EZH2 Enhances Ovarian Cancer Cisplatin Resistance By Mediating Cisplatin Induced CTR1 Degradation

Part OneEZH2 is associated with cDDP resistance in ovarian cancerObjectiveTo evaluate the association between EZH2 and ovarian cancer cisplatin resistance.Methods1?Compare the EZH2 level in tumor tissues from ovarian cancer patients sensitive or resistant to platinum-based chemotherapy using tissue micro-array and immunohistochemistry.2?Generate ovarian cancer resistant cell lines by repeated cDDP exposure(12 cycles),test cell cisplatin resistance using colony formation assay,measure the EZH2 protein dynamic change by WB.3?Transfect A2780 and ES2 cells with specific EZH2 lenti-virus shRNA(ShEZH2 vs ShNC),verify EZH2 protein down-regulation by WB and mRNA down-regulation by qRT-PCR.Test cellular sensitivity to cDDP after EZH2 depletion by MTT assay.4?Transfect ES2 cells with lenti-virus EZH2 over-expression plasmid(EZH2-08,EZH2-13 and EZH2-NC),verify EZH2 protein up-regulation by WB and mRNA up-regulation by qRT-PCR.Test cellular sensitivity to cDDP after EZH2 up-regulation by MTT assay.Results1?The tissue microarray and immunohistochemistry assays revealed that EZH2 is generally expressed in all the ovarian cancer tissues.EZH2 staining in the chemo-resistant group was much stronger than that in the chemo-sensitive group.2?We generated two cDDP-resistant cell sublines A2780C12 and ES2C12,as confirmed by colony formation assay,by exposing A2780 and ES2 parental cells to 12 cycles of cDDP treatment,respectively.A progressively increased expression of EZH2 during generation of both A2780C12 and ES2C12 was observed.3?Down-regulation of EZH2 dramatically increased the sensitivity of A2780 and ES2 cells to cDDP.4?Up-regulation of EZH2 decreased the sensitivity of ES2 cells to cDDP.ConclusionWe established a significant association between EZH2 and cDDP resistance based on generation of cellular cDDP-resistant models,the analysis of patient tissue samples and EZH2 expression modification assays.Part TwoEZH2 affects intracellular platinum accumulationObjectiveTo examine the effect of cellular EZH2 level on intracellular platinum accumulation.Methods1?Expose the shEZH2 and ShNC cells of A2780 and ES2 to cDDP treatement,submit the cells for cellular platinum measurement by GFAAS to evaluate cellular platinum accumulation,platinum influx and platinum efflux.2?Generate non-GFP tagged A2780 and ES2 cells with EZH2 depletion using liposomal mediated siRNA transfection.verify EZH2 protein down-regulation by WB and mRNA down-regulation by qRT-PCR.Measure cellular BODIPY-Pt(a BODIPY tagged cDDP analog,green)accumulation and influx by fluorescent microscopy.3?Measure cellular BODIPY-Pt influx and BODIPY-Pt/CTRl co-localization using confocal fluorescent microscopy.Results1?Platinum content was significantly higher in the EZH2 downregulated ShEZH2 cells compared to ShNC in both the A2780 and ES2 cell lines.Cellular platinum influx increased after EZH2 depletion while platinum efflux remained unchanged.2?EZH2 down-regulation by non-fluorescent tagged siEZH2 transfection in A2780 and ES2 cells was verified by WB and qRT-PCR.A fluorescence-modified cDDP analog,BODIPY-Pt,was used to track the process of cellular cDDP accumulation.Significant increases in intracellular BODIPY-Pt accumulation were observed in cells transfected with siEZH2 when compared with the siNC,which was remarkably consistent with the results from the platinum accumulation assessment in cells exposed to cDDP.EZH2 depletion increased cellular BODIPY-Pt influx3?Confocal fluorescent microscopy showed cellular CTR1 co-localized perfectly with BODIPY-Pt.ConclusionEZH2 is involved in cellular platinum accumulation and mainly affects drug influx.Part ThreeCTR1 is associated with ovarian cancer cDDP resistanceObjectiveTo evluate the association between cDDP transporter copper transporter 1(CTR1)expression and cDDP resistance.Methods1?Evaluate the relation of CTR1,copper transporter 2(CTR2),copper-transporting p-type adenosine triphosphatase 1 and 2(ATP7A and ATP7B)to overall survival(OS),progression-free survival(PFS)/disease-free survival(DFS)and treatment response(TR)of cancer patients who received chemotherapy:Literature and dataset search was perfomed using Pubmed,EMBASE,Wanfang Database,the Gene Expression Omnibus(GEO)and the Cancer Genome Atlas(TCGA)datasets.The inclusion criteria for identifying eligible studies included:(1)clinical studies measured CTR1,CTR2,ATP7A or ATP7B in solid tumors regardless of detection methods;(2)published prospective and retrospective