Suppression of heat shock protein 27 using OGX-427 induces endoplasmic reticulum stress and potentiates heat shock protein 90 inhibitors to delay castrate-resistant prostate cancer

Abstract

Background: Although prostate cancer responds initially to androgen ablation therapies, progression to castration-resistant prostate cancer (CRPC) frequently occurs. Heat shock protein (Hsp) 90 inhibition is a rational therapeutic strategy for CRPC that targets key proteins such as androgen receptor (AR) and protein kinase B (Akt); however, most Hsp90 inhibitors trigger elevation of stress proteins like Hsp27 that confer tumor cell survival and treatment resistance.

Objective: We hypothesized that cotargeting the cytoprotective chaperone Hsp27 and Hsp90 would amplify endoplasmic reticulum (ER) stress and treatment-induced cell death in cancer.

Design, setting, and participants: Inducible and constitutive Hsp27 and other HSPs were measured by real-time reverse transcription-polymerase chain reaction and immunoblot assays. The combinations of OGX-427 with Hsp90 inhibitors were evaluated in vitro for LNCaP cell growth and apoptosis and in vivo in CRPC LNCaP xenograft models.

Outcome measurements and statistical analysis: Tumor volumes were compared using the Kruskal-Wallis test. Overall survival was analyzed using Kaplan-Meier curves, and statistical significance was assessed with the log-rank test.

Results and limitations: Hsp90 inhibitors induced expression of HSPs in tumor cells and tissues in a dose- and time-dependent manner; in particular, Hsp27 mRNA and protein levels increased threefold. In vitro, OGX-427 synergistically enhanced Hsp90 inhibitor-induced suppression of cell growth and induced apoptosis by 60% as measured by increased sub-G1 fraction and poly(ADP-ribose) polymerase cleavage. These biologic events were accompanied by decreased expression of HSPs, Akt, AR, and prostate-specific antigen, and induction of ER stress markers (cleaved activating transcription factor 6, glucose-regulated protein 78, and DNA-damage-inducible transcript 3). In vivo, OGX-427 potentiated the anticancer effects of Hsp90 inhibitor PF-04929113 (orally, 25mg/kg) to inhibit tumor growth and prolong survival in CRPC LNCaP xenografts.

Conclusions: HSP90 inhibitor-mediated induction of Hsp27 expression can be attenuated by OGX-427, resulting in increased ER stress and apoptosis, and synergistic inhibition of CRPC tumor growth.

Patient summary: This study supports the development of targeted strategies using OGX-427 in combination with Hsp90 inhibitors to improve patient outcome in CRPC.

Keywords: Androgen receptor; Castration-resistant prostate cancer; Hsp27; Hsp90 inhibition; OGX-427.

Fig. 1

PF-04929113 and 17-AAG induce heat shock protein (HSP) expression in prostate cancer cells in vitro. PC-3 and LNCaP cells were treated with (a) 1 µM PF-04928473 or (c) 1 µM 17-AAG for the indicated time points. (b) In parallel, PC-3 and LNCaP cells were treated for 48 h with PF-04928473 for the indicated doses. Protein extracts were analyzed for Hsp27, Hsp70, and protein kinase B (Akt) by Western blot; vinculin was used as a loading control. (d) LNCaP tumor cells were treated for 24 h with 1 µM PF-04928473 or 1 µM 17-AAG. mRNA extracts were analyzed by real-time polymerase chain reaction for Hsp27, Hsp90, and Hsp70. *** p < 0.001. CT = control.

Fig. 2

PF-04928473 induces heat shock protein (HSP) expression in a prostate cancer xenograft model. Mice were treated for 6 wk with 50 mg/kg PF-04929113 or vehicle (control). Tumors were collected, and Hsp27 and Hsp70 were evaluated by immunohistochemical analysis. Specimens were scored and estimated in arbitrary units (AUs). *** p < 0.001.

