Metformin Regulates Epithelial-mesenchymal Transition And PD-L1 To Reverse PARP Inhibitors Resistance In Triple-negative Breast Cancer

Objective: Breast cancer has the highest incidence of all cancers in women worldwide and the leading mortality in 2018.Triple-negative breast cancer(TNBC)is the most aggressive subtype of breast cancer lacking estrogen and progesterone receptors and expressing low levels of HER2.The lack of these hormone receptors results in TNBC early relapse and metastasis.The treatment for TNBC is limited to surgery,chemotherapy and radiotherapy.Because TNBC frequently shows BRCA dysfunction and 80% of germline BRCA1 mutation the BRCA1/2 target therapy becomes a promising therapy of TNBC.Poly(ADP-ribose)polymerase(PARP)inhibitors have emerged as promising targeted therapies for BRCA-mutated cancers by blocking repair of DNA double-strand breaks and leading to synthetic lethality.However,resistance to PARP inhibitors have been described in some patients lowering overall response rates.The mechanisms underlying PARP inhibitor(PARPi)resistance are an area of active investigation.Methods: Using FDA approved PARPis(Olaparib and Rucaparib)to treat both PARPi-sensitive and PARPi-resistant TNBC cell lines,PARPi adaptive resistant clones were generated in triple-negative breast cancer cell lines.Through morphologic observation we observed the epithelial-mesenchymal transition(EMT)phenomenon.Through the floating cell assay,wound healing assay and Transwell invasion assay,we evaluated EMT function and changes in immune checkpoint programmed death-ligand 1(PD-L1).We also downregulated the expression of PD-L1 by shRNA to study the role of PD-L1 in PARPi resistance.Screening the TOPII inhibitors,Akt inhibitors,ERK inhibitors and metformin to reverse PARPi-induced resistance through western blot and cell culture.Results: 1 We demonstrated that inhibition of PARP reshaping the TNBC mesenchymal like,which induces fusiform and scattered cells.2 PARPi induced tumor cells expressing EMT markers by western blotting and qrt-PCR.3 PARPi treated tumor cells show stem like ability.4 PARPi induced EMT increases migration and invasion of tumor cells.5 PARPi treated cells upregulate the expression of PD-L1 by 2-3 fold in triple-negative breast cancer cells through phosphorylation of Akt at S473,and induce EMT.6 Metformin administration(10?M)was found to reverse EMT by blocking the p-Akt S473 axis through activation of AMPK,resulting in downregulation of PD-L1 expression and sensitizing PARPi-resistant cancer cells to cytotoxic T cells.7 In addition,PARPi–induced EMT occurred independent of PD-L1 upregulation in triplenegative breast cancer cells.Conclusions: In summary,we identified that induction of EMT and upregulation of PD-L1 are induced by using of PARPis,which is a new mechanism for PARP inhibitor resistance.Metformin was able to reverse both the PARPi-induced EMT and the upregulation of PD-L1 through inactivation of p-Akt.Besides,PARPi-induced EMT is an independent process of regulation of PD-L1.Metformin is able to reverse the PARPi induced adaptive resistance.Therefore a combination of metformin and PARP inhibitors may be a promising therapeutic strategy to increase the efficacy of PARP inhibitors and tumor sensitivity to immunity therapy.