IDH1R132H Promotes Malignant Transformation Of Benign Prostatic Epithelium And Its Potential Underlying Mechanisms

Prostate cancer(PCa)is the second leading malignancy in males and the fourth most common tumor type worldwide.In the current era of PSA(Prostate specific antigen)screening,nearly 90%of prostate cancers are clinically localized at the time of their diagnosis.But,this remains controversial due to the high rate of overdiagnosis and unnecessary prostate biopsies,despite evidence that it reduces mortality.With the increase of incidence,prostate cancer is becoming a serious threat to the health of old men disease.One of the major challenge for the clinical treatment is how to distinguish indolent from aggressive turner.Recent risk stratification systems combining the best currently available clinical and pathological parameters(such as Gleason score,PSA levels,clinical and pathological staging)have been developed.However,these tools still can not adequately predict outcome.Further risk stratification using molecular features to potentially help distinguish indolent from aggressive prostate cancer and guide selection of more precisely targeted therapeutic interventions will be important for prostate cancer.Isocitrate dehydrogenases(IDH)is a key homodimeric enzymes catalyze a redox reaction that converts isocitrate to a-ketoglutarate while reducing NADP to NADPH and liberating C02.Mutations in IDH has been identified in many human malignancies.Mutant IDH1 cause alterations in cellular metabolism,histone demethylation and DNA modification.Recently,researchers have proposed molecular taxonomy according to gene fusions,mutation and epigenetic alternations in prostate cancer.IDH1 mutation represents a methylator subtype and possesses fewer other common canonical genomic lesions in prostate cancers.IDH1R132H is the most common type.But,it is largely unknown in prostate cancer.So it will be significant to pay more attention to mutant IDH1 in prostate cancer.In this study,we show that expression of the IDH1R132H was noted in 0.6%prostate cancer patients.The in vitro experiment suggested that overexpressed IDH1R132H can promote non-transformed prostate cells(RWPE-1)proliferation under the situation of low cytokine,and migration.Mechanistically,IDH1R132H was a potent regulator promotes IGF 1R--AKT/STAT3 activation by downregulating a set of microRNAs.These microRNAs(miR-141-3p,miR-7-5p,miR-223-3p)negatively regulate IGF1R were repressed by the alteration of epigenetic modification—decrease the enrichment of active marker H3K4me3,or increase repressive marker H3K27me3 at their promoters.This is the first time to systematically explore the role and mechanism of IDH mutations in prostate cancer.Objects:1.To analyze the incidence rate and Clinicopathological characteristics of IDH1R132H in PCa patients.2.To study the function of IDH1R132H in PCa cells and non-transformed prostate epithelial cell lines.3.To explore the underlying mechanism of malignant transformation of non-transformed prostate epithelial cells.Methods:1.Mutational analysis of IDH1R132H in prostate cancer patients.1.1 Immunohistochemistry(IHC)assay was used to detect IDH1R132H patients.1.2 For immunohistochemistry positive cases,DNA was extracted from paraffin embedded tissue,PCR-based sequencing was used to confirm it.1.3 PCR-based sequencing was used to analyze IDH1R132H in prostate cancer cell lines.2.The function of IDH1R132H in prostate cancer and benign cell lines.2.1Lentiviral infection was used to construct and puromycin was used for selecting stable expressed cell lines.Western and RT-qPCR were used to confirm transfection efficiency.2.2 MTS assay were used to identify the capability of proliferation.2.3Transwell and wound healing assay were used to detect the capability of cell migration.2.4 RT-qPCR was used to determine the expression of associated stem cell marker.2.5 MTS assay and transwell assay were used to identify the capability of proliferation and migration after AGI5198 treatment,respectively.3.Mechanism of malignant transformation of non-transformed prostateepithelial cells.3.1 The altered microRNAs in IDH1R132H.1)MicroRNA array was used to screening the changed microRNAs.2)RT-qPCR was used to confirm the altered microRNAs in IDH1R132H.3)Chromatin immunoprecipitation(ChIP)was used to determine the enrichment of H3K4me3,H3K27me3 at the promoter of microRNAs.3.2 The altered target gene of microRNAs in IDH1R132H.1)TargetScan was used to forecast the target genes of changed microRNAs.2)Western Blot and Immunofluorescence were used to detect the protein level of IGF1R.3)Western Blot was performed to detect