CDK4/6 Inhibitor Reverses Acquired Drug Resistance Of PARP Inhibitor In Ovarian Epithelial Carcinoma By Restoring Homologous Recombination Deficiency

Objective:Ovarian cancer is the most malignant gynecological cancer and the eighth leading cause of cancer death in women worldwide(GLOBOCAN 2018).Among them,more than 90%of ovarian cancer originated from epithelium,and epithelial ovarian cancer accounted for the majority of ovarian cancer deaths.Therefore,it is an urgent need to explore effective treatment strategies for advanced epithelial ovarian cancer.Poly ADP-ribose polymerase inhibitor(PARPi)has changed the treatment pattern of epithelial ovarian cancer.PARPi irreversibly blocks the NAD~+binding site of PARP1,thus blocking the repair of DNA single-strand breaks by PARP1,and trapping PARP1 at the DNA break site.Inhibition of PARP1 produces a broken DNA single strand,and then the replication fork collapses.If the single strand break is not repaired,it can develop into a double strand break,resulting in replication blocking and chromosome deletion.The simultaneous inactivation of two non-lethal genes will lead to synthetic lethality(SL).On the one hand,PARPi has greatly improved the treatment options for epithelial ovarian cancer,but the acquired drug resistance caused by long-term use of PARPi has also limited the clinical benefits of the drug.The core mechanism of drug resistance is the re-repair of tumor cell homologous recombination repair pathway.Cyclin-dependent kinases(CDK)are serine/threonine protein kinase systems corresponding to the process of cell cycle,which interact with cyclin to control the G1/S checkpoint of cell cycle.CDK4/6 inhibitor(CDK4/6i)can block tumor cells in G1 phase and accumulate DNA single strand breaks.Homologous recombination repair(HRR)of cells requires the use of undamaged homologous DNA templates to guide the repair process,so the homologous recombination repair mainly plays a role in the late S and G2phases.The cells that remain in G1 phase cannot carry out homologous recombination repair,induce the cells to have homologous recombination deficiency(HRD),and then increase double strand breaks,so that the number of non-homologous end-joining(NHEJ)of cells increases,and the number of DNA missense repair of tumor cells increases,leading to cell apoptosis.To sum up,we propose a scientific hypothesis:CDK4/6i causes G1 phase arrest of primary tumor cells,thereby weakening the repair of homologous recombination induced by long-term treatment of PARPi,again causing the defect of homologous recombination of primary tumor,increasing double-stranded DNA breakage,promoting cell apoptosis,and reversing the drug resistance of PARPi.In this paper,we discuss the combination of PARPi and CDK4/6i in the treatment of epithelial ovarian cancer cell lines with acquired resistance to PARPi,so that the drug resistant cell lines can gain the sensitivity of PARPi again,reveal the internal mechanism of the combination of the two inhibitors in anti-tumor treatment,and provide potential new targets for targeted treatment of tumors.Methods:1.Cell culture:To construct stable epithelial ovarian cancer PARPi acquired drug resistance lines A2780-ola-r and SKOV-3-ola-r.After applying low concentration of Olaparib,Paldociclib and combined drugs to drug resistant cell lines,the results of cell proliferation experiment revealed that the combined treatment had the most significant effect on inhibiting tumor proliferation.2.Bioinformatics analysis:use the gene expression data of tumor cell lines and parent cell lines after PARPI treatment,and tumor cell lines and parent cell lines after CDK4/6i treatment in the GEO database to conduct differential expression analysis,and screen the related proteins in the differentially expressed genes and the common effects of PARPi and CDK4/6i,as well as the related signal pathways of drug effects.3.Flow cytometry was used to detect the effect of CDK4/6i on the cell cycle.4.Immunofluorescence double staining was used to detect the co-localization of p-γH2AX and RAD51 in A2780-ola-r and SKOV-3-ola-r cells treated with PARP inhibitors and CDK4/6 inhibitors to evaluate changes in the DNA repair pathway after treatment.5.Compared with the parental cell lines,the expression of homologous recombination repair pathway related proteins in drug-resistant cell lines increased significantly;After drug treatment,the levels of KNSTRN and CDK12 protein in drug-resistant cells were negatively correlated with CDK4/6i or combination therapy,while the levels of TRPC4AP protein were positively correlated with CDK4/6i or combination therapy.Results:1.Construction of stable epithelial ovarian cancer PARPi acquired drug resistance lines A2780-ola-r and SKOV-3-ola-r.After applying low concentration of Olaparib,Palbociclib and combined drugs to drug resistant cell lines,the results of cell proliferation experiment revealed that the combined treatment had the best effect in inhibiting tumor proliferation.2.Bioinformatic analysis was performed on gene expression data of tumor cell lines treated with PARPi and CDK4/6i from the GEO database,and enriched information of differentially expressed genes was obtained.Potential therapeutic target proteins including KNSTRN and TRPC4AP were identified as a potential therapeutic target protein.3.Flow cytometry was used to detect the effect of CDK4/6i on the cell cycle.Low concentrations of Olaparib,Palbociclib,and their combination were applied to A2780-ola-r and SKOV-3-ola-r cells,and it was found that Palbociclib and the combination treatment led to an increase in G1 phase cells and a decrease in S/G2 phase cells.Compared to the DMSO group,the combination treatment and Palbociclib alone significantly reduced nuclear RAD51 in the cells,while significantly increasing nuclear p-γH2AX in both groups.4.Compared with the parental cell lines,the expression of homologous recombination repair pathway related proteins in drug-resistant cell lines increased significantly;After drug treatment,the levels of KNSTRN protein in drug-resistant cells were negatively correlated with CDK4/6i or combination therapy,while the levels of TRPC4AP protein were positively correlated with CDK4/6i or combination therapy.Conclusion:In this experiment,compared with its parent cells,the drug resistant cell lines A2780-ola-r and SKOV-3-ola-r have enhanced homologous recombination and repair ability,and the combination of Olaparib and Palbociclib can significantly inhibit the proliferation of A2780-ola-r and SKOV-3-ola-r cells;Moreover,it is revealed that KNSTRN and TRPC4AP proteins can be used as indicators for evaluating the drug resistance level of drug-resistant cells or as potential therapeutic targets.