods for screening substances that modulate monoubiquitination of histone proteins.
  • the present application seeks to provide cancer therapeutic agents, diagnostic markers and therapeutic screening methods associated with abnormalities of RNF20 based on novel molecular mechanisms.
  • the present application relates to cancer associated with a reduction or deletion of RNF20, including a ring finger protein 20 (RNF20) gene, or a protein encoded by said gene, or a substance that increases expression of said gene, in particular solid cancer, in particular kidney cancer Or it provides a pharmaceutical composition for the prevention or treatment of liver cancer.
  • RNF20 ring finger protein 20
  • the expression of RNF20 is markedly reduced in renal and liver cancers, and tumors are suppressed upon overexpression through inhibition of demethylation or hypermethylation of the gene or protein in vivo and in vitro, or by demethylation of the endogenous RNF20 gene.
  • kidney cancer in which the composition according to the invention is used comprises kidney cancer, in particular not caused by VHL gene mutations.
  • the present invention also provides a composition for the diagnosis or prognosis of cancer, in particular solid cancer, in particular renal cancer or liver cancer, associated with a reduction or deletion of RNF20, comprising a substance for detecting ring finger protein 20 (RNF20).
  • RNF20 can be measured at the gene or protein level, and can be used to detect, judge, diagnose, or predict survival prognosis for kidney or liver cancer.
  • the present invention also relates to a method for screening or preventing or treating cancer, in particular solid cancer, more particularly renal or liver cancer, associated with a reduction or deletion of RNF20 targeting the RNF20-SREBP1c-PTTG1 signaling pathway.
  • the method comprises a first step of providing a cell with reduced or lacking expression of RNF20; A second step of treating said cells with a test substance that is expected to increase expression of RNF20; A third step of measuring expression of one or more of SREBP1c or PTTG1 in cells treated with the test substance; And comparing the expression of the SREBP1c or PTTG1 in the cells contacted with the test substance and the control cells not in contact with the test substance, so that the expression of the SREBP1c or PTTG1 in the cells contacted with the test substance is less than that of the control. And a fourth step of selecting it as a therapeutic agent candidate for cancer associated with abnormal expression of RNF20, if it has decreased in comparison with.
  • the third step instead of measuring expression in the SREBP1c, or in addition to measuring expression in SREBP1c, measuring the expression of a protein or a gene involved in lipid biosynthesis in which expression is promoted by the SREBP1c is measured. It may include a step. In this case, if the expression of the gene or protein involved in the lipid biosynthesis in the cell contacted with the test substance in the fourth step is reduced compared to the control cell not contacted with the test substance, the candidate substance is selected.
  • a first step of providing a cell with reduced or lacking expression of RNF20 A second step of treating said cells with a test substance that is expected to increase the expression of RNF20 or inhibit SREBP1c expression; A third step of measuring expression of PTTG1 in cells treated with the test substance; And comparing the PTTG1 expression in the cells contacted with the test substance and the control cells not in contact with the test substance, and when the expression of the PTTG1 in the cell contacted with the test substance decreased compared with the PTTG1 expression of the control. And a fourth step of selecting a candidate for treating a kidney cancer.
  • the third step measuring the expression of a protein or gene thereof involved in lipid biosynthesis that is promoted by SREBP1c
  • the fourth step Comparing the expression of the gene or protein involved in the lipid biosynthesis in the contacted cells and control cells not in contact with the test substance, the expression of the gene or protein involved in the lipid biosynthesis in the cell contacted with the test substance is If it is reduced compared to the expression of a gene or protein that promotes lipid biosynthesis, the step of selecting it as a candidate for the treatment of kidney cancer.
  • the present invention provides a method for detecting a prognosis of a RNF20 biomarker from a biological sample from a test subject to provide information necessary for the diagnosis or prognosis of renal or liver cancer; Comparing the detection result of the nucleic acid and / or protein level with the corresponding result of the corresponding marker of the control sample; And associating with a diagnosis or survival prognosis of kidney or liver cancer of the subject when there is a change in the nucleic acid or protein levels of the subject-derived sample compared to the control sample. Or a diagnosis or prognosis for renal or liver cancer.
  • the present disclosure provides RNF20-SREBP1c-PTTG1 signaling in kidney cancer cells in vitro, including a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that increases expression of the gene.
  • RNF20 ring finger protein 20
  • the present application provides a RNF20-SREBP1c-lipid biosynthesis pathway of renal cancer cells in vitro, including a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that increases expression of the gene.
  • RNF20 ring finger protein 20
  • Kits according to the invention can be used to modulate RNF20-SREBP1c-PTTG1, or RNF20-SREBP1c-lipid biosynthesis signaling in vitro for various purposes.
  • RNF20 according to the present application can be usefully used as a therapeutic agent and diagnostic marker for kidney cancer, and the new molecular mechanisms RNF20-SREBP1c-PTTG1 and RNF20-SREBP1c-lipid biosynthesis pathways identified herein can be usefully used to find a renal cancer therapeutic agent. have.
  • RNA-Seq database was obtained from TCGA. ## P ⁇ 0.01 compared to normal kidney tissue; ### P ⁇ 0.001 Compared with normal kidney tissue.
  • E Immunohistochemical staining pictures of tumors and normal kidney tissue near the tumor of the same kidney cancer patient are shown. RNF20 is a representative tissue photograph detected. Criteria represent 100 ⁇ m.
  • F The change in RNF20 mRNA expression according to the presence or absence of RG108 treatment in the ACHN kidney cancer cell line (+; 250 ⁇ M or ++; 1 mM) was measured by qRT-PCR. RNF20 mRNA expression level was corrected by Cyclophilin mRNA expression level. RNF20 mRNA expression was expressed relative to the control. The data represent the mean ⁇ standard deviation of three independent samples.
  • H and I Survival curve analysis results according to the VHL mutation status.
  • J After the infection of ACHN and A498 kidney cancer cell lines with GFP (Mock) or RNF20 expressing adenovirus, the cell proliferation rate was measured using CCK-8 technique. It was observed that RNF20 inhibited the proliferation of renal cancer cells by overexpression. The results represent the mean ⁇ standard deviation of five independent samples. CCK-8, Cell Counting Kit-8.
  • K Cell growth curves were measured by CCK-8 technique after treatment of ACHN and A498 kidney cancer cell lines with nonspecific control siRNA (siControl) or RNF20 specific siRNA (siRNF20). Inhibition of RNF20 increased the proliferation of renal cancer cells. * P ⁇ 0.05, ** P ⁇ 0.01.
  • Figure 2 shows that SREBP1 and liposynthetic genes are increased in the tumor tissue of kidney cancer patients, which is inversely correlated with the expression level of RNF20.
  • A As a result of qRT-PCR analysis of SREBP1c in tumors and normal kidney tissue of the same kidney cancer patient, it was observed that the amount of SREBP1c mRNA expression was increased in tumor tissues compared to normal kidney tissue of the same patient.
  • B Analysis of TCGA RNA-Seq database in tumors and normal kidney tissues of kidney cancer patients showed increased expression levels of SREBP1 in tumor tissues of kidney cancer patients.
  • C Analysis of SREBP1 expression according to the T stage of kidney cancer patients. RNA-Seq database was obtained from TCGA.
  • FIG. 3 shows that RNF20 inhibits SREBP1 inhibits the metabolic biosynthesis and renal cancer cell proliferation.
  • A ACHN kidney cancer cell lines were infected with adenoviruses expressing Myc-RNF20 or Flag-SREBP1c. Cell lysates were separated via SDS-PAGE and Western blotting was performed using antibodies. Validation of overexpression of RNF20 and SREBP1c.
  • B RNF20 or SREBP1c were continuously overexpressed using lentiviral in ACHN kidney cancer cell lines. Relative mRNA expression was measured by qRT-PCR. MRNA amount of each gene was corrected by mRNA expression amount of GAPDH gene. Each mRNA expression amount was expressed relative to the mock control.
  • Figure 4 shows the identification of PTTG1 as a new target gene of SREBP1c.
  • A Scatter plots of transcriptomes analyzed by RNA-Seq in livers of normal mice and mice lacking SREBP1c were expressed.
  • B PTTG1 mRNA expression levels in ACHN kidney cancer cell lines overexpressing RNF20 or SREBP1c were analyzed. PTTG1 mRNA expression was corrected by GAPDH and expressed as relative to the control.
  • C After transducing siRNF20 or siSREBP1 to ACHN kidney cancer cell lines, the amount of PTTG1 mRNA was analyzed by qRT-PCR.
  • PTTG1 mRNA expression level was corrected by GAPDH, and all mRNA expression levels were expressed relative to the control group.
  • siControl and siRNF20 were transduced into ACHN kidney cancer cells, and Western blotting was performed using antibodies. Inhibition of RNF20 increased SREBP1 and PTTG1 at protein levels. nSREBP1, nuclear SREBP1.
