FEN1 And Mutant P53 (R273H) Regulate Sensitivity Of PARP Inhibitors In BRCA Wild-type Triple Negative Breast Cancer

Objective: Breast cancer is the most common female malignant tumor which threatens the health of women seriously.Although early breast cancer patients receive standard treatment after operation,disease progression or relapse either occurred in most patients.Poly(ADP-ribose)polymerase(PARP)inhibitors are the first clinically approved drugs designed to exploit synthetic lethality,and were first introduced as a cancer-targeting strategy in 2005.Clinical studies have confirmed the validity of the synthetic lethality approach and two PARP inhibitors olaparib and talazoparib have been approved as monotherapies for BRCA-mutated HER2-negative metastatic breast cancer.As mentioned above,BRCA mutation remains the strongest biomarker of sensitivity to PARP inhibitors.However,BRCA-mutated tumors represent 5.3% of all breast cancers and 11.2% of triple negative breast cancers,which might restrict the therapeutic utility of PARP inhibitors monotherapy.In addition to BRCA mutation,PARP inhibitors and other genes in the DNA damage repair pathway also have synthetic lethality.For BRCA wild-type breast cancer,PARP inhibitors,in combination with drugs that target other DNA repair genes,may increase the efficacy of PARP inhibitors.In the first part of this study,low expression of FEN1 was found to be a marker of sensitivity to PARP inhibitors in BRCA wild-type breast cancer,while high expression of FEN1 decreased the sensitivity to PARP inhibitors.To investigate the mechanism of FEN1 in regulating the sensitivity to PARP inhibitors,we used MDA-MB-231 and MDA-MB-468 as models of BRCA wild-type triple negative breast cancer cell lines.In the second part,we found that the mutant p53 breast cancer cells were not sensitive to PARP inhibitors.We used the mutant p53 breast cancer cells MDA-MB-231,MDA-MB-468 and p53 wild-type breast cancer cells MCF-7 as models,to investigate the mechanism of mutant p53 in regulating the sensitivity to PARP inhibitors.Methods: 1.Download the TCGA and GEO breast cancer datasets to analyze gene function;2.Construction FEN1 silencing lentivirus plasmids which were stably transfected into MDA-MB-231 and MDA-MB-468 cells;3.Lipofectamin 2000 and si RNA transfection were used to silence target genes such as FEN1,PD-L1,p53 and FOXM1;4.Lipofectamin 2000 and c DNA plasmids transfection were performed to overexpress target genes such as mutant p53(R273H);5.Cell viability was measured by MTT assay;6.Cell cycle was evaluated by flow cytometry with PI(Propidium Iodide)staining;7.Cell apoptosis was evaluated by flow cytometry with Annexin V-FITC staining;8.Screening of proteins binding to FEN1 or PD-L1 by mass spectrometry;9.Formation of protein complex with FEN1 or PD-L1 were demonstrated by immunoprecipitation;10.Expression of FEN1,ATR,Top BP1,Chk1,p Chk1,RAD51,PD-L1,TOP2 A,p53?p-p53,FOXM1,BRCA2 and GAPDH proteins were analyzed by Western blot;11.Relative levels of ATR,Top BP1,Chk1,RAD51,PD-L1,BRCA2 and18 S m RNA were measured by RT-q PCR;12.All experimental data were expressed as mean ± SD and were analyzed by t test using Prism 7.0 software.P < 0.05 was considered statistically significant.Results: 1.High expression of FEN1 in triple negative breast cancer cells was less sensitive to PARP inhibitors.The expression of FEN1 was negatively correlated with HRD score,while low expression of FEN1 predicted sensitivity to PARP inhibitors.MTT assay showed that MDA-MB-231 cells with lower FEN1 expression were sensitive to olaparib,while MDA-MB-468 cells with higher FEN1 expression were less sensitive to olaparib.2.Inhibition of FEN1 increases the sensitivity of breast cancer cells to PARP inhibitors.MDA-MB-231 and MDA-MB-468 cells were transfected with lentiviral vectors to silence the expression of FEN1 protein.The results of MTT assay showed that the inhibitory effect of olaparib on the proliferation of FEN1-silenced cells was significantly enhanced.Compared with olaparib alone,olaparib combined with FEN1 inhibitor C20 can further inhibit cell activity.Calcusyn analysis of cell growth curve indicates that the combination of the two drugs has synergistic anti-proliferation effect.3.Inhibition of FEN1 increases apoptosis induced by PARP inhibitors.The FEN1 protein was down-regulated by FEN1 si RNA.Flow cytometry showed that olaparib increased apoptosis in MDA-MB-468 cells interfered by FEN1 si RNA.Compared with olaparib alone,olaparib combined with FEN1 inhibitor C20 could also increase apoptosis of MDA-MB-468 cells.4.Inhibition of FEN1 reduces G2/M phase arrest induced by PARP inhibitors.Flow cytometry showed that olaparib induced G2/M phase arrest in MDA-MB-231 and MDA-MB-468 cells.Si RNA down-regulated the expression of FEN1 protein or FEN1 inhibitors C20 reduced the G2 M phase arrest induced by olaparib.5.Inhibition of FEN1 reduces ATR/Chk1 activation induced by PARP inhibitors.IPA analysis suggested that FEN1 was involved in the regulation of G2/M DNA damage checkpoint.In MDA-MB-231 and MDA-MB-468 cells,Western blotting showed that olaparib activated the ATR/Chk1 pathway and promoted the expression of RAD51 involved in homologous recombination.Lentivirus silencing FEN1 protein