PRC2 Promotes DNA Double-strand Break Repair And Confers Chemo-Resistance In Ovarian Cancer Through Regulating BRCA1

ObjectivePolycomb repression complex 2?PRC2?inhibits gene expression through catalyzing tri-methylation of histone 3 at lysine 27?H3K27me3?by EZH2 that is a subunit of PRC2with histone methyltransferase activity.The purpose of this study was to investigate the role of PRC2 in BRCA1-associated DNA double-strand break?DSB?repair in ovarian cancer cells undergoing chemotherapy and the possible positive effects of PRC2 inhibitors on the response to cisplatin cDDP and PARP inhibitors in BRCA wild type ovarian cancers.Methods1.Epithelial ovarian cancer cell lines SKOV3 and OVCAR4?both are BRCA wild type?were exposed to cisplatin,and the DSB marker P-H2AX and histone markers H3K27me3,H3K4me3,H3K9me3,H3K36me3,H3K56ac,H3K362,H3K9me2 and EZH2 were detected by Western blotting?WB?.2.SKOV3 and OVCAR4 cell lines were treated with cisplatin or Saline.The binding and enrichment of H3K27me3,H3K4me3,H3K9me3,H3K36me3,H3K56ac,H3K36me2,H3K9me2,and EZH2 on the sites of DSB were detected by co-immunoprecipitation?Co-IP?.3.SKOV3 and OVCAR4 cell lines were pretreated with DMSO?control?or a PRC2inhibitor GSK126 for 48 hours followed by exposure to cisplatin for 24 hours.WB and qRT-PCR were used to detect the expression of BRCA1 protein and mRNA.4.CRISPR/cas9-sgRNA lenti-virus was used to knock down EZH2 in SKOV3 and OVCAR4 cells?SKOV3sg EZH2 and OVCAR4 sgEZH2?,and GSK126 was used to inhibit the methyltransferase activity of PRC2.The expression of H3K27me3 and EZH2 protein were detected by WB.5.Immunofluorescence assays were used to detect P-H2AX to evaluate the cisplatin-induced DSB in SKOV3 and OVCAR4 cells undergoing EZH2 inhibition.6.Flow cytometry was used to detect DR-GFP plasmid reporting system to evaluate the effect of PRC2 on homologous recombination repair.?I-Sec nuclease in DR-GFP reporter plasmid can cut DNA double strands and cause DSB.If the break can be repaired by HR?Homologous recombination repair?,the cells will express GFP?.7.SKOV3 and OVCAR4 cell lines were transfected with lenti-virus harboring siRNA targeting BRCA1?SKOV3siBRCA1 and OVCAR4siBRCA1?or pretreated with GSK126.The cytotoxicity of cDDP and cDDP combined with PARP inhibitor AZD?AZD2281,Olaparib?was assessed by flow cytometry assays for cell apoptosis and clony formation assays to evaluate whether GSK126 could achieve a cell-killing effect in BRCA^wt tumor cells similar to the synthetic lethality induced by genetic BRCA1 deficiency combined with PARP inhibitor.8.Flow cytometry cell apoptosis assays and colony formation assays were used to evaluate the effects of GSK126 on the sensitivity to cDDP and AZD in ovarian cancer cells.9.Flow cytometry cell apoptosis assays were used to evaluate the cytotoxicity of GSK126 and GSK126 combined with AZD in ovarian cancer cells.10.SKOV3 cells were subcutaneously injected into mice and the mice with established xenografts were randomly divided into four groups subjected to treatments as follows:?1?cDDP+solvent control,?2?cDDP+GSK126,?3?cDDP+AZD,or?4?cDDP+AZD+GSK126.Tumor diameter,body weight and mental state of the mice were monitored to evaluate the ani-tumor effects and the adverse effects of the regimens.11.The expression of EZH2,Ki67?nuclear proliferating antigen?,Cleaved-caspase 3?apoptosis marker?and H3K27me3 in the SKOV3 xenografts were detected by immunohistochemistry?IHC?to evaluate the anti-proliferation and pro-apoptotic effects of the drugs.Results1.Histone methylation modifications participate in DNA damage response induced by cisplatin.?1?P-H2AX,H3K27me3,H3K4me3,H3K9me3,H3K36me3,and H3K56ac were increased in ovarian cancer cells treated with cDDP in a time-dependent manner,but there were no significant changes in H3K36me2,H3K9me2 and EZH2 observed.?2?H3K27me3,H3K4me3,H3K9me3,H3K36me3,H3K56ac,H3K36me2,H3K9me2 and EZH2 were immunoprecipitated together with P-H2AX.2.PRC2 inhibitor antagonizes the up-regulation of BRCA1 mediated by cisplatin.The expression of BRCA1 protein was increased significantly in the parent ovarian cancer cells treated with cDDP,but not in the cells pretreated with GSK126 followed by cDDP administration.GSK126 alone did not affect the expression of BRCA1.3.Inhibition of PRC2 decrease the homologous recombination repair and increases DNA double-strand-break in ovarian cancer.?1?EZH2 and H3K27me3 levels were significantly decreased in the SKOV3 and OVCAR4 cells transfected with CRISPR/cas9-sgRNA targeting EZH2;GSK126 could decrease the expression of H3K27me3,but not influence the expression of EZH2.?2?The results of immunofluorescence assays showed that knocking down EZH2 or treatment with GSK126 could increase P-H2AX foci in ovarian cancer cells.?3?The results of DR-GFP plasmid reporting assays showed that knocking down EZH2 or treatment with GSK126 could inhibit the homologous recombination repair ability of ovarian cancer cells.4.Inhibition of PRC2 can increase the sensitivity of BRCA^wt ovarian cancer cells to cisplatin and Olaparib.?1?Compared with the control group,similar to the cells with BRCA1 deficiency induced by siRNA transfection,the cancer cells pretreated with GSK126 exhibited significantly increased sensitivity to cDDP and AZD as revealed by enhanced cell apoptosis and decreased colony formation.?2?Compared with cDDP alone,cDDP/GSK126 combination decreased the colony formation ability and increased the apoptosis rate in ovarian cancer cells;the cDDP/GSK126/AZD triple combination showed further enhanced pro-apoptotic effect and colony formation inhibition when compared to the cDDP/GSK126 and cDDP/AZD combinations.?3?GSK126 treatment alone or in combination with AZD had no obvious cytotoxicity to ovarian cancer cells.?4?The results of animal experiments showed that compared with the cDDP group the tumour volume of the triple combination group,the cDDP/GSK126 group and the cDDP/AZD group were decreased 92%,42%and 36%,respectively?P<0.01?,and there was no significant difference in the body weight of mouse among these groups.?5?IHC of the xenografts showed that H3K27me3 was significantly decreased in the groups treated with regimens containing GSK126.The triple combination group showed the lowest Ki67 and the highest cleaved-caspase 3 levels;the Ki67 was increased and cleaved-caspase 3 was decreased from the triple combination group,the cDDP/GSK126group,the cDDP/AZD group,to the cDDP group,ConclusionInhibition of PRC2 can antagonize the up-regulation of BRCA1 mediated by cisplatin in ovarian cancer cells,decrease homologous recombination,promote DSB,which in turn increase the sensitivity of BRCA^wtt ovarian cancer to cisplatin and Olaparib.