  • E ACHN kidney cancer cell lines were infected with SREBP1c adenovirus and transduced PTTG1 siRNA. Western blotting was performed using the antibody. It was observed that the amount of PTTG1 protein was increased by SREBP1c overexpression.
  • the HEK293 cell line was transduced with a luciferase reporter plasmid cloned with a PTTG1 promoter and a beta-galactosidase, RNF20, or SREBP1c expression vector. Cell lysates were analyzed by luciferase and beta-galactidase techniques. The data represent the mean ⁇ standard deviation of three independent samples. RLU, relative fluorescence (G) The qRT-PCR analysis of PTTG1 in tumors and normal kidney tissues of the same kidney cancer patients showed that PTTG1 mRNA expression was increased in kidney cancer tumor tissues.
  • RNA-Seq database in tumors and normal kidney tissues of kidney cancer patients showed increased expression of PTTG1 in tumor tissues of kidney cancer patients.
  • PTTG1 expression level according to T stage of kidney cancer patients was analyzed.
  • RNA-Seq database was obtained from TCGA. ### P ⁇ 0.001 compared with normal height; ### P ⁇ 0.001 Compared with normal height.
  • J The correlation between RNF20 and PTTG1 mRNA expression in the TCGA database was calculated using Pearson's correlation analysis. Kaplan-Meier survival curves were analyzed in the group divided by the number of patients (n), Pearson's correlation coefficient (r), and P value (P)
  • K PTTG1 expression. P values were calculated via log-rank test. The survival prognosis of renal cancer patients with high PTTG1 expression was observed poorly.
  • FIG. 5 shows that betulin, an SREBP inhibitor, inhibits kidney cancer cell proliferation.
  • A ACHN and A498 kidney cancer cell lines were treated with various concentrations of betulin for 12 hours, and then cell lysates were separated by SDS-PAGE to perform western blotting. It was observed that betulin reduced the amount of SREBP1, fat metabolism biosynthesis, and cell cycle regulatory proteins. pSREBP1, precursor SREBP1; nSREBP1, nuclear SREBP1.
  • B and C ACHN and A498 kidney cancer cell lines were treated with betulin and cell proliferation was measured via CCK-8 technique. Betulin treatment inhibited renal cancer cell proliferation. The data represent the mean ⁇ standard deviation of five independent samples.
  • FIG. 6 shows that SREBP1c affects cell growth in kidney cancer by regulating cell cycle and fat metabolism.
  • A An experimental design schematic for verifying the inhibitory effect of PTTG1 or FASN inhibition through siRNA and the fat biosynthesis gene (FASN, ACC) through the drug is shown.
  • B and C PTRE1 siRNA was transduced into SREBP1c overexpressing ACHN kidney cancer cell line via lentiviral. After 48 hours, mRNA expression of adipose biosynthesis and cell cycle regulators was measured by qRT-PCR and corrected by GAPDH mRNA expression. The data represent the mean ⁇ standard deviation of three independent samples.
  • D Relative proliferation of the cells described in (B and C) was measured by CCK-8 technique.
  • ACHN kidney cancer cell lines were treated with ACC inhibitor Topa (TOFA, 10 ⁇ g / ml) or FASN inhibitor C75 (10 ⁇ g / ml) for 24 hours and then the amount of triglycerides in the cells was measured. The effect of fat metabolism reduction by inhibiting fat biosynthesis was verified.
  • (F) ACHN kidney cancer cell lines were treated with topa (10 ⁇ g / ml) or C75 (10 ⁇ g / ml) for 24 hours and then PTTG1 mRNA expression was measured by qRT-PCR. mRNA amount was expressed relative to the control. The amount of PTTG1 mRNA expression did not change by the control of fat biosynthesis.
  • FIG. 7 shows that RNF20 overexpression inhibits tumor proliferation in xenograft mice.
  • A BALC / c nude mice were injected subcutaneously with Mock or RNF20 overexpressing ACHN kidney cancer cells to generate tumors. Each group represents 10 tumors from five mice. Representative photographs showing tumor size at the end of the experiment show the effect of RNF20 overexpression on xenograft tumor growth in an individual. Validation of anticancer effect by RNF20 overexpression in vivo. Criteria represent 10 mm.
  • B Xenograft tumor volume of ACHN kidney cancer cells following RNF20 overexpression was measured over 35 days. The plot shows the mean volume plus standard error.
  • C Tumor weights were measured and expressed at the end of the experiment.
  • ACHN xenograft tumor sections overexpressing Mock or RNF20 were stained with hematoxylin eosin (H / E) or Oil red O and representative pictures were taken. Xenograft tumor sections were stained with Ki67 and TUNEL. TUNEL, terminal deoxynucleotide transferase dUTP niche terminal label. RNF20 overexpression demonstrated the effects of tumor growth inhibition and fat metabolism accumulation. Standard means 100 ⁇ m.
  • FIG. 8 shows a model (summary model) suggesting SREBP1-dependent adipose biosynthesis of RNF20 and renal tumor suppression function through cell cycle control in kidney cancer.
  • Decreased expression of RNF20 and increased expression of SREBP1 in kidney cancer patients are associated with poor survival prognosis.
  • RNF20 inhibition leads to the activation of SREBP1 leading to renal cancer oncogenesis.
  • SREBP1 regulates the cell cycle through a new target gene, PTTG1.
  • Betulin, a SREBP1 inhibitor also inhibits renal cancer cell proliferation through adipose biosynthesis control and G1 cell cycle arrest. Taken together, reduced RNF20 expression promotes tumorigenicity of renal cancer through SREBP1-dependent adipose biosynthesis and cell proliferation activity.
  • RNF20 shows tumor suppression function in kidney cancer cells, but does not affect cell proliferation of normal kidney cells.
  • A Western blotting technique for the expression of RNF20 protein in human primary renal cortical epithelial (HRCE) cell lines, normal kidney cell lines such as HEK293, and kidney cancer cell lines such as ACHN, A498, and Caki-2. Analyzed.
  • B RG108 (+; 250 ⁇ M or ++; 1 mM) was treated in HEK293 cell line for 48 hours, and RNF20 mRNA expression was measured by qRT-PCR. The expression level of RNF20 mRNA was corrected by the expression level of Cyclophilin mRNA and expressed as a relative value to the control group.
  • the data represent the mean ⁇ standard deviation of three independent samples. n.s., not significant.
  • C ACHN and A498 kidney cancer cell lines were infected with GFP (Mock) or Myc-RNF20 expressing adenovirus. After 24 hours, the cell lysate was separated by SDS-PAGE, followed by Western blotting using an antibody.
  • D After transducing siControl and siRNF20 into ACHN kidney cancer cell lines, RNF20 expression was measured by Western blotting.
  • E HRCE and HEK293 cell lines were infected with Mock or Myc-RNF20 expressing adenovirus and cell lysates were isolated via SDS-PAGE, followed by Western blotting using antibodies.
  • Figure 10 shows that the expression of liposynthetic enzymes in renal cancer tumor tissue is increased and inversely correlated with RNF20 expression.
  • A qRT-PCR analysis of FASN in tumors and normal kidney tissue of the same kidney cancer patient. mRNA expression level was corrected to normal kidney tissue of the same patient.
  • B shows the corrected FASN RNA-Seq expression in tumors and normal kidney tissue of kidney cancer patients.
  • C Analysis of FASN expression according to renal cancer T stage.
  • D qRT-PCR analysis of SCD1 in tumors and normal kidney tissue of the same kidney cancer patient. mRNA expression level was corrected to normal kidney tissue of the same patient.
  • E Corrected SCD1 RNA-seq expression levels are shown in renal cancer tumors and normal kidney tissues.
  • FIG. 11 shows that RNF20 inhibition in kidney cancer cells promotes lipobiosynthesis and cell proliferation.
  • siRNF20 or siSREBP1 were transduced into ACHN kidney cancer cell lines. After 48 hours, cell lysates were separated on SDS-PAGE and Western blotting was performed using antibodies. nSREBp1, nuclear SREBP1.
  • B and C After knocking down RNF20 or SREBP1 by siRNA in ACHN kidney cancer cell lines, mRNA expression levels of adipose biosynthesis-related genes were analyzed using qRT-PCR. mRNA expression level was corrected by GAPDH mRNA expression level and expressed as relative value to Mock control group.
  • Figure 12 shows that PTTG1 is induced by SREBP1c, the expression level is increased in the tumor tissue of kidney cancer patients.
  • Kidney, liver and adipose tissues of normal mice and mice lacking SREBP1c were isolated. After extracting RNA from various tissues, mRNA expression levels of SREBP1c and PTTG1 were analyzed by qRT-PCR. mRNA expression level was corrected by TATA-binding protein (TBP) expression level.
  • TBP TATA-binding protein
  • EAT visceral adipose tissue, IAT; Subcutaneous adipose tissue; BAT; Brown adipose tissue ** P ⁇ 0.01, *** P ⁇ 0.001.
  • B SRE motif and E-BOX sequence present in PTTG promoter in various species.
