EZH2 Induces The Cisplatin Resistance In Ovarian Cancer Stem Cells Through CHEK1 Pathway

Objective Our preliminary results showed that EZH2(enhancer of zeste homolog 2)played an essential role in maintaining the stemness of ovarian cancer cells and was associated with cisplatin resistance in ovarian cancer stem cells(CSCs).A RT2 Profiler PCR array analysis revealed that CHEK1(checkpoint kinase 1)might be a key downstream factor that mediates the contribution of EZH2 to cisplatin resistance in CSCs.The aim of this study was to investigate whether EZH2 promotes cisplatin resistance in CSCs through CHEK1.Methods1.Overexpression plasmids and lentiviral CRISPR/Cas9 sgRNAs were used to up-regulate and knock-down EZH2 expression in an ovarian cancer cell line SKOV3 and its stem cell subline SK3rd(CSCs derived from SKOV3 by in vivo cisplatin administration combined with in vitro culture of cell spheroids),respectively.2.After transfection with EZH2 overexpression plasmids or sgRNAs,the expression levels of EZH2 and CHEK1 were detected by using western blotting and quantitative real-time PCR(qRT-PCR)assays.3.Dual luciferase reporter assays were used to detect the transcriptional activity at the CHEK1 promoter region in SKOV3 and A2780 cells with up-or down-regulated EZH2 levels.4.Chromatin immunoprecipitation assays were used to detect the direct interaction between EZH2 and the promoter region of CHEK1 in SKOV3 and A2780 cells.5.An overexpression plasmid was used to up-regulate the CHEK1 expression in the SK3rd-sgEZH2 cells(SK3rd-sgEZH2/CHEK1).Western blotting assays were used to confirm the manipulation of EZH2 and CHEK1 expression.6.The sphere formation ability of ovarian cancer cells was measured by serum-free suspension culture conditions.7.After treated with cisplatin,the levels of p-CHEK1,p-CDC25 C and yH2 AX in ovarian cancer cells were detected by using western blotting.8.Flow cytometry was used to detect cell cycle distribution.9.The apoptosis rates were detected by using flow cytometry.10.MTT and colony formation assays were used to evaluate the cytotoxicity of cisplatin in CSCs.11.Subcutaneous xenograft mouse models derived from SK3 rd cells used to compare the anti-tumor effects of intraperitoneal administration of cisplatin,cisplatin combined with an EZH2 inhibitor DZNEP,cisplatin combined with a CHEK1 inhibitor AZD7762,and cisplatin combined with both DZNEP and AZD7762(the triple therapy group).12.Immunohistochemistry was used to detect the levels of EZH2,p-CHEK1,Ki67,Cleaved-caspase 3,and γH2AX in the xenografts.Results1.The EZH2 overexpression in ovarian cancer cells significantly promoted the CHEK1 expression,and vice versa.2.The results of dual luciferase reporter assays showed that EZH2 overexpression stimulated the transcription of CHEK1 in SKOV3 and A2780 cell lines,and vice versa.3.The chromatin immunoprecipitation assays revealed that EZH2 bound to the CHEK1 promoter region.4.In the SK3 rd cells,the EZH2 knockdown induced by the CRISPR/Cas9 sgRNAs and the CHEK1 overexpression induced by expression plasmids were confirmed.5.Knockdown of EZH2 expression in SK3 rd cells significantly decreased the spheroid formation capacity;while upregulation of CHEK1 expression in SK3rd-sgEZH2 cells significantly increased the spheroid formation capacity.6.Western blotting results showed that the phosphorylation level of CHEK1 and CDC25 C in SK3 rd cells treated with ciplatin were significantly increased,while yH2 AX level was not detectable or only slightly evident,showing a considerable increase in the DNA damage response.However,compared with SK3 rd cells,down-regulation of EZH2 in SK3 rd cells can significantly inhibit the DNA damage response;Interestingly,CHEK1 overexpression rescued the sgEZH2-induced inhibition of DNA damage response in the SK3 rd cells exposed to cisplatin.7.The results of flow cytometry showed that treatment of cisplatin in SK3 rd cells resulted in a considerable decrease in the proportion of G1 phase cells,while the proportions of S and G2/M phase cells were significantly increased.However,compared with SK3 rd cells,down-regulation of EZH2 can inhibit the cell cycle arrest.Interestingly,CHEK1 overexpression rescued the sgEZH2-induced reduction of the cell cycle arrest in the SK3 rd cells exposed to cisplatin.8.The results of flow cytometry analyses showed that CHEK1 overexpression rescued the sgEZH2-induced reduction of apoptosis rate in the SK3 rd cells exposed to cisplatin.9.The MTT and colony formation assays showed that the EZH2 knockdown increased the cytotoxicity of cisplatin in the SK3 rd cells,while the CHEK1 overexpression rescued the sgEZH2-induced cisplatin resistance.10.The in vivo experiments revealed that the tumor volumes were decreased by 26%(P=0.25),47%(P=0.057),81%(P<0.001)and the tumor weights were decreased by 33%(P=0.34),49%(P=0.045),72%(P<0.001)in the cisplatin/DZNEP group,the cisplatin/AZD7762 group,and the triple therapy group,respectively,when compared to the cisplatin group.11.The immunohistochemistry staining of the xenografts showed that theintraperitoneal administration of DZNEP and AZD7762 could effectively decrease the expression of EZH2 and pCHEK1 in vivo and enhance the anti-proliferation and pro-apoptosis effects of cisplatin.Conclusion EZH2 induces the cisplatin resistance in ovarian cancer stem cells by directly binding to and activating the transcription of CHEK1,and therapies targeting EZH2 and CHEK1 can resensitize the CSCs to cisplatin chemotherapy,which might provide new ideas for the treatment of chemo-resistant ovarian cancer.