Abstract 1180: Targeting the HSP40/HSP70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer
Authors: Moses Michael A. , Kim Yeong Sang , Rivera-Marquez Genesis , Watson Matthew J. , Lee Sunmin , Kravats Andrea , Wickner Sue , Gestwicki Jason , Trepel Jane , Neckers Len 
Published: July 2017

Abstract
Castration-resistant prostate cancer (CRPC) is frequently characterized by elevated expression of nuclear receptors able to at least partially maintain the androgen receptor (AR) transcriptional program. Elevated expression of a number of constitutively active AR splice variants lacking the ligand binding domain (LBD) (e.g., ARv7, which is ligand-independent and correlates with poor prognosis, reduced survival, and resistance to existing LBD-targeted standard of care therapy) is a frequent occurrence in CRPC. Thus, alternative approaches to disrupt AR signaling in CRPC are of great clinical importance, and a single strategy able to target AR and ARv7 remains a critical unmet need. As a steroid hormone nuclear receptor, the AR exists in an interactive and dynamic cycle with the molecular chaperones (heat shock proteins, HSPs) HSP40/HSP70/HSP90 for proper folding and remodeling of the AR LBD to bind ligand. Notably, HSP90 inhibitors promote AR degradation and display efficacy in prostate cancer xenograft models. Although it has been shown that ARv7 functions independently of HSP90, additional chaperone requirements of LBD-deficient ARv7 are not known. Thus, we tested the hypothesis that both AR and ARv7 are dependent on HSP40/HSP70 and that targeting these chaperones with specific inhibitors (C86 and JG98, respectively) will lead to AR/ARv7 destabilization and loss of transcriptional activity in models of CRPC. To determine if AR proteins associate with HSP40/HSP70, 22Rv1 CRPC cells (expressing endogenous AR and ARv7) were first transfected with FLAG-HSP40 or FLAG-HSP70. Immunoprecipitation with FLAG beads revealed AR and ARv7 associated with both chaperones, indicating potential functional dependence of these nuclear receptors on HSP40/HSP70. To further characterize these interactions, 22Rv1 lysate was probed with biotinylated-C86 and subjected to IP with streptavidin beads. C86 bound a significant fraction of HSP40 complexed with HSP70, AR, and ARv7. Excess unlabeled C86 or JG98 effectively competed away binding of HSP40/HSP70 to biotinylated-C86 with concomitant loss of associated AR and ARv7. Treatment of 22Rv1 cells with C86 or JG98 led to a time and dose-dependent decrease in AR and ARv7 protein, concomitant with a significant loss of viability. We also observed that HSP40/HSP70 inhibition markedly reduced AR and ARv7 transcriptional activity, as indicated by decreased AR (KLK3, TMPRSS2) and ARv7 (UBE2C) target gene expression. Finally, treatment of mice bearing 22Rv1 xenografts with JG231 (an analog of JG98 with enhanced PK properties) led to significantly smaller tumors relative to vehicle treated mice. Together, these data confirm the continued dependence of AR and ARv7 on HSP40/HSP70 molecular chaperones and they demonstrate the feasibility of targeting the HSP40/HSP70 axis to abrogate sustained AR-mediated signaling in CRPC.
Citation Format: Michael A. Moses, Yeong Sang Kim, Genesis Rivera-Marquez, Matthew J. Watson, Sunmin Lee, Andrea Kravats, Sue Wickner, Jason Gestwicki, Jane Trepel, Len Neckers. Targeting the HSP40/HSP70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1180. doi:10.1158/1538-7445.AM2017-1180