  • FIG. 13 shows that betulin effectively lowers kidney cancer cell proliferation.
  • siControl and siRNF20 were transduced into ACHN kidney cancer cell lines. After 24 hours of betulin treatment, qRT-PCR was used to measure mRNA expression of adipose biosynthesis and cell cycle regulators. mRNA expression level was corrected by GAPDH expression level. The data represent the mean ⁇ standard deviation of three independent samples. # P ⁇ 0.05 compared with control, ## P ⁇ 0.01 compared with control, ### P ⁇ 0.001 compared with control, ** P ⁇ 0.01, *** P ⁇ 0.001. Growth curves of kidney cancer cell lines described in (C and D) (A and B) were measured using the CCK-8 technique. P ⁇ 0.05, *** P ⁇ 0.001, ns, not significant; CCK-8, Cell Counting Kit-8.
  • Figure 14 shows that SREBP1c regulates renal cancer cell proliferation through two pathways (lipid biosynthesis and cell cycle regulation).
  • a and B ACHN kidney cancer cell lines were treated with Topa (10 ⁇ g / ml) or C75 (10 ⁇ g / ml) for 24 hours. MRNA expression of adipose biosynthesis and cell cycle regulators was corrected by GAPDH mRNA expression and expressed as relative to control.
  • C The growth rate of the cells described in (A and B) was measured by CCK-8 technique.
  • RNF20 controls apoptosis and apoptosis genes in kidney cancer xenograft tumors.
  • the effect of RNF20 overexpression on apoptotic gene expression control in ACHN kidney cancer xenograft tumors was analyzed using qRT-PCR. Relative mRNA expression was corrected by GAPDH mRNA expression and expressed as relative to the mock control. ** P ⁇ 0.01, *** P ⁇ 0.001.
  • FIG. 16 shows the relative expression levels of RNF20 and SREBP in HCC, Huh-7 and HepG2 cell lines, which are liver cancer cell lines, as measured by gene (left) and protein (level), and compared with the control group (HEK293T) in liver cancer cell line. The amount of expression is decreased, and the SREBP is increased.
  • HEK293T human embroynic kidney cell; non-cancer cell
  • Huh-7 human hepatocellular carcinoma cell; mutant p53-Y220C
  • HepG2 human hepatoblastoma cell; wild-type p53. * P ⁇ 0.05 vs. HEK293T, ** P ⁇ 0.01 vs. HEK293T.
  • FIG. 17 shows that cell proliferation was reduced in hepatocellular carcinoma cell lines by RNF20 expression.
  • the cell lines were measured using CCK-8 (cell counting kit-8) 48 hours after transfer to plasmid expressing RNF20.
  • FIG. 18 is a Western blot showing the decrease of the concentration of SREBP1c protein in liver cancer cell line by RNF20 expression. Analysis was performed 48 hours after delivery of the cell line to the RNF20 expressing plasmid.
  • Figure 19 is a quantitative RT-PCR results showing that the expression of adipogenetic genes in liver cancer cell lines by RNF20 expression, the analysis was performed 48 hours after delivery of the cell line to the RNF20 expressing plasmid. * P ⁇ 0.05 vs. Mock, # P ⁇ 0.05.
  • FIG. 21 shows that SREBP1 protein concentration was increased in liver cancer cell lines which inhibited the expression of RNF20 using siRNF20. Analysis was performed 72 hours after treatment with siRNF20.
  • Figure 22 is a quantitative RT-PCR results showing the increase in the expression of adipogenic genes in hepatocarcinoma cell lines that inhibited the expression of RNF20 using siRNF20, the analysis was performed 72 hours after treatment with the cell line siRNF20. * P ⁇ 0.05, ** P ⁇ 0.01.
  • the present application shows that the expression level of ring finger protein 20 (RNF20) is decreased in tumor tissues of renal and liver cancer patients, and the overexpression of RNF20 is associated with the proliferation and fat metabolism activity of liver and kidney cancer cells in liver or kidney cancer cell lines and xenograft models.
  • RNF20 as a key regulator, it was found to regulate the RNF20-SREBP1c-PTTG1 and RNF20-SREBP1c-lipid biosynthesis pathway.
  • the present application for the prevention or treatment of kidney cancer or liver cancer including a ring finger protein 20 (RNF20) gene or a protein or a functional equivalent thereof, or a substance that increases the expression through methylation inhibition of the RNF20 gene To a composition.
  • RNF20 ring finger protein 20
  • Ring finger protein 20 included in the composition according to the present invention is a type of E3 ubiquitin ligase that plays a variety of roles in transcriptional regulation, DNA damage response, stem cell differentiation and lipogenesis, and contributes to histone remodeling. Promote monoubiquitination of H2B (Minsky et al., 2008, Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells. Nat Cell Biol 10 , 483-488; Shema et al., 2011, supra).
  • RNF20 included in the composition according to the present invention can be used in various forms and derived so long as the effect according to the present application is achieved.
  • RNF20 or a functionally equivalent variant from mammals, in particular humans is used, the genes and protein sequences of which are known as NCBI Entrez Gene ID: 56254 and 34878777, respectively.
  • SEQ ID NO: 1 and 2 respectively, the RNF gene and protein sequence is used.
  • Genomic DNA and cDNA can be prepared according to methods known in the art.
  • Genomic DNA for example, extracts genomic DNA from cells having the gene of interest and constructs a genomic library (vectors may be used, for example, plasmids, phages, cosmids, BACs, PACs, etc.) and viewed the library.
  • Colony or plaque hybridization is performed using a probe made on the basis of DNA encoding the protein of the invention (eg, SEQ ID NO: 2), or DNA encoding the protein of the present invention (eg, SEQ ID NO: 2).
  • cDNA synthesizes a cDNA based on mRNA extracted from a cell having an RNF20 gene, inserts the synthesized cDNA into a vector such as ⁇ ZAP, prepares a cDNA library, and expands the cDNA library.
  • a vector such as ⁇ ZAP
  • cDNA library can be prepared by colony or plaque hybridization or by PCR.
  • the term "functionally equivalent variant” refers to a variant compared to a wild type protein or gene sequence thereof derived from a particular species, but having the efficacy identified herein.
  • the RNF20 protein of the present invention or a gene encoding the same may be a mutants encoding a protein derived from a human sequence consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted, Derivatives, alleles, variants and homologues.
  • variations in nucleotide sequences may not be accompanied by variations in amino acids in proteins (degeneracy variants), and such degeneracy mutants are also included in the gene of the present invention.
  • the RNF20 protein and the gene encoding the functionally equivalent protein are known in the art, for example PCR methods (Saiki et al., Science, 230: 1350-1354, 1985; Saiki et al., Science, 239: 487-491, 1988).
  • those skilled in the art will be able to isolate primers from a variety of mammals having high homology with the RNF20 gene by designing primers capable of specific hybridization thereof from known RNF20 gene sequences.
  • the gene isolated as described above has high homology with the amino acid sequence of the human-derived RNF20 protein at the amino acid level.
  • High homology refers to the identity of at least 50%, more preferably at least 70%, more preferably at least 90% (eg, at least 95%) sequences throughout an amino acid sequence.
  • the homology of amino acid sequences or nucleotide sequences is based on BLAST (Proc. Natl. Acad. Sci. USA, 90, 5873-5877, 1993), a program called BLASTN or BLASTX (Altschul et al, J. Mol. Biol. , 215, 403-410, 1990) and specific methods are known on the following website (http: //www,ncbi.nlm.nih.gov.).
  • the RNF20 protein or gene included in the composition according to the present invention includes all of full length or truncated forms.
  • sequence variations according to a specific individual, region, environment, etc. even if they are derived from the same host, for example, humans, and of course, some sequences have been modified (deleted, substituted, added), but all functionally equivalent variants are present. It can be used in the present invention. In one embodiment it is of human origin and is as previously mentioned.
  • the gene encoding the protein is RNF20 in a target cell, for example, a tumor cell of mammalian origin, in particular a kidney cancer cell, according to known methods such as recombinant DNA technology.
  • a target cell for example, a tumor cell of mammalian origin, in particular a kidney cancer cell
  • the gene may be introduced and used as a linear DNA, a plasmid vector or other DNA delivery vector.
  • Such vectors are preferably non-viral and are not particularly limited as long as they can express the RNF20 gene in eukaryotic cells, and reference may be made to those described in the Examples herein.
  • the RNF20 gene of the present invention may be introduced into an expression vector used in a gene therapy system or the like, for example, a viral vector, and then included in a viral particle as a carrier according to a known method.
  • the viral vector is not limited as long as the protein of the present invention can be introduced into a desired cell or tissue, and is preferably an adenovirus vector, and the viral vector is capable of expressing a gene contained therein in a eukaryotic cell. It is not particularly limited.
  • the vector may be purified and used after transfection into an appropriate cell in a cell culture system according to a known method.
