Constitutively-active androgen receptor variants function independently of the HSP90 chaperone but do not confer resistance to HSP90 inhibitors

Abstract

The development of lethal, castration resistant prostate cancer is associated with adaptive changes to the androgen receptor (AR), including the emergence of mutant receptors and truncated, constitutively active AR variants. AR relies on the molecular chaperone HSP90 for its function in both normal and malignant prostate cells, but the requirement for HSP90 in environments with aberrant AR expression is largely unknown. Here, we investigated the efficacy of three HSP90 inhibitors, 17-AAG, HSP990 and AUY922, against clinically-relevant AR missense mutants and truncated variants. HSP90 inhibition effectively suppressed the signaling of wild-type AR and all AR missense mutants tested. By contrast, two truncated AR variants, AR-V7 and ARv567es, exhibited marked resistance to HSP90 inhibitors. Supporting this observation, nuclear localization of the truncated AR variants was not affected by HSP90 inhibition and AR variant:HSP90 complexes could not be detected in prostate cancer cells. Interestingly, HSP90 inhibition resulted in accumulation of AR-V7 and ARv567es in both cell lines and human tumor explants. Despite the apparent independence of AR variants from HSP90 and their treatment-associated induction, the growth of cell lines with endogenous or enforced expression of AR-V7 or ARv567es remained highly sensitive to AUY922. This study demonstrates that functional AR variant signaling does not confer resistance to HSP90 inhibition, yields insight into the interaction between AR and HSP90 and provides further impetus for the clinical application of HSP90 inhibitors in advanced prostate cancer.

Figure 1. HSP90 inhibitors dose-dependently reduce transactivation activity of wtAR and gain-of-function missense mutants

PC-3 cells were transfected with plasmids expressing wtAR or AR mutants and a probasin-luciferase reporter for 4 h prior to a 20 h treatment with 1 nM DHT. The doses of HSP90 inhibitor are shown to the right of the graphs. Luciferase activity values are expressed relative to 0 nM HSP90 inhibitor (set to 100%) and represent the mean (± SEM) of two independent experiments.

Figure 2. Truncated AR variants exhibit resistance to HSP90 inhibitors

Transactivation assays were performed as described in Figure 1 with low doses of HSP90 inhibitors (A) or high doses of HSP90 inhibitors (B) (keys are shown below the graphs). Lysates not analyzed for luciferase activity were pooled from replicate wells and immunoblotted for AR and GAPDH to visualize steady-state protein levels.

Figure 3. HSP90 inhibition does not affect the nuclear localization of truncated AR variants

PC-3 cells grown in androgen-depleted media (phenol-red free RPMI + DCC-FBS) were transfected with GFP-tagged forms of wtAR (A), AR v567es (B) or AR-V7 (C) and treated with DMSO (vehicle control), 1 nM DHT or 1 nM DHT and 100 nM AUY922. Nuclei were stained with DAPI and cytoskeletons with tubulin. Representative images are shown for five color channels (GFP, DAPI, tubulin, merged GFP/DAPI and merged GFP/tubulin).

Figure 4. Truncated AR variants do not form stable complexes with HSP90

(A) DHT promotes dissociation of wild-type AR from HSP90. PC-3 cells grown in androgen-depleted media (phenol-red free RPMI + DCC-FBS) were transfected with FLAG-tagged wtAR or empty plasmid and treated with 1 nM DHT or vehicle control. FLAG-tagged wtAR protein complexes were immunoprecipitated and immunoblotted for HSP90. (B) Truncated AR variants do not associate with HSP90. PC-3 cells grown in androgen-depleted media were transfected with FLAG-tagged wtAR, ARv567es or AR-V7 or an empty plasmid control and grown in an androgen-depleted environment. FLAG-tagged AR protein complexes were immunoprecipitated and immunoblotted for HSP90.

Figure 5. AUY922 modulates the levels of wtAR and AR-V7 transcript and protein

(A) VCaP cells were treated for 24 h with the indicated doses of AUY922 or vehicle DMSO. Wild-type AR and AR-V7 mRNA was measured by quantitative RT-PCR (normalized to reference gene GAPDH; DMSO set to 1). Values are the mean (± SEM) of triplicate samples; results are representative of two independent experiments. (B) Lysates from cells treated in part A were collected and immunoblotted for wtAR (AR N-20; AR C-19), HSP70 or AR-V7. GAPDH was used as a loading control. (C) Human prostate tumor explants were cultured in medium containing 500 nM AUY922 or vehicle DMSO for 48 h. Wild-type AR, AR-V7 and ARv567es mRNA was measured by quantitative RT-PCR (normalized to reference gene GAPDH; DMSO set to 1). Statistically significant differences compared to DMSO control treatment were assessed using one-sample t tests (*, p < 0.05; **, p < 0.01).

Figure 6. Prostate cancer cells expressing ARVs are sensitive to HSP90 inhibition

(A) AUY922 reduces cell viability of VCaP prostate cancer cells. Proliferation of cells cultured with increasing concentrations of AUY922 was assessed by trypan blue exclusion assays. Cells were grown in media containing 10% FBS + 0.1 nM DHT. (B) AUY922 reduces cell viability of 22Rv1 prostate cancer cells, as assessed by trypan blue exclusion assays. Cells were grown in the presence of androgen (10% FBS; left) or in androgen-depleted media (10% DCC-FBS; right). (C) Induction of FLAG-ARVs by cumate in stably-transduced LNCaP cells. Western blots with FLAG antibody (M2; top) or AR antibody (N-20; bottom) are shown. (D) AUY922 reduces cell viability of LNCaP cells stably transfected with inducible expression constructs: empty (C), wtAR (wt), AR-V7 (V7) and ARv567es (v567). Cells were cultured in 10% FBS and treated with cumate to induce protein expression or ethanol control (EtOH), followed by AUY922 or DMSO (vehicle control). Counts were performed as described above.