Potent activity of the Hsp90 inhibitor ganetespib in prostate cancer cells irrespective of androgen receptor status or variant receptor expression

Abstract

Androgen ablation therapy represents the first line of therapeutic intervention in men with advanced or recurrent prostate tumors. However, the incomplete efficacy and lack of durable response to this clinical strategy highlights an urgent need for alternative treatment options to improve patient outcomes. Targeting the molecular chaperone heat shock protein 90 (Hsp90) represents a potential avenue for therapeutic intervention as its inhibition results in the coordinate blockade of multiple oncogenic signaling pathways in cancer cells. Moreover, Hsp90 is essential for the stability and function of numerous client proteins, a number of which have been causally implicated in the pathogenesis of prostate cancer, including the androgen receptor (AR). Here, we examined the preclinical activity of ganetespib, a small molecule inhibitor of Hsp90, in a panel of prostate cancer cell lines. Ganetespib potently decreased viability in all lines, irrespective of their androgen sensitivity or receptor status, and more effectively than the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Interestingly, while ganetespib exposure decreased AR expression and activation, the constitutively active V7 truncated isoform of the receptor was unaffected by Hsp90 inhibition. Mechanistically, ganetespib exerted concomitant effects on mitogenic and survival pathways, as well as direct modulation of cell cycle regulators, to induce growth arrest and apoptosis. Further, ganetespib displayed robust antitumor efficacy in both AR-negative and positive xenografts, including those derived from the 22Rv1 prostate cancer cell line that co-expresses full-length and variant receptors. Together these data suggest that further investigation of ganetespib as a new therapeutic treatment for prostate cancer patients is warranted.

Figure 1.

Ganetespib treatment destabilizes full-length AR receptor expression and activity, as well as multiple client proteins, in AR-positive cancer cell lines. (A), LNCaP cells were exposed to increasing concentrations of ganetespib or 17-AAG as indicated for 24 h. Cell lysates were immunoblotted using antibodies against AR, IGF-IR, phosphorylated EGFR (p-EGFR), phosphorylated AKT (p-AKT), total AKT and p70 S6K as shown. Cleaved PARP expression was included as a marker of apoptosis. Total protein levels were determined using GAPDH. (B), LNCaP cells were cultured in charcoal-stripped medium for 24 h and then treated with 250 nM ganetespib, 1 μM geldanamycin (GA), or vehicle for 24 h in the absence or presence of 10 nM androgen (R1881). Prostate specific antigen (PSA) and transmembrane protease, serine 2 (TMPRSS2) mRNA levels were measured and normalized to 18S mRNA values. Experiments were performed in triplicate. Androgen-inducible transcriptional activation was significantly inhibited in the presence of either Hsp90 inhibitor (^*P<0.001). (C), HeLa cells were transiently transfected with 3 ng of pCR3.1-AR or 0.5 ng of pCR3.1-ARV7 plasmid to induce expression of the full-length and V7 truncated AR proteins, respectively (arrowheads). Twenty-four hours following infection, cells were treated with 10 nM R1881, 1 μM GA, or 250 nM ganetespib as indicated. Cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-AR antibody. Total protein levels were determined using an anti-actin antibody. (D), To determine the effect of Hsp90 inhibitors on AR and variant activity, HeLa cells were transiently transfected with 250 ng of GRE-luciferase reporter, 30 ng of pCR3.1 β-galactosidase, 3 ng of pCR3.1-AR, or 0.03 ng of pCR3.1-V7 and treated with vehicle (ethanol and DMSO), R1881 (10 nM), GA (1 μM), and/or ganetespib (250 nM) for 24 h. Luciferase and β-galactosidase activities were measured and luciferase levels were normalized to β-galactosidase levels. Experiments were performed in triplicate.

Figure 2.

Ganetespib inhibits multiple Hsp90-dependent signaling pathways in AR-negative DU145 prostate cells to induce apoptosis. (A), DU145 cells were exposed to graded concentrations of ganetespib as indicated for 24 h. Cell lysates were immunoblotted using antibodies against phosphorylated EGFR (p-EGFR), total EGFR, phosphorylated STAT3 (p-STAT3), total STAT3, phosphorylated AKT (p-AKT), total AKT, phosphorylated SRC (p-SRC), IGF-IR, RAF1, phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 as shown. Cleaved PARP expression is included as a marker of apoptosis. Total protein levels were determined using GAPDH. (B), DU145 cells were treated with ganetespib (10, 100 or 500 nM), 17-AAG (500 or 1000 nM) or control (DMSO) for 24 h. Cells were harvested, stained with a fluorescent conjugated anti-Annexin V antibody and apoptosis measured by flow cytometry.

Figure 3.

Comparative kinetics and potency of Hsp90 client protein destabilization by ganetespib and 17-AAG in prostate cell lines. Effect of Hsp90 inhibition by: 1, ganetespib or 2, 17-AAG in LNCaP (A), DU145 (B) or PC3 (C) cells was assessed. Cell lines were exposed to 100 nM concentrations of either inhibitor and harvested at 1, 3, 6, 18, 24 and 48-h post-treatment. Cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.

Figure 4.

Ganetespib modulates cell cycle protein expression and induces growth arrest in prostate cancer cells. (A), DU145 and PC3 cells were treated with increasing concentrations of ganetespib as indicated. Cell cycle distribution was determined in each line by flow cytometry 24-h post-treatment. (B), DU145 and PC3 cells were treated with increasing concentrations of ganetespib for 24 h as in (A). Cell lysates were immunoblotted using antibodies against CDK1, CHK1 and GAPDH. (C), VCaP cells were treated with ganetespib at 0, 10, 50 and 100 nM for 24 h. Cell lysates were immunoblotted using antibodies against AR, IGF-IR, phosphorylated AKT (p-AKT), total AKT, p27^Kip1, phosphorylated-CDK1 (p-CDK1), total CDK1, phosphorylated (p-)histone H2AX, PARP and GAPDH.

Figure 5.

Suppression of in vivo prostate tumor growth by ganetespib. (A), Nude mice bearing established PC3 prostate xenografts were i.v. dosed with ganetespib (150 mg/kg) or vehicle (n=8 mice/group) on a weekly dosing schedule as indicated (arrowheads) for 4 weeks. (B), SCID mice bearing established 22Rv1 xenografts were i.v. dosed with ganetespib (150 mg/kg) or vehicle (n=8 mice/group) on a weekly dosing schedule as indicated (arrowheads). Tumor volumes were measured by caliper. Results are presented as mean ± SEM. The reduction in tumor volume in ganetespib-treated animals for both studies was significant (^*P<0.05, ANOVA).