EURURO-5447; No. of Pages 11 EUROPEAN UROLOGY XXX (2014) XXX–XXX

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Prostate Cancer

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 Franc¸ois Lamoureux a, Christian Thomas a, Min-Jean Yin b, Ladan Fazli a, Amina Zoubeidi a, Martin E. Gleave a,* a

The Vancouver Prostate Centre, University of British Columbia, Vancouver, BC, Canada; b Oncology Research, Pfizer Worldwide Research & Development,

San Diego, CA, USA

Article info

Abstract

Article history: Accepted December 13, 2013 Published online ahead of print on December 29, 2013

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 inhibitorinduced 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, 25 mg/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. # 2013 European Association of Urology. Published by Elsevier B.V. All rights reserved.

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

* Corresponding author. The Vancouver Prostate Centre, 2660 Oak Street, Vancouver, BC, V6H 3Z6, Canada. Tel. +1 604 875 5686; Fax: +1 604 875 5654. E-mail address: [email protected] (M.E. Gleave).

0302-2838/$ – see back matter # 2013 European Association of Urology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.eururo.2013.12.019

Please cite this article in press as: Lamoureux F, et al. 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. Eur Urol (2014), http://dx.doi.org/10.1016/j.eururo.2013.12.019

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1.

Introduction

Although early detection and treatment of localized prostate cancer (PCa) has improved survival rates, many men still die of metastatic disease. Although 80% of patients initially respond to androgen-deprivation therapy, most progress to metastatic castrate-resistant prostate cancer (CRPC) [1,2]. To significantly improve survival in men with PCa, new therapeutic strategies targeting the molecular basis of CRPC and treatment resistance are required. Molecular chaperones, including heat shock proteins (HSPs), help tumor cells cope with stress-induced misfolded proteins and play prominent roles in cellular signaling and transcriptional regulatory networks. In particular, Hsp90 and Hsp27 are highly expressed in various cancers, including CRPC, and induce tumor cell survival or treatment resistance [3]. Hsp90 is an ATPase-dependent chaperone required for protein folding, maturation, and conformational stabilization of many client proteins [4]. Hsp90 interacts with several proteins involved in CRPC including growth factor receptors, cell cycle regulators, and signaling kinases, including protein kinase B (Akt) or androgen receptor (AR) [5]. Tumor cells express higher Hsp90 levels and activity than benign cells [6,7], and Hsp90 inhibition has emerged as a target in CRPC and other cancers. Many Hsp90 inhibitors were developed that target the ATPase pocket, including natural compounds such as geldanamycin and its analog 17-allylamino-17-demethoxy-geldanamycin (17-AAG), or synthetic compounds including PF-04928473. These agents inhibited Hsp90 function and induced apoptosis in preclinical studies of cancers of the colon, breast, and prostate, among others [7,8]. While promising, treatment resistance emerges early due to compensatory mechanisms involving activation of heat shock factor (HSF) 1, which induces increased expression of HSPs, including Hsp70 and clusterin [9]. Interestingly, the upregulation of these chaperones plays a role in cellular recovery from stress by restoring protein homeostasis and promoting thermotolerance and cell survival [10]. Among them, Hsp27 is a stress-activated chaperone that interacts with many key apoptosis-associated proteins to regulate a cell’s apoptotic rheostat through both intrinsic and extrinsic pathways. We previously reported that knocking down Hsp27 using a specific inhibitor, OGX-427, induces apoptosis and potentiates the effect of anticancer drugs both in vitro and in vivo in CRPC and bladder cancer [11]. OGX-427 is currently in a multicenter phase 2 clinical trial in CRPC and metastatic bladder cancer (NCT01454089 and NCT01120470) [12,13]. Molecular chaperones play key roles in endoplasmic reticulum (ER) stress responses, thereby regulating protein homeostasis. Disruption of proteostasis induces ER stress, which, in turn, leads to the unfolded protein response (UPR), a prosurvival process induced to restore normal ER function. The UPR is distinguished by the action of three signaling proteins localized on the ER membrane: pancreatic ER kinase (PKR)-like ER kinase (PERK), inositol requiring enzyme (IRE) 1, and activating transcription factor (ATF) 6 that are kept inactive through the association of their luminal domain with the ER chaperone binding