  • proteins of the invention include, for example, purified proteins, water soluble proteins, or those in fusion with protein or amino acid residues in a form bound to a carrier for delivery or administration to a target cell.
  • Substances that increase the expression of RNF20 included in the composition according to the present application is a substance that inhibits methylation of the promoter region of the gene, particularly an endogenous gene, that is, the cell itself, in particular, a substance that inhibits methylation in CpG islands. Inhibition of methylation promotes the transcription of RNF20, resulting in increased expression of RNF20 in cells. Therefore, various materials which exhibit this effect can be included in the compositions according to the present application. In one embodiment a DNA methyltransferase (DNMT) inhibitor is used.
  • DNMT DNA methyltransferase
  • DNA methyltransferase (DNMT) inhibitors include, but are not limited to, nucleoside analogs as epigenetic gene-modulating drugs such as, for example, azacytidine, decitabine, and zebularine, and may include various substances including the effects described above.
  • Non-nucleoside analogs may also be used, including hydralazine, RG109, procainnamide, procaine, SCI-1027, and the like.
  • Non-nucleoside analogs interfere with the binding of DNMT to the CpG island, which in turn inhibits methylation, and in the process specifically interacts with DNMT.
  • RG108 is used.
  • Kidney cancer in which the composition according to the present invention is used includes renal cell carcinoma that occurs in the parenchyma of the kidney (consisting of the medulla and the cortex as a collection of urine-forming cells in the kidney), and clear cell type renal cell carcinoma (clear cell cancer) RCC, ccRCC), Papillary RCC, Chromophobe RCC, Medullary RCC, Unclassified RCC, Kidney transitional cancer cell carcinoma (TCC), renal eosinophilic granuloma (ranal oncocytoma). In one embodiment, it is ccRCC.
  • HCC hepatocellular carcinoma
  • composition according to the present invention exhibits an effect irrespective of von Hippel-Lindau (VHL) variation or condition of kidney cancer, and thus can be used for the treatment and diagnosis of kidney cancer of more various causes.
  • VHL von Hippel-Lindau
  • compositions and methods herein can be used for the treatment and diagnosis of kidney cancer independently of VHL mutations.
  • the kidney cancer in which the compositions and methods herein are used may or may not include VHL variations.
  • the compositions and methods herein can be used in kidney cancer that is not caused by VHL mutations.
  • treatment is a concept that includes inhibiting, eliminating, alleviating, alleviating, ameliorating, and / or preventing a disease or symptom or condition resulting from the disease.
  • composition of the present application may further contain a compound which maintains / increases the solubility and / or the absorbency of at least one active ingredient or an active ingredient exhibiting the same or similar function in addition to RNF20.
  • pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, drug treatment and biological response modifiers for the treatment or prevention of kidney cancer.
  • composition of the present invention may be prepared by including at least one pharmaceutically acceptable carrier in addition to the above-mentioned active ingredient.
  • Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, seeding, and one or more of these components, as necessary.
  • Other conventional additives such as buffers and bacteriostatic agents can be added.
  • diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs.
  • Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used.
  • it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.
  • the method of administration of the composition of the present application is not particularly limited thereto, and known administration methods may be applied, and parenteral administration (for example, intravenous, subcutaneous, intraperitoneal, or topical) or oral, depending on the desired method.
  • parenteral administration for example, intravenous, subcutaneous, intraperitoneal, or topical
  • it can be administered through a patch-type, nasal / respirator attached to the skin, and administration by intravenous injection is preferable to obtain a rapid therapeutic effect.
  • the dosage may vary widely depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient.
  • Parenteral administration may be preferred for protein preparations comprising the RNF20 gene or polypeptide, but does not exclude other routes and means.
  • dosage units include, for example, about 0.01 mg to 100 mg but do not exclude the below and above ranges.
  • the daily dose may be about 1 ⁇ g to 10 g, and may be administered once to several times
  • the present application relates to the use of RNF20 as a biomarker, and more particularly to a composition or kit for diagnosing or prognosticting liver or kidney cancer comprising a substance for detection thereof.
  • diagnosis refers to determining the susceptibility of a subject's disease to a particular disease or condition, determining whether he or she currently has a particular disease or condition, prognosis of the subject having a particular disease or condition ( prognosis (eg, identifying a transitional cancer state, determining stage or progression of a cancer, or determining the responsiveness of a cancer to treatment) or to determine therametrics (eg, to provide information about treatment efficacy). Monitoring the state of an object).
  • prognosis eg, identifying a transitional cancer state, determining stage or progression of a cancer, or determining the responsiveness of a cancer to treatment
  • therametrics eg, to provide information about treatment efficacy
  • diagnostic biomarker or diagnostic marker is a substance that can diagnose cancer-causing tissues or cells from normal cells or appropriately treated tissues or cells and diagnose the diseased tissues or regions as compared to normal samples.
  • Protein or nucleic acid showing an increase or decrease in Renal cancer diagnostic marker herein is an RNF20 protein or a nucleic acid or gene encoding the same that reduces expression in kidney cancer tissue.
  • Biological sample as used herein refers to a substance or mixture of substances that includes one or more components capable of detecting a biomarker and includes, but is not limited to, cells, tissues or body fluids, such as whole blood, plasma, and serum from an organism, in particular humans. It is not. It also includes cells or tissues derived directly from an organism as well as cultured in vitro. Various samples may be used for the detection of liver or kidney cancer markers according to the present disclosure, but are not limited thereto. In one embodiment, whole blood, serum and / or plasma may be used. In other embodiments, renal tissue / cells or in vitro cell cultures obtained from, or suspected of developing or likely to develop, liver cancer or kidney cancer may be used. It also includes fractions or derivatives of the blood, cells or tissues. When using a cell or tissue, the cell itself or a fusion of the cell or tissue may be used.
  • RNF20 protein and nucleic acid sequences thereof are known herein and are also as previously mentioned, but not limited to, including functional equivalents thereof.
  • Detection herein includes quantitative and / or qualitative analysis, including the detection of presence, absence, and expression level detection. Such methods are well known in the art and those skilled in the art will select appropriate methods for carrying out the present application. Can be.
  • the markers according to the invention can be detected at the level of the detection of the presence of nucleic acids, in particular of mRNA and / or protein, and / or their expression levels themselves, changes in expression levels, differences in expression amounts, through quantitative or qualitative analysis.
  • Such markers for diagnosing liver or kidney cancer according to the present application are based on their functional and / or antigenic characteristics.
  • the activity, function or activity of a protein can be detected using agents that specifically interact at the nucleic acid encoding the protein, particularly at the mRNA and / or protein level.
  • the present application also includes an antibody or aptamer specifically recognizing a nucleic acid sequence of each biomarker, a nucleic acid sequence complementary to the nucleic acid sequence, a fragment of the nucleic acid sequence, or a protein encoded by the nucleic acid sequence. It relates to a marker for diagnosing kidney cancer.
  • detecting substance is a substance capable of detecting the marker of the present invention quantitatively or for the presence of a protein or nucleic acid such as mRNA level.
  • Methods for the analysis of the amount and presence of the protein expression patterns are known, for example Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, oukterroni Ouchterlony) immune diffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like.
  • Reagents for detecting protein or nucleic acid levels are known, for example, the former may be an antigen-antibody reaction, a substrate that specifically binds to the marker, a nucleic acid or peptide aptamer, a receptor that specifically interacts with the marker or It can be detected through reaction with a ligand or cofactor, or a mass spectrometer can be used.
  • Reagents or materials that specifically interact with or bind to the markers of the present disclosure may be used with chip or nanoparticles.
  • the diagnostic composition according to the present disclosure may comprise reagents necessary for the detection of an RNF20 marker at a protein or nucleic acid such as at the mRNA level.
  • reagents detectable at the protein level may include monoclonal antibodies, polyclonal antibodies, substrates, aptamers, receptors, ligands, cofactors, and the like. Such reagents can be incorporated into nanoparticles or chips as needed. Proteins can also be detected using mass spectrometry.
  • RNA levels, expression levels or patterns using transcription polymerase chain reaction (RT-PCR) / polymerase chain reaction, competitive RT-PCR, real-time RT-PCR RNase protection assays, chips or Northern blots Methods can be used, and such assays are known and can also be performed using commercial kits, and one skilled in the art will be able to select the appropriate one for the practice herein.
  • a detection reagent in a method for measuring the presence and amount or pattern of the mRNA by RT-PCR for example, a primer specific for mRNA of the marker of the present application is included.
  • primer is meant a nucleic acid sequence having a free 3 'hydroxyl group capable of complementarily binding to a template and allowing reverse transcriptase or DNA polymerase to initiate replication of the template.
  • the detection reagent used herein may be conjugated with a coloring material such as a fluorescent material for signal detection.
  • Detection herein includes quantitative and / or qualitative analysis, including the detection of presence, absence, and expression level detection. Such methods are well known in the art, and those skilled in the art will recognize methods suitable for the practice of the present application. You will be able to choose.