the IGF1R in RWPE-1 after transfection with mimic/inhibitor or their controls of microRNAs.4)PmirGLO vector carried wild type or mutant 3'UTR of IGF 1R gene,mimic of microRNA were co-transfected in HEK293T cells.Cells were harvested 48h after transfection.Dual-Luciferase Report Assay System was used to detect the renilla and firefly luciferase activity.5)Gene Expression Microarrays was used to determine the altered gene,bioinformatics was used to analyze IGF1R associated genes.3.3 IGFIR is required for IDH1R132H mediated malignant transformation.1)Western Blot was conducted to detect AKT,STAT3 protein levels.2)Western Blot were used to detect AKT,STAT3 protein levels after knocking down IGF1R.3)MTS assay and transwell assay were used to identify the capability of proliferation and migration after knocking down IGF1R.Results:1.The analysis of IDH1R132H in prostate cancer patients.1.1 Immunohistochemical staining about IDH1R132H,the most common mutation,were performed in PCa samples.Totally,four TMAs including 336 samples were tested.15 were immunopositive using the IDH1R132H MAb,of the 15 cases,4 showed strong staining,11 showed weak staining.The staining was both nuclear and cytoplasm.1.2 PCR-based sequencing confirm IDH1R132H positive rate is 0.6%(2/336).IDH18132-mutant tumors possess fewer other canonical genomic lesions that are common to most other prostate cancers.1.3 Besides,we also tested the mutation situations of prostate cancer cell lines RWPE-1,LNCAP,VCAP,22RV1.No IDH1 mutations were found in these cell lines.2.IDH1R132H promotes prostate non-transformed epithelial cell RWPE-1 malignant transformation.2.1 RWPE-1 cells expressed IDH1R132H have obvious growth advantage in the absence of cytokine,wound healing assay and transwell assays demonstrated that IDH1R132H promoted migration ability of RWPE-1cells.2.2IDH1R132H decreased proliferation and migration in LNCAP cells.2.3 RT-qPCR revealed that IDH1R132H promotes CD133 expression at both RWPE-1 and LNCAP cells.2.4 AGI-5198 treatment at 20 ?M inhibited proliferation in the absence of cytokine in IDH1R132H-expressing RWPE-lcells.Transwell assay also show that AGI-5198 signficantly inhibit migration3.Mechanism of malignant transformation of normal prostate epithelial cells.3.1 IDH1R132H downregulates miR-141-3p/miR-7-5p/miR-223-3p in Prostate Cells1)MicroRNA array shown,together analyzing IDH1R13 2H/WT and IDH1R132H/VECTOR,we identified 4 and 68 microRNAs that were overlapped upregulated or down-regulated in IDH1R132H.By reading literature,we chosed 9 microRNAs of 68 overlapped down-regulated in IDH1R132H and analyzed their expression in stable infected RWPE-1 cell line.MiR-223,miR-141 and miR-7-1 were the most obvious downregulated microRNAs.2)Our data show that H3K4me3 was specifically reduced at the P3,P4 promoter of miR-141,P2,P3,P4 promoter of miR-223 and P2,P4 promoter of miR--7-1 in R132H RWPE-1cells.H3K27me3 was increased at P2 promoter of miR-223 and P2 promoter of miR-7-1.3.2 IDH1R132H-downregulated microRNAs lead to increased IGF1R expression1)TargetScan prediction software was used to the potential target of the microRNAs is IGF1R.2)Western Blot and Immunofluorescence show that IDH1R132H enhanced IGF1R protein expression compared with IDH1WT and VECTOR.3)Exogenous mimics of miR-141-3p,miR-7-5p,miR-223-3p reduced IGF1R expression in protein level in IDH1R132H-RWPE-1cells.4)HEK293T cells were co-transfected with a pGL3-promoter vector carrying the wt or mutant IGF1R 3'UTR and corresponding mimics of miR-141-3p,miR-7-5p,miR-223-3p,respectively.The relative LUC activity levels was inhibited by mimics of the microRNAs,such effects were not observed with the mutant construct of IGF1R3'-UTR.5)Gene Set Enrichment Analysis(GSEA)indicated that genes signatures of IGF1R up-regulated(GSE5225)were significantly enriched in R132H RWPE-1 cells.3.3 IGF1R is required for IDH1R132H mediated malignant transformation.1)p-AKT and p-STAT3 were up regulated in IDH1R132H RWPE-lcells.2)The protein level of P-AKT,P-STAT3 were decreased after knocking down IGF1R in IDH1R132HRWPE-1 cells.3)The ability of migration and proliferation of IDH1R132H RWPE-1 cells were decreased after knocking down IGF1R.Conclusions:1.The incidence rate of IDH1R132H in prostate cancer patients is 0.6%.IDH1 R132-mutant tumors possess fewer other canonical genomic lesions that are common to most other prostate cancers.2.IDH1R132H can promote non-transformed prostate cells(RWPE-1)malignant transformation---proliferation under the situation of low cytokine,and migration.3.IDH1R132H promotes IGF 1R--AKT/STAT3 activation by downregulating miR-141-3p,miR-7-5p,miR-223-3p.