Castration-resistant prostate cancer (CRPC) is characterized by reactivation of androgen receptor (AR) signaling, in part by elevated expression of AR splice variants (ARv) including ARv7, a constitutively active, ligand binding domain (LBD)-deficient variant whose expression has been correlated with therapeutic resistance and poor prognosis. In a screen to identify small-molecule dual inhibitors of both androgen-dependent and androgen-independent AR gene signatures, we identified the chalcone C86. Binding studies using purified proteins and CRPC cell lysates revealed C86 to interact with Hsp40. Pull-down studies using biotinylated-C86 found Hsp40 present in a multiprotein complex with full-length (FL-) AR, ARv7, and Hsp70 in CRPC cells. Treatment of CRPC cells with C86 or the allosteric Hsp70 inhibitor JG98 resulted in rapid protein destabilization of both FL-AR and ARv, including ARv7, concomitant with reduced FL-AR- and ARv7-mediated transcriptional activity. The glucocorticoid receptor, whose elevated expression in a subset of CRPC also leads to androgen-independent AR target gene transcription, was also destabilized by inhibition of Hsp40 or Hsp70. , Hsp40 or Hsp70 inhibition demonstrated single-agent and combinatorial activity in a 22Rv1 CRPC xenograft model. These data reveal that, in addition to recognized roles of Hsp40 and Hsp70 in FL-AR LBD remodeling, ARv lacking the LBD remain dependent on molecular chaperones for stability and function. Our findings highlight the feasibility and potential benefit of targeting the Hsp40/Hsp70 chaperone axis to treat prostate cancer that has become resistant to standard antiandrogen therapy. These findings highlight the feasibility of targeting the Hsp40/Hsp70 chaperone axis to treat CRPC that has become resistant to standard antiandrogen therapy. .

Heat shock protein 90 (Hsp90) is an essential eukaryotic molecular chaperone. To properly chaperone its clientele, Hsp90 proceeds through an ATP-dependent conformational cycle influenced by posttranslational modifications (PTMs) and assisted by a number of co-chaperone proteins. Although Hsp90 conformational changes in solution have been well-studied, regulation of these complex dynamics in cells remains unclear. Phosphorylation of human Hsp90α at the highly conserved tyrosine 627 has previously been reported to reduce client interaction and Aha1 binding. Here we report that these effects are due to a long-range conformational impact inhibiting Hsp90α N-domain dimerization and involving a region of the middle domain/carboxy-terminal domain interface previously suggested to be a substrate binding site. Although Y627 is not phosphorylated in yeast, we demonstrate that the non-conserved yeast co-chaperone, Hch1, similarly affects yeast Hsp90 (Hsp82) conformation and function, raising the possibility that appearance of this PTM in higher eukaryotes represents an evolutionary substitution for HCH1.

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