Abstract 2112: AR splice variants ARv7 and ARv567 utilize different mechanisms of cytoplasmic trafficking and nuclear translocation: Therapeutic implications for PC

Abstract

Abstract Prostate cancer (PC) is one of the most prevalent cancers among men worldwide. While androgen deprivation therapy (ADT) is initially effective in the treatment of PC, resistance emerges resulting in castration-resistant prostate cancer (CRPC) disease, which is driven by continuous signaling from the androgen receptor (AR). Several new drugs designed to inhibit AR signaling (abiraterone and enzalutamide) have been recently approved for PC treatment. However resistance to these next-generation AR inhibitors inevitably occurs and is mediated by adaptive mechanisms that restore AR function. Overexpression of AR splice variants (ARv567 and ARv7), which lack the ligand-binding domain (LBD), was shown to mediate resistance to abiraterone and enzalutamide. Following ADT resistance, CRPC patients are treated with the taxanes Docetaxel (DTX) and Cabazitaxel as first and second line chemotherapy. Our recent work showed that AR binds to microtubules (MTs) utilizing them as tracks for its nuclear translocation, and that taxanes inhibit AR signaling by sequesting AR in the cytoplasm downstream of MT stabilization. We showed that MT binding is mediated by the Hinge region of AR, which is present in ARv567 but missing in ARv7 variant. In contrast to ARv567, ARv7 does not bind MTs and its nuclear localization is not affected by taxane treatment and its expression confers taxane resistance in vitro and in vivo. Since the main nuclear localization signal (NLS) of AR is in the hinge region we hypothesized that ARv7 has a different nuclear translocation mechanism than Arv567 or AR-WT. Co-immunoprecipitation experiments revealed that AR-WT and ARv567 interact with importin-α, and that DTX treatment inhibits this interaction. In contrast, ARv7 does not interact with importin-α and is insensitive to DTX. To monitor the dynamics of AR variant nuclear accumulation we performed live cell confocal microscopy using PC3 cells microinjected with GFP-AR-WT, -ARv7 or -ARv567. Our results showed that both AR-WT and AR-v567 were sensitive to inhibition of the importin pathway, using the importin-β inhibitor importazole, while ARv7 was unaffected. FRAP experiments of M12 cells stably expressing GFP-tagged AR-WT, or ARvs revealed that the recovery after photobleaching of ARv7 nuclear fraction was much faster than that of ARv567 or AR-WT, suggesting enhanced motility of ARv7. FRAP experiments in DTX or importazole treated cells further confirmed the insensitivity of ARv7 to perturbations of the MT network and importin pathway. Taken together our results show that AR-WT and ARv567 utilize MTs and, the importin-α/β pathway for nuclear translocation; while ARv7 is MTs and importin independent. Elucidation of the mechanisms of cytoplasmic trafficking and nuclear translocation of ARv7 variant will allow us to identify new druggable targets in order to revert resistance to taxanes and next generation AR targeting drugs. Citation Format: Luigi Portella, Paraskevi Giannakakou. AR splice variants ARv7 and ARv567 utilize different mechanisms of cytoplasmic trafficking and nuclear translocation: Therapeutic implications for PC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2112. doi:10.1158/1538-7445.AM2014-2112