expression or FEN1 inhibitor C20 inhibited olaparib-induced Chk1 activation.The results showed that inhibition of FEN1 increased the sensitivity of olaparib by inhibiting the activation of ATR/Chk1 pathway and thus the homologous recombination repair.6.FEN1 forms a complex with ATG5 to activate autophagy and modulate Chk1 activation.Mass spectrometry indicated that FEN1 was bound to ATG5,and co-immunoprecipitation confirmed that FEN1 and ATG5 could form complex in MDA-MB-231 and MMB-468 cell lines.The results of Western blotting showed that the expression of LC3 II decreased after silencing of FEN1 or FEN1 inhibitor C20,which demonstrated that FEN1 inhibited autophagy and ultimately inhibited ATR/Chk1 pathway activation.7.Down-regulation of PD-L1 m RNA and protein levels by FEN1 inhibition.The expression of PD-L1 protein in MDA-MB-231 and MDA-MB-468 cells was detected by Western blotting.The results showed that PD-L1 protein was overexpressed in MDA-MB-231 cells,but not in MDA-MB-468 cells.RT-q PCR and Western blot showed that after FEN1 protein was down-regulated by si RNA or C20,PD-L1 m RNA and protein levels were significantly down-regulated in MDA-MB-231 cells.8.Down-regulation of PD-L1 protein increases apoptosis induced by PARP inhibitors.Western blotting showed that the expression of PD-L1 increased after olaparib treated MDA-MB-231 cells.Flow cytometry showed that olaparib increased apoptosis in PD-L1 down-regulated MDA-MB-231 cells.Co-immunoprecipitation showed that PD-L1 could form complex with TOP2 A,and the complex of PD-L1 and TOP2 A increased after olaparib treated.The results of Western blotting showed that the level of TOP2 A protein in MDA-MB-231 cells decreased significantly after down-regulation the expression of PD-L1 protein,and the decrease of TOP2 A protein was reversed when MG132 was added into MDA-MB-231 cells.It is suggested that PD-L1 and TOP2 A form a complex to stabilize TOP2 A protein.The results suggest that PD-L1 was resistant to olaparib-induced apoptosis by forming a complex with TOP2 A.9.Down-regulation of mutant p53(R273H)protein increases sensitivity to PARP inhibitors.MDA-MB-231 and MDA-MB-468 cells were transfected with p53 si RNA to down-regulate the expression of p53 protein.MTT assay showed that down-regulating the expression of p53 protein in MDA-MB-231(R280K mutation)cells did not change the sensitivity to olaparib,however,in MDA-MB-468(R280K mutation)cells,the down-regulation of p53 protein expression increased the sensitivity to olaparib,cell apoptosis also increased.10.Up-regulation of mutant p53(R273H)reduces the sensitivity to PARP inhibitors.MTT assay showed that up-regulation of mutant p53(R273H)in MCF-7 cells decreased the sensitivity to olaparib.11.Mutant p53(R273H)upregulates FOXM1 expression.Western blotting showed that down-regulating p53 protein expression did not change FOXM1 protein expression in MDA-MB-231(R280K mutation)cells,however,FOXM1 protein was down-regulated in MDA-MB-468(R273H mutation)cells.The overexpression of P53(R273H)was confirmed by Western blotting and the expression of FOXM1 was up-regulated.Western blotting showed that the expression of FOXM1 increased after up-regulation of mutant p53(R273H)in MCF-7 cells.12.Down-regulation of FOXM1 protein expression increases sensitivity to PARP inhibitors.MDA-MB-231 and MDA-MB-468 cells transfected with FOXM1 si RNA down-regulated FOXM1 protein expression.MTT assay showed that down-regulated FOXM1 protein expression could increase the sensitivity to olaparib.The results of enrichment analysis indicated that the target genes of FOXM1 were involved in cell cycle,DNA replication and repair.Among these target genes,ATR,Top BP1,Chk1,RAD51 and BRCA2 were co-expressed with FOXM1.The expression of FOXM1 protein was down-regulated in MDA-MB-231 and MDA-MB-468 cells.Western blot and RT-q PCR showed that the protein and m RNA levels of ATR,Top BP1,Chk1,RAD51 and BRCA2 were down-regulated.These results showed that FOXM1 decreased the sensitivity of PARP inhibitors,while mutant p53(R273H)decreased the sensitivity of breast cancer cells to PARP inhibitors by up-regulating FOXM1 expression.13.Reactivation of mutant p53 enhances the efficacy of PARP inhibitors.In MDA-MB-468 cells,p53 reactivators APR-246 and olaparib were used in combination.MTT assay and flow cytometry showed that olaparib combined with APR-246,compared with olaparib alone,enhance the tumor inhibition,so that the increase of cell apoptosis.Conclusion: 1.The expression of FEN1 was negatively correlated with HRD score.High expression of FEN1 decreased the sensitivity of PARP inhibitors in BRCA wild-type triple negative breast cancer.2.FEN1 forms a complex with ATG5 that promotes autophagy,activates ATR/Chk1 and promotes homologous recombination repair,ultimately reducing the sensitivity to PARP inhibitors.3.By up-regulating the expression of PD-L1,FEN1 promotes the formation of PD-L1-TOP2 A complex and resists apoptosis induced by PARP inhibitors.4.FEN1 and PARP inhibitors have synthetic lethality in BRCA wild type breast cancer.5.Mutant p53(R273H)decreases the sensitivity to PARP inhibitors by up-regulating FOXM1.6.Combination of p53-reactivating molecule in p53 mutant breast cancer increases the efficacy of PARP inhibitors.