  • the original detection reagent comprises an antibody
  • the RNF20 protein detection of the present application is carried out using a monoclonal antibody that specifically binds thereto.
  • Antibodies that can be used herein are polyclonal or monoclonal antibodies, preferably monoclonal antibodies.
  • Antibodies may be commonly used in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) Or phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol., 222: 58, 1-597 (1991)).
  • fusion methods Kellow and Milstein, European Journal of Immunology, 6: 511-519 (1976)
  • recombinant DNA methods US Pat. No. 4,816,56
  • the immunoassay format may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbant assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunity. Including but not limited to fluorescent staining and immunoaffinity purification.
  • the immunoassay or method of immunostaining is described in Enzyme Immunoassay, E. T.
  • the detection reagent is a reagent for RNA analysis
  • the detection according to the present application is carried out at the mRNA level.
  • mRNA detection is usually carried out by Northern blot or reverse transcriptase PCR (polymerase chain reaction).
  • the RNA of a sample is specifically isolated from mRNA, and then cDNA is synthesized therefrom, and then a specific gene or a combination of primers and probes is used to detect a specific gene in the sample. Or it is a method which can determine the expression amount.
  • the present application provides a therapeutic agent for cancer associated with abnormal expression of RNF20 targeting RNF20-SREBP1c (Sterol Regulatory Element Binding Transcription Factor 1c) -PTTG1 (Pituitary Tumor Transforming 1) or RNF20-SREBP1c-lipidogenesis promoting protein signaling pathway. It relates to a screening method.
  • the low expression or suppression or lack of expression of RNF20 is associated with the development of renal cancer or liver cancer, and elucidated the mechanism of the various cancers, if abnormal expression of RNF20, in particular, low expression or lack of, led to the development of cancer.
  • Therapeutic agents for can be discovered by the screening methods herein. Examples of such cancers are not particularly limited to those including kidney cancer or liver cancer.
  • kidney cancer, particularly kidney cancer that is not caused by von Hippel-Lindau (VHL) gene mutations, as described above, may or may not include VHL mutations and includes VHL mutations. Even if these mutations are not the cause of kidney cancer.
  • VHL von Hippel-Lindau
  • the method according to the present invention comprises a first step of providing a cell with reduced or lacking expression of RNF20; A second step of treating said cells with a test substance that is expected to increase expression of RNF20; A third step of measuring expression of one or more of SREBP1c or PTTG1 in cells treated with the test substance; And comparing the expression of the SREBP1c or PTTG1 in the cells contacted with the test substance and the control cells not in contact with the test substance, so that the expression of the SREBP1c or PTTG1 in the cells contacted with the test substance is less than that of the control. And a fourth step of selecting it as a therapeutic agent candidate for cancer associated with abnormal expression of RNF20.
  • the reduction or lack of expression of RNF20 is reduced or inhibited by the mutation or epigenetic regulation of the RNF20 gene itself, or the expressed protein is epigenetic even if it does not involve abnormality of the gene.
  • the desired function is not performed by regulation or the like, those skilled in the art will be able to determine the extent to which expression is reduced or inhibited with reference to normal cells and those described in the Examples herein. For example, if gene expression (or transcription) of RNF20 is inhibited, leading to a decrease in protein expression (or translation), or if it is expressed as a protein but the protein does not function as desired due to gene mutation, the gene is methylated. And when the expression of the gene is suppressed. Examples of cases lacking include the case where the gene of RNF20 is present but not expressed by epigenetic regulation such as methylation.
  • cells or cell lines derived from renal or hepatic cancer tissue are used as the cells with reduced or lacking the expression of RNF20.
  • Expression of RNF20 refers to a decrease or lack in gene and / or protein levels, wherein a decrease or lack of expression is reduced in comparison to using a normal kidney cell line as a control. Based on the level, the reduced level can be judged.
  • the cells with reduced expression of RNF20 used in the methods according to the present disclosure include, but are not limited to, ACHN, A498, or Caki-2 as a kidney cell line, Huh-7 or HepG2 as a liver cancer cell line.
  • the method comprises a first step of providing a cell with reduced or lacking expression of RNF20; A second step of treating with a test substance expected to increase the expression of RNF20 or inhibit SREBP1c expression of said cells; A third step of measuring expression of PTTG1 in cells treated with the test substance; And comparing the PTTG1 expression in the cells contacted with the test substance and the control cells not in contact with the test substance, and when the expression of the PTTG1 in the cell contacted with the test substance decreased compared with the PTTG1 expression of the control. And a fourth step of selecting a candidate for treating a kidney cancer.
  • the candidate material in the third step, instead of or in addition to measuring the expression of SREBP1c, measuring the expression of a gene or a protein thereof involved in lipid biosynthesis in which expression is promoted by the SREBP1c.
  • the candidate material in the fourth step, has a decreased expression of a gene or protein involved in the lipid biosynthesis in comparison with control cells not in contact with the test material in cells in contact with the test material.
  • the third step measuring the expression of a protein and / or a gene involved in lipid biosynthesis in which expression is increased, enhanced, or promoted by the SREBP1c
  • the fourth step instead, the expression of the proteins and / or genes involved in the lipid biosynthesis in the cells in contact with the test substance and in control cells not in contact with the test substance was compared to determine the lipid biosynthesis in the cells in contact with the test substance. If the expression of the involved protein is reduced compared to the expression of the protein involved in the lipid biosynthesis of the control group comprising the step of selecting it as a candidate for the treatment of kidney cancer.
  • RNF20-SREBP1c-lipid biosynthesis promoting pathway was found, wherein the gene or the protein involved in lipid biosynthesis is that its transcription is increased by SREBP1c and its expression to the protein is increased.
  • the increase increases or promotes the synthesis of lipids in the cell. Therefore, as long as these characteristics are not particularly limited, substances that are involved in lipid metabolism in kidney cancer are FAS (Fatty acid synthase, or gene is FASN), SCD1 (Stearoyl-CoA desaturase-1), or ELOVL6 (Elongation of very Long chain fatty acids protein 6) can be selected by measuring the expression of the gene or protein.
  • each protein and its gene sequence are for example FASN NCBI gene ID (2194), protein DB (41872631); ACC1 Gene ID (31), Protein DB (38679960); SCD1 gene ID (6319), protein DB (53759151); And ELOVL6 Gene ID (79071), Protein DB (195539343), will be readily apparent to those skilled in the art, based on the sequences and disclosed herein, the expression of the protein or gene. It is also not intended to exclude the measurement of other genes known as target genes of SREBP1c that are involved in lipid biosynthesis in addition to these genes.
  • the expression of the protein involved in the lipid metabolism in the cell contacted with the test substance decreases compared with the expression of the protein involved in the lipid metabolism of the control group, it may be selected as a candidate for treating cancer of the kidney.
  • the molecular targets are RNF20 and / or SREBP1c, and the substance which increases the expression of the former or the substance which decreases the expression of the latter can be used as a test substance for screening a therapeutic agent for kidney cancer.
  • the test substance used in the method of the present invention is a substance which is expected to modulate the expression and / or activity of the target gene or protein described above.
  • the compound may have a low molecular weight therapeutic effect.
  • compounds of about 1000 Da in weight such as 400 Da, 600 Da or 800 Da can be used.
  • such compounds may form part of a compound library, and the number of compounds constituting the library may vary from tens to millions.
  • Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and low molecular compounds based on templates such as benzodiazepines, hydantoin, biaryls, carbocycles and polycycle compounds (such as naphthalene, phenoty) Azine, acridine, steroids, and the like), carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is merely illustrative. It is not limited to this.
  • PTTG1 has disclosed its sequence, and may refer to GenBank ID: NM_004219 for human cDNA sequence and NP_001269311 for protein sequence. Expression of PTTG1 can be detected at the protein or gene level, for which reference can be made above.
  • Candidates compared the PTTG1 expression in cells in contact with the test substance and control cells not in contact with the test substance, whereby the expression of the PTTG1 in the cells in contact with the test substance decreased compared to the PTTG1 expression of the control. If this can be selected as a candidate candidate for the treatment of kidney cancer.
  • increasing the expression of RNF20 or suppressing SREBP1c expression by the treatment of the test substance can be determined by measuring the expression of proteins involved in lipid metabolism in the cells.
  • the type of cell used in the present method and the amount and type of test substance vary depending on the specific test method used and the type of test substance, and those skilled in the art will be able to select an appropriate amount.
  • a substance which results in a decrease in the expression or activity of the protein in the presence of the test substance is selected as compared with the control group which is not in contact with the test substance.
  • the present disclosure provides a method for detecting a cancer or kidney cancer, comprising: detecting a level of nucleic acid and / or protein of a RNF20 biomarker from a biological sample from a test subject to provide information necessary for the diagnosis or prognosis of liver or kidney cancer; Comparing the detection result of the nucleic acid and / or protein level with the corresponding result of the corresponding marker of the control sample; And when there is a change in the nucleic acid or protein level of the subject-derived sample compared to the control sample, correlating with a renal cancer diagnosis or survival prognosis of the subject. .
