Androgen Receptor Splicing Variant 7（ARv7） Promotes The DNA Damage Response In Prostate Cancer Cells

Since the androgen receptor pathway promotes the ability of cells to repair DNA damage,androgen deprivation therapy（ADT）can significantly enhance the efficacy of radiotherapy in the treatment of patients with locally advanced prostate cancer（PCa）.This synergistic therapy is widely used in clinic.However,some patients are not sensitive to this synergistic treatment initially,and many patients with desirable responses will relapse after a period,which seriously impairs the final effect of this combination treatment.It is worth noting that ADT promotes alternative splicing of AR to generate androgen receptor alternatively spliced variants（ARVs）.Unlike AR,most of the ARVs discovered so far lack the ligand-binding domain so that ARVs can localize to the nucleus in the presence or absence of androgen,causing androgen-independent activation of AR signaling pathways.Recent studies have shown that when the AR pathway is inhibited,ARVs will compensatively promote the non-homologous end joining（NHEJ）pathway activity,leading to patients’ resistance to this DNA damage-based synergistic therapy.Obviously,this possible mechanism provides a reasonable basis for explaining the treatment resistance of some patients.However,the functions of specific ARVs and the specific mechanisms by which ARVs regulate the DNA damage response（DDR）pathway are still largely unknown.In addition,some recent articles have suggested that ARVs may be involved in regulating the activity of the DDR pathway independently of AR,especially by promoting the expression of certain genes involved in the process of cellular DNA damage repair.This unique regulatory effect has led us to further explore its specific function.Studies have shown that when poly（ADP-ribose）polymerase（PARP）is inhibited,cancer cells with defective homologous recombination（HR）pathway will die.This finding provides a theoretical basis for applying PARP inhibitors in cancer therapy.Up to now,17 PARP family members have been discovered,including PARP1,PARP2,etc.Of note,the ideal phase III clinical trial results leaded to the approval of PARP1 inhibitor Olaparib（Ola）in treating m CRPC patients with mutated HR genes.However,the research and use of PARP inhibitors in PCa are limited,and the molecular mechanisms that regulating the sensitivity of patients to PARP inhibitors are not yet known.Thus,further research is needed regarding this novel therapeutic approach.The most abundant ARVs is Androgen Receptor Splicing Variant 7（ARv7）.Based on previous research results on AR signaling and DDR pathway,we speculate that ARv7 may mediate the recovery of HR pathway,resulting in patients being insensitive to PARP inhibitors even under the combined hormone therapy.Therefore,deciphering the specific role of ARv7 in the DDR pathway may be crucial for screening the suitable population of AR inhibitor and PARP inhibitor combination therapy.This study utilized a lentiviral system to change the expression level of ARv7 in various PCa cells.By studying the changes of DDR in PCa cells under the action of ionizing radiation and doxorubicin（Dox）,and the survival rate of PCa cells under the combined treatment of AR inhibitors and PARP inhibitors,the role of ARv7 in regulating DNA damage repair and the synergistic effect between AR antagonists and PARP inhibitors were respectively investigated.Overall,this study provides a novel mechanism for overcoming radiotherapy resistance in prostate cancer and an essential reference index for evaluating the effect of novel PARP inhibitors-based therapy.Methods:1.In this study,lentiviral vectors were used to construct prostate cancer cell lines C4-2,PC-3（overexpression of ARv7）,and 22RV1（to silence endogenous ARv7）with constant ARv7 expression.2.After the establishment of the cell models,immunofluorescence,comet assay（single-cell electrophoresis）,western blot（WB）,clone formation,and other indicating assays were performed to explore whether ARv7 is involved in the regulation of the DDR pathway under the action of ionizing radiation and Dox as well as cellular sensitivity to DNA damage.3.Secondly,we explored whether ARv7 can promote DDR independently of parental AR by detecting the changes of AR expression and nuclear localization after ARv7 modifications.In addition,we also checked whether ARv7 still has the ability to regulate DDR after AR is significantly inhibited.4.Transcriptome sequencing of the prostate cancer cell lines C4-2（ARv7overexpressing）,22RV1（silencing endogenous ARv7）with constant altered ARv7 expression was performed using bioinformatics analysis.KEGG and GSEA were used to explore which pathways in cells could be changed by altering ARv7 expression,and q PCR was used to validate that ARv7 regulates certain genes in these pathways.5.Subsequently,IP and other experiments were used to further verify whether ARv7 interacts with PARP1 and DNA-PKcs,and WB was used to verify the effect of ARv7 on the activities of PARP1 and DNA-PKcs,and q PCR assays were performed to investigate whether the transcriptional activity of ARv7 is affected when PARP1 was inhibited by Olaparib.6.Finally,PARP inhibitor olaparib（Ola）and AR inhibitor enzalutamide（Enz）were used in combination to explore whether the high expression of ARv7 has an impact on the effect of this combination through in vitro cell experiments.Result:1.Overexpression of ARv7 promoted the DNA damage repair of cells under ionizing radiation and Dox-induced DSBs,while silencing endogenous ARv7 inhibited the DNA damage repair of cells under DSBs.2.After silencing or overexpressing ARv7,there was no significant change in AR expression in total protein or cytoplasmic and nuclear fractions.After ARv7 was overexpressed in PC-3 cells without AR system,the rate of DSBs repairing was significantly accelerated.After we inhibited AR activity with enzalutamide（Enz）,the rate of DSBs repairing in ARv7-overexpressed C4-2 cells was not significantly reduced.3.KEGG and GSEA analysis showed that after changing the expression of ARv7,the HR pathway was dramatically changed,and the two had a strong correlation.After silencing or overexpressing ARv7,the expression levels of some key genes involved in the HR pathway were correspondingly decreased or increased.The results of the HR activity assay showed that the number of cells repairing DNA damage by HR in the ARv7 overexpression group was significantly higher than that in the control group.4.Similarly,the number of cells repairing DNA damage through non-homologous end joining（NHEJ）in the ARv7-overexpressing group was higher than that in the control group.ARv7 could bind to DNA-PKcs and promote the phosphorylation of DNA-PKcs.Transcriptome sequencing and PCR results showed that after overexpression/silencing of ARv7,the expression of RNA polymerase II elongation factor（ELL2）,a key gene involved in the initiation of the NHEJ repair pathway,was significantly increased/decreased,respectively.In addition,ARv7 also interacts with PARP1.After olaparib（Ola）inhibits PARP1 activity,the genes whose transcription levels are up-regulated by ARv7 overexpression return to normal levels,and the phosphorylation level of ATM decreases.5.The combination index showed that the combination of PARP1 inhibitor Ola and AR antagonist Enz had enhanced ability to kill prostate cancer cells,and cells with high ARv7 expression could attenuate the combined killing effect of Ola and Enz.Conclusion:1.The expression level of ARv7 is closely related to the DSBs repair of PCa cells,and ARv7 can function independently of full-length AR.2.ARv7 promotes the DSBs repair in prostate cancer cells by regulating the HR and NHEJ pathways.3.ARv7-PARP1 forms a positive regulatory loop to mediate DDR.4.ARv7 can weaken the synergistic effect of AR inhibitors and PARP inhibitors.