Fig. 3

Heat shock protein (Hsp) 27 protects tumor cells from Hsp90 inhibition-induced endoplasmic reticulum (ER) stress. (a) Hsp27 overexpressing stable LNCaP cells (LNCaP-Hsp27) and control vector-transfected cells (LNCaP-Empty) were treated with indicated concentrations of PF-04928473 for 48 h. Cell growth was determined by crystal violet staining. (b) Schematic picture of the ER stress/unfolded protein response pathway. (c) LNCaP cells were treated with 1 µM PF-04928473 or 17-AAG for 24 h (real-time polymerase chain reaction [PCR]) or 48 h (Western blot). Cells were harvested; Western blotting analyses were performed on the cell lysates for Hsp27, glucose-regulated protein (Grp) 78, activating transcription factor (ATF) 4, DNA-damage-inducible transcript 3 (CHOP), and vinculin; or quantitative real-time PCR analysis was performed on RNA for splicing X-box binding protein (sXBP) 1. (d) LNCaP cells were treated with 5 µM carbobenzoxy-Leu-Leu-leucinal (MG132) for 48 h. Western blotting analyses were performed for Hsp27, Grp78, P-eiF2α, ATF4, CHOP, ATF6, cleaved ATF6, and vinculin expression. (e) LNCaP cells were treated twice with 50 nM OGX-427 or control ScrB ASO. Cells were harvested, and Western blotting analyses were performed for indicated antibodies or quantitative real-time PCR analysis for sXBP1. Means of at least three independent experiments were done in triplicate. *** p < 0.001; * p < 0.05. IRE1 = inositol requiring enzyme 1; BCL2 = B-cell CLL/lymphoma 2; ROS = c-ros oncogene 1, receptor tyrosine kinase.

Fig. 4

Cotargeting heat shock protein (Hsp) 90 and Hsp27 amplifies endoplasmic reticulum (ER) stress response and treatment-induced apoptosis. LNCaP cells were treated twice with 50 nM OGX-427 or control ScrB ASO, followed by 1 µM PF-04928473 for 48 h. (a) Cells were harvested and Western blotting analyses were performed for Hsp27, glucose-regulated protein (Grp) 78, DNA-damage-inducible transcript 3 (CHOP), and vinculin. (b) Cells were harvested and quantitative real-time polymerase chain reaction analysis was performed on RNA for splicing X-box binding protein (sXBP) 1. (c) Dose-dependent effects and combination index (CI) values were assessed in LNCaP cells treated for 48 h with OGX-427 alone, PF-04928473 or 17-AAG alone, or with combined treatment at the indicated concentration with a constant ratio design between both drugs. The CI values for effective dose (ED)₅₀ and ED₇₅ were, respectively, 0.05 and 0.1 for PF-04928473, and 0.18 and 0.95 for 17-AAG, indicating a synergistic effect (<1) of Hsp27 inhibition combined with Hsp90 inhibitors. (d–f) LNCaP cells were treated twice with 50 nM OGX-427 or control ScrB ASO, followed by 1 µM PF-04928473 for 48 h. (d) The proportion of cells in subG₁, G₀–G₁, S, G₂–M was determined by propidium iodide staining. Cells were harvested, caspase-3 activity was determined on the cell lysates, and the results expressed in arbitrary units (AUs) and corrected for (e) protein content, or (f) Western blotting analyses were performed for Hsp27, poly ADP ribose polymerase (PARP), protein kinase B (AKT), androgen receptor (AR), prostate-specific antigen (PSA), and vinculin expression. All experiments were repeated at least three times. *** p < 0.001; ** p < 0.01. s.d. = standard deviation; RFLU = relative fluorescence unit.

Fig. 5

OGX-427 potentiates PF-04929113 activity in a LNCaP castrate-resistant prostate cancer xenograft model. (a) The mean tumor volume, (b) the percentage of tumor progression, and (c) the serum prostate-specific antigen (PSA) level were compared among the four groups plus or minus the standard error of the mean (n = 8). (d) PSA doubling time and velocity. (e) Using the Kaplan-Meier curve, cancer-specific survival was compared among the four groups over 56 d. The control group corresponds to the mice bearing tumor that did not receive any treatment. This group only received vehicle. *** p < 0.001.

Fig. 6

OGX-427 potentiates PF-04929113-induced apoptosis in castrate-resistant prostate cancer LNCaP tumors. (a) Tumors were collected after 56 d and heat shock protein (Hsp)27, Ki67, androgen receptor (AR), glucose-regulated protein (Grp) 78, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were evaluated by immunohistochemical analysis (original magnification: ×200). (b) Specimens were scored and estimated in percentage of positive cells. The control group corresponds to the mice bearing tumor that did not receive any treatment. This group only received vehicle. *** p < 0.001.