  • Detection of the amount of expression of a marker used in the method according to the invention can be determined at the protein and / or mRNA expression level, as mentioned above.
  • Subject biological samples may also refer to the foregoing.
  • the control group is a normal control group, and when the level of the marker decreases in the subject compared to the level of the marker determined in the normal control group, the subject agent has a poor renal cancer or a poor survival prognosis.
  • Survival prognosis refers to 5-year survival after diagnosis of kidney cancer, which can be determined by combining pathological, clinical and molecular biological findings. Survival rates are especially associated with cancer stages, which are generally 88-100% in stage 1, 63-88% in stage 2, 34-59% in stage 3 and 0-20 in stage 4 %, Which means lower than that.
  • the step of associating a sample of the subject with a normal control group sets a threshold value for diagnosing the onset of each marker, and then detects the detection result of the subject with the threshold value. Can be compared. Threshold settings may refer to, for example, the methods described in the Examples herein.
  • non-protein clinical information i.e., non-marker clinical information
  • non-marker clinical information includes, but is not limited to, for example, one or more of age, sex, weight, diet, body mass, underlying disease, ultrasound, computed tomography (CT) of the patient.
  • CT computed tomography
  • the present disclosure provides RNF20-SREBP1c-PTTG1 signaling in cells using a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that results in increased expression of the gene in vitro or in an animal.
  • RNF20 ring finger protein 20
  • the present disclosure also provides RNF20-SREBP1c-lipid biosynthesis of cells using a ring finger protein 20 (RNF20) gene, or a protein encoded by the gene, or a substance that results in increased expression of the gene in vitro or in an animal.
  • RNF20 ring finger protein 20
  • kits and methods and the regulatory mechanism using the same may be usefully used as treatments for kidney or liver cancer, existing drug tests, drug development, research tools, but are not limited thereto.
  • ACHN, A498, HEK293, Caki-2 and human primary renal cortical epithelial (HRCE) cell lines were obtained from the American Type Culture Collection (ATCC) and cultured according to the supplier's manual .
  • ATCC American Type Culture Collection
  • ACHN and A498 cell lines were cultured in Eagle's minimum essential medium (MEM) medium containing 10% fetal bovine serum (FBS), penicillin (100 U / ml) and streptomycin (100 ⁇ g / ml).
  • HEK293 and Caki-2 cell lines were cultured in Dulbecco's modified Eagle medium (DMEM) medium containing 10% FBS, penicillin and streptomycin.
  • DMEM Dulbecco's modified Eagle medium
  • HRCE cell line 0.5% FBS, 10 nM triiodothyronine, 10 ng / ml epidermal growth factor, 100 ng / ml hydrocortisone, 5 ⁇ g / ml insulin, 1 ⁇ M epinephrine, 5 ⁇ g / ml transferrin, 2.4 mM L-alanine-L-glutamine Cultured in renal epidermal cell culture medium containing penicillin and streptomycin. All cells were cultured in 5% carbon dioxide, 37 °C conditions. Betulin and BODIPY 493/503 were purchased via Sigma-Aldrich. C75 and TOFA were purchased from Abcam. Propidium iodine compounds were purchased from BD Biosciences.
  • Tumor tissue and normal kidney tissue specimens of liver and kidney cancer patients were provided by Seoul National University Hospital. The study was approved by a review institution at Seoul National University Hospital (approved number: H-1501-011-636). Due to the retrospective nature of this study, the patient's consent was not required.
  • RNA sequencing results for 533 kidney cancer tumor tissues and 72 normal kidney samples were analyzed using the 1-99th percentile (bar), 25-75th percentile (box), and median (line within the box). Indicated. Patients were ranked according to gene expression from TCGA RNA-Seq data for survival analysis. Patients with higher than average gene expression were defined as "high” and the rest as "low”. Overall survival curves were estimated by Kaplan-Meier survival analysis and survival differences between the two groups were compared by log-rank analysis.
  • tissue microarray included 50 kidney cancer tumor tissue sections and 9 normal kidney tissues.
  • streptavidin-biotin complex technique was used to detect RNF20 and SREBP1, and each antibody was purchased from Abcam and BD Biosciences.
  • Adenovirus plasmids were constructed as reported in previous papers (1). Briefly, cDNAs encoding rat SREBP1c amino acids 1-403 and the entire mouse RNF20 were cloned into an AdTrack-CMV shuttle vector and constructed via an Ad-Easy adenovirus vector system. In all experiments, adenovirus encoding GFP was used as a control. Adenovirus was propagated in HEK293A cell line and purified using the previously reported CsCl density gradient centrifugal method (2).
  • RNF20 and SREBP1c cDNA were cloned into the lentiviral vector pLVX-EF1 ⁇ -AcGFP1-N1.
  • the lentiviral vector and pAX2 and pMD2.G vectors were transduced into HEK293T cell line using lipofectamine 2000. 48 hours after transduction, the virus was collected and filtered through a 0.45- ⁇ m filter. Thereafter, the cells were incubated for 18 hours in a culture medium containing 8 ⁇ g / ml polybrene and virus in an ACHN cell line. Infected cells were recovered for 48 hours and only colonies resistant to puromycin were selected for use in the experiment.
  • Cells and tissues were radioimmunoprecipitation assay containing 150 mM sodium chloride, 50 mM tris-sulfate, pH 7.4, 1% NP-40, 0.25% sodium dioxycholate, 1 mM EDTA, 1 mM PMSF, and a protease inhibitor mixture. Dissolved in (RIPA) buffer. Equal amounts of protein in each sample were separated on an SDS-PAGE gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was then added to TBS solution containing 0.1% Tween-20 to suppress nonspecific reactions by adding 5% nonfat milk or 3% bovine serum albumin.
  • PVDF polyvinylidene difluoride
  • Antibodies that bind -tag, Flag-tag or ⁇ -actin were bound to the PVDF membrane. After binding the horseradish peroxidase-conjugated secondary antibody to the PVDF membrane, the protein was visualized by chemiluminescence detection using a LuminoImager (LAS-3000) machine.
  • RNA was extracted with TRIzol lysis reagent.
  • CDNA was synthesized from the same amount of RNA using RevertAid reverse transcriptase.
  • Relative mRNA expression was measured by quantitative real-time PCR analysis (qRT-PCR) and calculated after correction with GAPDH or Cyclophilin mRNA.
  • SiRNA double strands of RNA20, SREBP1, PTTG1 and FASN were synthesized in Bioneer (South Korea). SiRNA transduction was performed to ACHN kidney cancer cell lines using the lipofectamine RNAiMAX system according to the provider instructions.
  • Cell proliferation was measured using a previously reported Cell Counting Kit-8 (CCK-8) system (3).
  • cell coloration curves were obtained by performing colorimetric analysis to detect metabolites of living cells.
  • ACHN cell lines with lentivirus overexpressing RNF20 or SREBP1c were cultured in 6-well plates. Cells were incubated at 5% carbon dioxide at 37 ° C. for 7 days, then fixed with formaldehyde and stained with crystal violet.
  • Trypsin-treated cells were washed with phosphate buffered saline (PBS) and fixed at 70 ° C. for 30 minutes at 4 ° C.
  • the fixed cells were washed twice with phosphate buffered saline, and then rinsed in a solution containing 0.1% nonniche P-40, 100 ⁇ g / ml RNA degrading enzyme and 2.5 ⁇ g / ml propidium iodide (PI) for 30 minutes.
  • Stained. Stained cells were analyzed by flow cytometry using the FACS Canto II machine, and the number of cells in each cell cycle was calculated using the ModFit LTTM cell cycle analysis program.
  • Intracellular triglycerides were measured by colorimetric analysis as previously reported and expressed in mg of lipid per mg of protein in cells (1). Briefly, the cell lysate was extracted using 5% Triton X-100, and then immersed in an 80 ° C. water bath and immersed in ice twice. After centrifugation at 12,000 rpm for 5 minutes, the supernatant was collected and the amount of intracellular triglyceride was measured using Infinity triglyceride assay. The measured value was corrected by the total protein amount analyzed by the BCA protein quantitative kit.
  • the ACHN kidney cancer cell line was incubated for 24 hours with or without 10 ⁇ M betulin, washed twice with phosphate buffered saline, and then fixed for 4 minutes with 4% paraformaldehyde.
  • the immobilized cells were washed twice with phosphate buffered saline containing Tween-20 and stained with fluorescein isothiocyanate (FITC) bound to bodiphy 493/503 for 1 hour in the dark. .
  • Samples were stained with a betashield solution containing 4 ′, 6-diamidino-2-phenylindole (DAPI) and observed using a Zeiss LSM 700 confocal microscope. All pictures were observed and analyzed under the same conditions.
  • DAPI 6-diamidino-2-phenylindole
  • PTTG1 promoter region (transcription start point -908 to +25 nucleotides) was cloned into pGL3-basic vector.