immunoglobulin protein/glucose-regulated protein (BiP/ GRP) 78 [14]. Increasing levels of misfolded proteins in the ER lumen release the three ER stress sensors from BiP/ GRP78, allowing the activation of their signaling functions and the transcription of UPR target genes such as activating transcription factor (ATF) 4, X-box binding protein (XBP) 1, and DNA-damage-inducible transcript 3 (CHOP). However, excessive ER stress leads to mitochondrial apoptosis to eliminate damaged cells [15], which is mainly controlled by the pro-apoptotic transcription factor CHOP [16]. Therefore, cotargeting molecular chaperones regulating ER homeostasis may enhance cancer control by overwhelming a cancer cell’s ability to regulate misfolded protein burden. In this regard, Hsp90 modulates the UPR by interacting and stabilizing two of three ER stress sensors, IRE1 and PERK [17], so that Hsp90 inhibition induces ERstress-mediated apoptosis [18,19]. We recently reported that Hsp27 plays an important role in ER homeostasis and that knocking down Hsp27 using OGX-427 induces ER stress [11]. We set out to test the hypothesis that Hsp90 inhibition induction of Hsp27 functions to inhibit treatment-induced apoptosis and enhance emergence of treatment resistance. It follows that cotargeting Hsp27 (using OGX-427) will potentiate effects of Hsp90 inhibitors (17-AAG and PF04928473) by amplifying ER stress and leading to apoptosis in CRPC. 2.

Materials and methods

2.1.

Cell culture experiments

The human PCa cell lines PC-3 and LNCaP were maintained in Dulbecco modified Eagle medium and Roswell Park Memorial Institute medium 1640, respectively (Life Technologies Corp, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum and 2 mmol/l L-glutamine in a humidified 5% carbon dioxide/air atmosphere at 37oC. Hsp90 inhibitor PF-04928473 [4-(6,6-dimethyl-4-oxo-3-trifluoromethyl4,5,6,7-tetrahydro-indazol-1-yl)-2-(4-hydroxy-cyclohexylamino)-benzamide] and its prodrug PF-04929113 (Pfizer Inc, New York, NY, USA) were used for in vitro and in vivo studies, respectively. These compounds are synthetic inhibitors that bind the N-terminal adenosine triphosphate binding site of Hsp90; PF-04929113 is orally bioavailable. Stocks and dilutions of PF-04928473 and 17-AAG (US National Institutes of Health, Bethesda, MD, USA) were made in dimethyl sulfoxide. Cells were quantified using crystal violet staining. The sequence of OGX-427 (OncoGenex Pharmaceuticals, Bothell, WA, USA) corresponds to the human Hsp27 translation initiation site (50 -GGGACGCGGCGCTCGGTCAT-30 ). Scrambled antisense oligonucleotide (ScrB ASO; ISIS Pharmaceuticals, Carlsbad, CA, USA) was used as a control. 2.2.

Protein detection

Western blots were probed with antibodies anti-Hsp70, antiAR, anti-PSA, anti-GRP78, anti-ATF4, and anti-CHOP (Santa

Please cite this article in press as: Lamoureux F, et al. 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. Eur Urol (2014), http://dx.doi.org/10.1016/j.eururo.2013.12.019

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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 mM PF-04928473 or (C) 1 mM 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 mM PF-04928473 or 1 mM 17-AAG. mRNA extracts were analyzed by real-time polymerase chain reaction for Hsp27, Hsp90, and Hsp70. *** p < 0.001. CT = control.

Cruz Biotechnology, Dallas, TX, USA); anti–phospho-eIF2a (Life Technologies Corp, Carlsbad, CA, USA); anti-ATF6 (ImgenexCorp, San Diego, CA, USA); anti-HSP27 (Enzo Life Sciences Inc, Farmingdale, NY, USA); anti-PARP and anti-Akt (Cell Signaling Technology Inc, Beverly, MA, USA); and antivinculin (Sigma-Aldrich Corp, St. Louis, MO, USA). For immunohistochemistry, paraffin-embedded, formalin-fixed tissue sections were antigen retrieved, incubated with the primary antibody followed by secondary antibodies (Life Technologies Corp, Carlsbad, CA, USA). 2.3.

RNA quantification

For real-time quantitative polymerase chain reaction, total RNA was extracted and cDNA quantified by the ABI PRISM 7900HT Sequence Detection System (Life Technologies Corp, Carlsbad, CA, USA). Relative expression was calculated using the delta-Ct method. 2.4.

