PARP Inhibitors Paradoxically Increase Stromal Fibroblast Activation Through Promoting CCL5 Autocrine Expression In Ovarian Cancer

Objective: By inducing synthetic lethality,PARP inhibitors can effectively kill ovarian cancer cells with defects in homologous recombination repair.In this context,approximately 50% of ovarian cancer patients may benefit from PARP inhibitors.However,little is known about the effects of PARP inhibitors on tumor microenvironment.The primary objective of this study was to extend the effect of PARP inhibitors from ovarian cancer epithelial cells to stromal fibroblasts,and to explore their role in the activation of stromal fibroblasts and their possible mechanisms.Methods: CCK8 assay was used to evaluate the sensitivity difference between ovarian cancer cell lines and ovarian cancer stromal fibroblasts to PARP inhibitors.Immunofluorescence and cell cycle experiments were conducted to confirm the effects of PARP inhibitors on DNA damage repair and cell cycle distribution of stromal fibroblasts.RNAseq,immunofluorescence,collagen contraction experiments,and western blotting experiments were performed to confirm the effects of PARP inhibitors on the activation of stromal fibroblasts in vitro.The protein microarray technique was used to detect the supernatants of fibroblasts treated with PARP inhibitors,and bioinformatics techniques were used to analyze the relationship between the candidate cytokines and fibroblast activation.Enzyme linked immunosorbent assay and western blotting were performed to verify the expression changes of this cytokine in tumor stromal fibroblasts before and after PARP inhibitor treatment.Human recombinant cytokine and neutralizing antibody were used to determine the effect of this cytokine on PARP inhibitor-induced activation of stromal fibroblasts.Bioinformatics,western blotting,and collagen contraction experiments were used to investigate mechanisms by which PARP inhibitors promoted this cytokine secretion.The mouse tumor model was used to verify the adaptive response of fibroblasts to PARP inhibitors and the potential value of anti-fibroblast activation therapy in combination with PARP inhibitors for the treatment of ovarian cancer.The changes in amount and activation degree of stroma components in tumor tissues were detected by masson’s staining,picrosirius red staining,and immunohistochemical staining.Results: Our results indicated that stromal fibroblasts were relatively insensitive to PARP inhibitors compared to ovarian cancer cell lines.PARP inhibitors could cause DNA damages and G2/M phase arrest in fibroblasts.With the repair of DNA damages,cell cycle arrest gradually recovered.The results of RNAseq and in vitro experiments showed that fibroblasts treated with PARP inhibitors exhibited a more activated state,a more stretched spindle appearance and a stronger ability to contract ECM.After PARP inhibitors treatment,the secretion spectrum of stromal fibroblasts changed significantly.CCL5,MIP-3α,MCP3,CCL11 and ENA-78 were all obviously increased in fibroblasts treated with two PARP inhibitors,Olaparib and Niraparib.Bioinformatics analysis showed that the m RNA level of CCL5 was significantly correlated with the stromal activation scores.After Olaparib treatment,the expression of CCL5 in fibroblasts was significantly escalated,and CCL5 neutralizing antibodies could significantly reversed the increase of fibroblast activation degree caused by PARP inhibitors in vitro.Further experiments showed that increased CCL5 secretion caused by PARP inhibitors was mainly mediated by NF-κB signaling pathway,and the inhibition of NF-κB signaling pathway not only reduced secretion of CCL5 in fibroblasts but also attenuated fibroblast activation degree.Animal experiments showed that Olaparib could significantly inhibit tumor growth,but the stromal components ratio and stromal fibroblast activation marker such as α-SMA were also significantly increased.The addition of CCL5 neutralizing antibody or NF-κB inhibitor could obviously reverse the increase of fibroblast activation caused by PARP inhibitors and further enhance the inhibitory effect of PARP inhibitors on mouse tumor growth.Conclusion: PARP inhibitors could promote CCL5 autocrine of stromal fibroblasts by activating NF-κB signaling pathway,which in turn maintained and promoted the activation of stromal fibroblasts.This study complements our understanding about PARP inhibitors in the treatment of ovarian cancer and provides a theoretical basis for exploring new and more rational PARP inhibitor applications.