  • DNA plasmids were transduced into HEK293 cell line using the previously reported calcium-phosphate method (4). After 36 hours of incubation, the transduced cells were harvested and lysed using a buffer containing 25 mM tris-phosphate (pH 7.8), 10% glycerol, 2 mM EDTA, 2 mM DTT, and 1% Triton X-100. Was extracted. Luciferase and beta-galactidase activity were measured according to the instructions of the provider. Luciferase activity was corrected to beta-galactosidase activity.
  • mice Five weeks after transplantation, the mice were euthanized by inhalation of carbon dioxide and xenograft tumors were taken out and analyzed.
  • Xenograft tissue samples were fixed in 4% paraformaldehyde and placed in 30% sucrose and then in OCT solution.
  • Xenograft tumor tissue sections were subjected to hematoxylin / eosin (H / E) and oil red o staining and immunohistochemistry (5). Briefly, Ki67 protein and TUNEL techniques were performed using a streptavidin-biotin complex system. Photographs were observed using an EVOS ORIGINAL microscope and NIKON TMS inverted microscope.
  • Results were expressed as mean ⁇ standard deviation or mean ⁇ standard error ( Figures 7B and 7C). Multigroup comparisons were performed by one-way ANOVA or two-way ANOVA if two conditions exist. Differences between the two groups were analyzed by both Student's t-tests. Statistical analysis was performed using the Prism graph pad and the difference was significant when P ⁇ 0.05.
  • Ectopic lipid accumulation is significantly upregulated in clear cell Renal Cancer Carcinmoa (Rezende et al., 1999, Differential diagnosis between monomorphic clear cell adenocarcinoma of salivary glands and renal (clear) cell carcinoma.Am J Surg Pathol) 23 , 1532-1538; Valera and Merino, 2011, Misdiagnosis of clear cell renal cell carcinoma.Nat Rev Urol 8 , 321-333).
  • RNF20 is known to act as a negative regulator of angiogenesis by inhibiting SREBP1c (Lee et al., 2014, Ring finger protein20 regulates hepatic lipid metabolism through protein kinase A-dependent sterol regulatory element binding protein1c degradation.Hepatology 60 , 844- 857)
  • SREBP1c Ring finger protein20 regulates hepatic lipid metabolism through protein kinase A-dependent sterol regulatory element binding protein1c degradation.Hepatology 60 , 844- 857)
  • the inventors investigated whether RNF20 is dysregulated in ccRCC tumors. As shown in FIG. 1A, RNF20 mRNA expression is significantly downregulated in ccRCC tumors compared to normal kidney tissue from the same patient.
  • RNA-Seq data from the Cancer Genome Atlas showed a significant decrease in RNF20 mRNA expression in ccRCC tumors, indicating that low RNF20 expression is closely associated with advanced tumor stages ( 1C).
  • immunohistochemistry (IHC) analysis showed that RNF20 protein expression was lower in ccRCC tumors than adjacent normal kidney tissues (FIG. 1D).
  • RNF20 staining data obtained from normal kidney and tumor tissues of the same patient also showed reduced RNF20 expression in ccRCC (FIG. 1E).
  • RNF20 expression was also reduced in ccRCC cell lines A498, Caki-2, and ACHN as compared to human primary renal cortical epithelial (HRCE) and HEK293 normal kidney cells (FIG. 9A). As shown in FIG. 16, RNF20 was also reduced in liver cancer cells at the protein and gene levels.
  • siRNA-mediated inhibition of RNF20 increased cell growth in ccRCC cell lines and liver cancer cell lines, including ACHN and A498 cells (FIGS. 1K and 9D, FIG. 20).
  • RNF20 was not overexpressed or siRNA-mediated knocked down, the levels of RNF20 did not affect the growth of high HRCE and HEK293 normal kidney cells (FIG. 9E-H).
  • the data means that RNF20 can act as a tumor suppressor in ccRCC cells independently of VHL status.
  • SREBP1 and adipose biosynthesis genes are expressed at high levels, which is also positively correlated with malignant progression and bad outcomes (Guo et al., 2009, EGFR signaling through an Akt-SREBP-1-dependent, rapamycin-resistant pathway sensitizes glioblastomas to antilipogenic therapy.Sci Signal 2, ra82; Huang et al., 2012, Activation of androgen receptor, lipogenesis, and oxidative stress converged by SREBP -1 is responsible for regulating growth and progression of prostate cancer cells.Mol Cancer Res 10 , 133-142; Sun et al., 2015, SREBP1 regulates tumorigenesis and prognosis of pancreatic cancer through targeting lipid metabolism.Tumor Biol 36 , 4133-4141 ).
  • SREBP1 and lipophilic genes are associated with ectopic lipid storage in ccRCC. Therefore, the present inventors analyzed the expression pattern of the adipose biosynthetic gene in normal kidney and ccRCC tumor tissue. As shown in FIG. 2A, SREBP1c mRNA was significantly upregulated in ccRCC tumors compared to normal samples of the same patient (patient-matched). In addition, the TCGA RNA-Seq data indicated that SREBP1 was upregulated in ccRCC tumors (FIG. 2B) and had a positive correlation with advanced tumor stages (FIG. 2C).
  • mRNA levels of SREBP1 target genes for FASN and SCD1 were elevated in ccRCC tumors (FIGS. 10A-F).
  • the protein expression of SREBP1 and the lipophilic enzymes FASN and SCD1 increased simultaneously in ccRCC tumors compared to normal kidney tissue of the same patient, while RNF20 protein was downregulated (FIG. 2D).
  • mRNA expression of RNF20 is inversely correlated with that of the SREBP1c target genes FASN and ELOVL6 (FIGS. 10G and H), and FASN mRNA expression is positively associated with low survival (FIG. 2G).
  • mRNA levels of SREBP2 and its target gene, HMGCR, a gene that limits the rate of cholesterol synthesis were reduced in ccRCC tumors in qRT-PCR and TCGA RNASeq assays (FIG. 10 I-L).
  • intracellular triglyceride accumulation was greater in SREBP1c-overexpressing ACHN cells than in control ACHN cells (FIG. 3C).
  • inhibition of RNF20 increased intracellular triglyceride levels (FIG. 11D).
  • Inhibition of RNF20 using siRNF20 as described above was found to increase the SREBP1 protein concentration and increase the fat biosynthesis in liver cancer cell lines (FIGS. 21 and 22).
  • SREBP1c overexpression promoted mRNA expression of cell cycle regulators including PCNA, cyclin A, D1, and E in ccRCC cells (FIG. 3D), and further, RNF20 overexpression reduced the effect of overexpressed SREBP1c (FIG. 3D).
  • RNF20 knockdown also promoted cell cycle gene expression in ccRCC cells (FIG. 11E).
  • RNF20 overexpression reduced colony formation whereas ectopic SREBP1c expression increased colony formation in ACHN cells (FIG. 3E).
  • SREBP1c overexpression enhanced ccRCC cell proliferation (FIG. 3F) and knockdown of SREBP1 decreased ccRCC cell proliferation in both control and RNF20-inhibited cells (FIG. 11F). The same results were observed in liver cancer cell lines, and the results are described in FIGS. 17, 18 and 19.
  • RNF20 inhibits ccRCC cell proliferation by inhibiting SREBP1c-induced fat biosynthesis and cell cycle progression, indicating that RNF20 can be usefully used as a therapeutic agent for kidney cancer.
  • SREBP inhibitor betulin inhibits ccRCC cell proliferation by inhibiting SREBP1 and adipose biosynthesis irrespective of the VHL gene mutation (FIGS. 5A-C).
  • betulin eliminated cell proliferation and increased lipogenic activity after inhibition of RNF20 (FIGS. 13A-D).
  • SREBP1 and lipogenesis were overactivated with RNF20 downregulation in ccRCC (FIGS. 1 and 2), free SREBP1 was shown to promote ccRCC tumor development through activating lipogenesis.
  • SREBP1 and adipose biosynthesis pathways can be therapeutic targets for ccRCC.
  • SREBP1c As transcriptional activators, SREBP1c has been reported to stimulate fatty acid metabolism and cell cycle progression (Bengoechea-Alonso and Ericsson, 2006, Cdk1 / cyclin B-mediated phosphorylation stabilizes SREBP1 during mitosis. Cell Cycle 5 , 1708-1718; Jeon et. al., 2013, An SREBP-responsive microRNA operon contributes to a regulatory loop for intracellular lipid homeostasis.Cell Metab 18, 51-61; Williams et al., 2013, An essential requirement for the SCAP / SREBP signaling axis to protect cancer cells from lipotoxicity.Cancer Res 73 , 2850-2862).
  • PTTG1 was identified as a new target gene of SREBP1c (FIG. 4A).
  • PTTG1 expression was significantly reduced in kidney, liver and adipose tissue of SREBP1c deficient mice compared to wild type mice.
