Role And Mechanistic Study Of Downregulating PKM2 On Increasing Sensitivity Of Olaparib To BRCA1 Wild-type Ovarian Cancer

Purpose: Poly(ADP-ribose)polymerase(PARP)inhibitors have shown significant clinical benefits as maintenance therapy in advanced homologous recombination(HR)deficiency ovarian cancer by impairing repair of DNA.Recent studies have shown that Pyruvate kinase M2(PKM2)integrates with DNA damage by directly promoting DNA double-strand break repair.However,whether PKM2 regulates PARP inhibitor’s sensitivity of ovarian cancers remains to be elucidated.In this study,si RNA and small molecule inhibitor are used to down-regulate PKM2.The effects and mechanisms of downregulating PKM2 on the sensitivity of olaparib to ovarian cancer will be explored at the cellular and animal levels.Methods: Three types of ovarian cancer cells(A2780,SKOV3 and OVCAR3)were treated with a combination of PKM2-targeting si RNA,PKM2 inhibitor shikonin and olaparib.The expression of PKM2,p-ATM,γH2AX and BRCA1 proteins were detected to investigate the molecular mechanism by western blotting and immunofluorescence.Inhibitory efficiencies were determined in ovarian cancer cell by MTT,clone formation,transwell assay and flow cytometry.Xenograft animal models were performed by treating with a combination of shikonin and olaparib.Combined effects were determined using tumor volume,tumor weight analysis and tumormarker detection.Body weight measurement and main organ(liver and kidney)tissues pathological analysis were used to assess drug toxicity.The expressions of PKM2,γH2AX and BRCA1 were detected by immunochemistry.Results: At the protein level,PKM2-targeting si RNA or PKM2 inhibitor shikonin significantly activated the expression of p-ATM andγH2AX and inhibited the expression of HR repair protein BRCA1.Furthermore,immunofluorescence experiments showed that si RNA/shikonin amplified olaparib-induced γH2AX and p-ATM activation and interfered with BRCA1 accumulation at nucleus.More importantly,si PKM2/shikonin synergized with olaparib in reducing cell growth,colony formation,migration and inducing cells apoptosis.In vivo experiments showed that,treatment of shikonin in xenograft nude mice profoundly enhanced the efficacy of olaparib and accompanied by increasing γH2AX expression and reducing BRCA1 and PKM2 expression.Conclusions: Downregulating PKM2 promotes DNA double-strand breaks and disrupt HR pathways.Downregulating PKM2 amplifies olaparib-induced γH2AX and p-ATM activation.Either si PKM2 or inhibitor shikonin enhances anti-cancer activity of olaparib in ovarian cancer cells.This study may provide a new strategy for improving the efficacy of olaparib.