cohort studies;(3)studies provided information of survival and chemotherapy response.The inclusion criteria for eligible datasets included:(1)cancer patients received chemotherapy;(2)the sample size should be more than 40;(3)gene array was performed using Affymetrix platform.Literuatre exclusion criteria included:(1)the patients enrolled did not receive chemotherapy;(2)studies focused on gene polymorphisms or DNA methylation;(3)studies investigated different end-points;(4)studies did not provide sufficient data.Hazard ratios(HRs)were pooled to evaluate OS and PFS,odds ratios(ORs)were pooled to evaluate TR using random-effect models.Subgroup analysis and sensitivity analysis were conducted;heterogeneity and publication bias were assessed.Results1?Results of meta-analysis:(1)Electronic search:Twelve literatures and eight datasets with 2149 patients were included.(2)Meta-analysis:High CTR1 expression was associated with favorable OS,PFS,DFS and TR in cancer patients who underwent chemotherapy with acceptable heterogeneity.(3)Subgroup-analysis:The relationship of CTR1 to cancer prognosis remained significant in the subgroup of patients who underwent platinumbased chemotherapy,the patients with ovarian cancer and those with lung cancer.The significance of these relationships was not influenced by geological region of publication,data origin or detection method.(4)No significant correlation was found between CTR2,ATP7A or ATP7B to OS,PFS,DFS or TR.(5)Test of publication bias and sensitivity analysis suggested a robustness of all the summary effect estimates.2?The tissue microarray and immunohistochemistry assays revealed that CTR1 is generally expressed in all the ovarian cancer tissues.CTR1 staining in the chemo-sensitvie group was much stronger than that in the chemo-resistant group.3?Cellular CTR1 expression in A2780 and ES2 went through a time-dependent decrease when exposed to cDDP as measured by WB.cDDP induced CTR1 degradation went to a greater extend in cDDP resistant cell ACP comparing to its sensitive counterpart A2780.ConclusionCTR1 expression level and cDDP induced CTR1 degradation were associated with ovarian cancer cDDP resistance.Part FourEZH2 accelerates cDDP-induced proteasomal CTR1 degradationObjectiveTo determine the underlying mechanism for EZH2 mediated cellular cDDP accumulation and investigate the influence of EZH2 expression level on cDDP-induced CTR1 degradation.Methods1?Down-regulate EZH2 gene expression using specific EZH2 lenti-virus shRNA in A2780 and ES2,measure cellular H3K27Me3 and CTR1 protein alteration by WB.2?Down-regulate EZH2 gene expression using specific EZH2 lenti,virus shRNA in A2780 and ES2,inhibit EZH2 function by specific EZH2 inhibitor GSK126 and DZNEP,compare cDDP induced CTR1 degradation before and after EHZ2 depletion using EZH2 specific shRNA and EZH2 inhibitors by WB.3?Down-regulate EZH2 gene expression using specific EZH2 lenti-virus shRNA in A2780 and ES2,inhibit protein synthesis by CHX,measure CTR1 stability before and after EHZ2 depletion by WB.4?Down-regulate EZH2 gene expression using specific EZH2 lenti-virus shRNA in A2780 and ES2,inhibit protein synthesis by CHX,measure cDDP induced CTR1 degradation before and after EHZ2 depletion by WB.5?Expose A2780 and ES2 to cDDP treatment,inhibite protein degradation by MG132,down-regulate cellular EZH2 gene expression using specific EZH2 lenti-virus shRNA,immuno-precipitate CTR1 with cDDP exposure,analyze CTR1 ubiquitination by WB before and after EZH2 depletion.6?Identify cDDP induced CTR1 degradation associated proteins mediated by EZH2 with LC/MS.Results1?Cellular CTR1,CTR2,ATP7A and ATP7B remained unchanged in EZH2 depleted A2780 and ES2 cells.2?Either depletion of EZH2 gene expression by lenti-virus shRNA transfection or inhibition of EZH2 function by specific inhibitors alleviated cDDP induced CTR1 degradation.3?The half-life of CTR1 in A2780 and ES2 cells remained similar before and after EHZ2 depletion mesaured by WB.4?Cellular EZH2 depletion alleviated cDDP-induced CTR1 degradation when exposed to cDDP after cellular protein synthesis inhibition by CHX.5?cDDP exposure significantly increased cellular CTR1 mono-and poly-ubiquitination level in A2780 and ES2.EZH2 depletion reduced cDDP mediated CTR1 ubiquitination.6?The proteins identified by LC/MS from ES2ShNC-CDDP and ES2ShEZH2-CDDP were mainly proteins from translation,protein folding,transport,organelle targeting and metabolic pathways.ConclusioncDDP is responsible for CTR1 proteasomal degradation,EZH2 accelerate cDDP induced CTR1 ubiquitination.