  • SREBP1c-overexpressing ACHN cells we examined PTTG1 expression in SREBP1c-overexpressing ACHN cells and found that ectopic SREBP1c expression increased PTTG1 mRNA expression, while RNF20 co-expression attenuated this effect (FIG. 4B).
  • RNF20 did not inhibit PTTG1 mRNA (FIG. 4C) or protein (FIG. 4D) expression.
  • SREBP1c promoted PTTG1 protein expression in ccRCC cells (FIG. 4E, lane 3).
  • siRNA mediated inhibition of PTTG1 did not affect SREBP1c or FASN protein expression (FIG. 4E, lane 4).
  • SREBP1c directly modulates PTTG1 transcription
  • the inventors have identified an EBOX motif and a potential SER (binding regulatory site), a binding site for SREBP1c at the proximal promoter site of PTTG1 gene in humans, monkeys, dogs, mice and rats. element) was analyzed (FIG. 12B).
  • PTTG1 is also involved in cell cycle progression and tumorigenesis in ccRCC, acting as a novel target gene for SREBP1c. It was also found that high expression of PTTG1 is closely associated with advanced tumor stage and low survival in ccRCC patients (FIGS. 4I and K). In addition, SREBP1 strongly stimulated mRNA and protein expression of PPTTG1, and several cell-cycle regulators, resulting in cancer cell proliferation in ccRCC (FIGS. 3 and 4). In contrast, RNF20 overexpression inhibited PTTG1 in both ccRCC cells and xenograft tumors (FIG. 4B; FIGS. 7D and E), and mRNA and protein levels of PTTG1 were increased by RNF20 inhibition (FIGS. 4C and D).
  • Betulin is a pharmacological inhibitor that exhibits a lipid-lowering effect by inhibiting the proteolytic process of SREBP protein (Soyal et al., 2015, Targeting SREBPs for treatment of the metabolic syndrome.Trends Pharmacol Sci 36 , 406-416; Tang et al., 2011, Inhibition of SREBP by a small molecule, betulin, improves hyperlipidemia and insulin resistance and reduces atherosclerotic plaques.Cell Metab 13 , 44-56). Betulin also attenuates the growth of various cancers by inhibiting various tumor factors, including cell-cycle regulators (Chintharlapalli et al., 2007, Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors.
  • Betulin inhibits lung carcinoma proliferation through activation of AMPK signaling.Tumor Biol 35 , 11153-11158).
  • the present inventors investigated the effect of betulin on SREBP inhibition to determine the anti-tumor effect on ccRCC cells.
  • Betulin treatment of VHL wild-type ACHN and VHL-depleted A498 ccRCC cells resulted in a decrease in nuclear SREBP1 protein in a dose-dependent manner, whereas the precursor morphology of SREBP1 was not affected (FIG. 5A). Means to suppress processing.
  • protein levels of cell cycle regulators including PTTG1 and cyclins B1 and E, were reduced in betulin-treated ccRCC cells (FIG. 5A).
  • RNF20 can inhibit the growth of ccRCC cells through mediating SREBP1c control and regulating lipobiosynthesis and cell cycle regulatory gene expression.
  • PTTG1 inhibition did not alter the mRNA expression of SREBP1c or FASN (FIG. 6B), whereas ectopic SREBP1c expression did not affect cell-cycle regulators including PCNA, cyclin A, D1, and E in ccRCC cells and mRNA of PTTG1. Expression was promoted (FIGS. 6B and C). In contrast, inhibition of PTTG1 expression in ccRCC cells downregulated these cell-cycle proteins (FIG. 6C). In addition, inhibition of PTTG1 inhibited cell proliferation in both control and SREBP1c-overexpressing ACHN cells (FIG. 6D).
  • FIG. 6A we measured PTTG1 expression and cell proliferation in the presence or absence of the ACC inhibitor TOFA or FASN inhibitor C75 (FIG. 6A).
  • TOFA and C75 as shown in FIG. 6E reduced intracellular triglyceride accumulation in ACHN cells.
  • lipophilic activity by TOFA or C75 did not significantly affect the mRNA expression of PTTG1 or cell-cycle regulator PCNA, Cyclin A, D1 and E (FIG. 6F; FIGS. 14A and B).
  • siRNA-mediated inhibition of FASN did not significantly alter the mRNA levels of PTTG1 and cell-cycle regulatory genes (FIGS. 14D and E).
  • ccRCC cell proliferation was significantly reduced in both cases where FASN was C75-mediated pharmacologically inhibited or siRNA-induced FASN knocked down (FIGS. 14C and F). Furthermore, the pharmacogenetic inhibition of FASN reduced the effect of SREBP1c overexpression on cell growth in ACHN cells, meaning that adipose biosynthesis inhibition could attenuate ccRCC cell proliferation via the SREBP1c-dependent pathway ( 6G and F).
  • PTTG1 expression is inversely correlated with RNF20 expression, reflecting the regulation of SREBP1c in ccRCC tumor tissue (FIGS. 4J and 8) and RNF20 downregulation, in part, upwards PTTG1 Regulation promotes ccRCC development and progression.
  • siRNA knockdown of PTTG1 resulted in decreased mRNA expression of cell cycle regulatory genes without altering adibiosynthetic activity (FIGS. 6C and E).
  • PTTG1 inhibition weakened the effect of activated SREBP1c on ccRCC cell proliferation (FIG. 6D).
  • RNF20-SREBP1c-PTTG1 axis is the center of ccRCC cell proliferation and tumorigenesis.
  • SREBP1 regulates fat biosynthesis by significantly increasing new fat biosynthesis. Therefore, the present inventors tested whether liposynthesis is associated with PTTG1 expression in the presence of novel lipophilic genetic pharmacological inhibitors, and the mRNA expression of PTTG1 in ACHN ccRCC cells was evaluated by pharmacological inhibition of lipobiosynthesis using TOFA or C75. 6F) or siRNA mediated knockdown of FASN (FIG. 14D). This means that PTTG1 is induced independent of the mechanism of lipophilic activation by SREBP1c. Thus, SREBP1c can influence different lipogenesis and cell cycle progression by regulating different target gene families, ultimately promoting tumor development in ccRCC.
  • RNF20 attenuated mRNA expression of SREBP1c, cell-cycle regulators, and lipophilic genes in xenograft tumors (FIG. 7E).
  • H & E staining ACHN tumors with high RNF20 expression showed a reduced number of cells with clear cell morphology (FIG. 7F).
  • oil red O staining lipid accumulation was reduced by ectopic RNF20 expression (FIG. 7F).
  • cell proliferation was reduced in xenograft tumors by exogenous RNF20 expression in Ki67 staining assay (FIG. 7F).
  • apoptosis was induced in xenograft tumors by RNF20 overexpression in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (FIG. 7F).
  • TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling
  • mRNA expression of pro-apoptotic genes including Bax, Bid, and caspase-3, is increased after ectopic RNF20 expression, whereas anti-apoptotic genes Bcl-2, cIAP-2, and XIAP -MRNA expression of apoptotic genes was observed to decrease (Fig. 15).
  • results indicate that overexpression of RNF20 can inhibit the growth of cancer by inhibiting SREBP1c expression and adipose biosynthesis (FIG. 7).
  • results also indicate that redirection of RNF20 in ccRCC leads to increased expression of SREBP1c, which in turn leads to increased expression of PTTG1 and lipophilic genes in ccRCC (FIG. 8), leading to tumor growth and progression.
  • RNF20 acts as a tumor suppressor by inhibiting SREBP1c-mediated adipose biosynthesis and cell cycle regulation (FIG. 8).
  • the data indicate that RNF20 downregulation promotes tumorigenesis by activating SREBP1c in ccRCC tumors, and down expression of RNF20 may act as a marker of cancer.
  • SREBP1c induced a PTTG1 in ccRCC to identify a new mechanism for stimulating cell cycle progression, and RNF20 can regulate the SREBP1c-lipid biosynthesis axis and the SREBP1c-PTTG1 axis.
  • the present invention demonstrates that the new therapeutic approaches that target RNF20-SREBP1c-lipid biosynthesis axis and RNF20-SREBP1c-PTTG1 axis pathways in cancer and RNF20 are novel tumor suppressors in ccRCC. It is present.

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Abstract

La présente demande concerne l'utilisation du RNF20 à titre d'agent thérapeutique contre le cancer du rein ou du foie et à titre de marqueur pour diagnostiquer un cancer du rein ou du foie. Un procédé de criblage pour identifier des agents thérapeutiques contre un cancer associé à une anomalie du RNF20 à l'aide d'un nouveau mécanisme moléculaire, basé sur la voie de la lipogenèse RNF20-SREBP1c-PTTG1 et RNF20-SREBP1c, est en outre décrit.
PCT/KR2017/011172 2016-10-20 2017-10-11 Utilisation du rnf20 pour le diagnostic et le traitement du cancer du rein ou du foie et criblage pour identifier des agents thérapeutiques contre le cancer du rein ou du foie Ceased WO2018074770A1 (fr)

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