Telomeres protect the ends of linear chromosomes from being recognized as damaged DNA, and telomere stability is required for genome stability. Here we demonstrate that telomere stability in androgen receptor (AR)-positive LNCaP human prostate cancer cells is dependent on AR and androgen, as AR inactivation by AR antagonist bicalutamide (Casodex), AR-knockdown, or androgen-depletion caused telomere dysfunction, and the effect of androgen-depletion or Casodex was blocked by the addition of androgen. Notably, neither actinomycin D nor cycloheximide blocked the DNA damage response to Casodex, indicating that the role of AR in telomere stability is independent of its role in transcription. We also demonstrate that AR is a component of telomeres, as AR-bound chromatin contains telomeric DNA, and telomeric chromatin contains AR. Importantly, AR inactivation by Casodex caused telomere aberrations, including multiple abnormal telomere signals, remindful of a fragile telomere phenotype that has been described previously to result from defective telomere DNA replication. We suggest that AR plays an important role in telomere stability and replication of telomere DNA in prostate cancer cells, and that AR inactivation-mediated telomere dysfunction may contribute to genomic instability and progression of prostate cancer cells.

Prostate cancer is the most commonly diagnosed non-skin cancer, and second leading cause of cancer deaths, in American men. The androgen receptor (AR) plays a critical role in the survival of prostate cancer (PCa) cells. Therefore, treatments that inactivate AR, either by decreasing the androgen level or by inhibiting AR directly, are used to treat advanced PCa. However, these treatments are not curative, and disease progression generates castration-resistant prostate cancer (CRPC). Our studies point to a novel role of AR in telomere stability that, in our opinion, can be exploited to enhance the effectiveness of AR-targeted therapies for CRPC. Telomere stability is essential for cell proliferation and survival. We previously reported that AR plays a role in maintaining telomere stability in prostate cancer cells. Since telomere dysfunction activates the DNA damage response (DDR) signaling pathway which can activate cell cycle checkpoints, repair damaged DNA, and promote cell survival, we tested a hypothesis that AR-inactivated telomere dysfunction activates a DDR signaling pathway protein, called ATM (ataxia telangiectasia mutated) kinase, and ATM inhibitor potentiates cell death by averting telomeric DNA repair in AR-inactivated PCa cells. ATM pathway was assessed by the phosphorylation of ATM and its downstream target Chk2 through Western blotting, inhibition of telomere DNA repair was evaluated by counting RPA (DNA replication protein A) foci at damaged telomeres, and the cell viability was determined by PARP cleavage and colony formation assays. We observed that telomere dysfunction by AR-antagonists (Casodex or MDV3100) or AR-siRNA was associated with a dramatic increase in phosphorylation of ATM and Chk2 and the presence of phosphorylated ATM at telomeres, indicating the activation of DNA damage response signaling at telomeres. Moreover, Casodex washout led to the reversal of telomere dysfunction, indicating repair of damaged telomeres. ATM inhibitor blocked ATM phosphorylation and induced PARP cleavage, suggesting that ATM inhibitors induce apoptosis in AR-inactivated cells by blocking the repair of damaged DNA at telomere. This was further corroborated by a conspicuous localization of RPA at damaged telomeres; presence of RPA foci is indicative of unrepaired stretch of single-stranded DNA at telomeres. Finally, colony formation assay revealed a dramatic decrease in the survival of cells co-treated with Casodex and ATM inhibitor as compared with those treated with either Casodex or ATM inhibitor alone. These observations indicate that inhibitors of DDR signaling pathways may offer a unique opportunity to enhance the potency of AR-targeted therapies for the treatment of androgen-sensitive as well as castration- resistant prostate cancer. Citation Format: Sahn-Ho Kim, Vidyavathi Reddy, Min Wu, Evelyn R. Barrack, G. Prem-Veer Reddy. ATM inhibition potentiates death of androgen receptor-inactivated prostate cancer cells with telomere dysfunction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1815.