Animal treatment

For xenograft experiments, 2  106 LNCaP cells (suspended in 0.1 ml Matrigel; Becton, Dickinson and Co, Franklin Lakes, NJ, USA) were rapidly injected subcutaneously into the flank region of athymic male mice (Harlan Sprague-Dawley, Inc, Indianapolis, IN, USA). The mice were castrated once tumors reached between 300 mm3 and 500 mm3 or the prostatespecific antigen (PSA) level increased >50 ng/ml. Once tumors progressed to castrate resistance, mice were randomly assigned to vehicle PF-04929113, PF-04929113 plus ScrB ASO, or PF-04929113 plus OGX-427. Tumor volume was measured twice weekly. All animal procedures were performed according to the guidelines of the Canadian

Council on Animal Care and appropriate institutional certification. 2.5.

Statistical analysis

All in vitro data were assessed using the Student t test and Mann-Whitney test. Tumor volumes of mice 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. Levels of statistical significance were set at p < 0.05. Additional methods are presented in the Supplement.

3.

Results

3.1.

Heat shock protein 90 inhibitors induce heat shock protein

expression in prostate cancer

Both 17-AAG and PF-04928473 increased Hsp27 and Hsp70 protein levels up to threefold in a dose- and time-dependent manner in LNCaP and PC-3 cells (Fig. 1A–1C) and were accompanied by a decline of total Akt expression. mRNA levels of Hsp27, Hsp70, and Hsp90 also increased after Hsp90 inhibitor treatment (Fig. 1D). Next, the effects of PF-04928473 on Hsp27 expression in CRPC LNCaP xenografts were assessed using immunohistochemistry (Fig. 2). Once tumors became palpable, mice were treated with Hsp90 inhibitors. Hsp27 expression increased 2.5-fold after treatment with PF-04929113 compared with vehicle (Fig. 2). Similarly, Hsp70, a pharmacodynamic measure of Hsp90 inhibition [8,20], increased 2.3-fold after treatment with PF-04929113 (Fig. 2).

Please cite this article in press as: Lamoureux F, et al. 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. Eur Urol (2014), http://dx.doi.org/10.1016/j.eururo.2013.12.019

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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 PF04929113 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.

3.2.

Heat shock protein 27 protects tumor cells from heat shock

protein 90 inhibition-induced endoplasmic reticulum stress

Since Hsp90 inhibition using PF-04928473 or 17-AAG induces Hsp27 expression (Figs. 1 and 2), Hsp27 overexpression protects tumor cells from Hsp90 inhibitor treatment (Fig. 3A), and key proteins initiating ER stress, such as PERK or IRE1, are client proteins of Hsp90 [17]. We consequently analyzed if PF-04928473 induces ER stress and UPR in PCa cell lines (Fig. 3B and 3C). PF-04928473 treatment increased expression of the ER resident chaperone Grp78, a marker of ER stress induction (Fig. 3C). Hsp90 inhibition induced upregulation of ATF4, involved in the PERK signaling of the UPR, and upregulation of XBP1 splicing, suggesting IRE1 and/or ATF6 signaling activation (Fig. 3C). CHOP expression, a major component of the ER stress-mediated apoptosis, was also increased after Hsp90 inhibitor treatment. These results indicate that Hsp90 inhibition induces ER stress in PCa cells by activating several signaling pathways of the UPR associated with the upregulation of Hsp27 expression (Fig. 3C). We previously reported that Hsp27 plays an important role in ER stress regulation [11]. The proteasome inhibitor MG132 is known to induce ER stress by inhibiting the UPR [21]. MG132 treatment induced ER stress and UPR by increasing Grp78, p-eIF2a, ATF4, cleaved ATF6, and CHOP expression (Fig. 3D, Supplemental Fig. 1a). This upregulation of ER stress and UPR signaling was also associated with increased Hsp27 expression. Hsp27 induction was also observed after Hsp90 or proteasome inhibitor treatments (Fig. 3C and 3D, Supplemental Fig. 1a), which both lead to ER stress and UPR induction, identifying Hsp27 as a key component in the ER stress and UPR. We next evaluated the effect of OGX-427-induced Hsp27 knockdown on ER stress and UPR induction. Interestingly,

OGX-427 induced both ER stress and UPR activation by increasing Grp78, p-eIF2a, ATF4, cleaved ATF6, and CHOP expression, as well as XBP1 splicing (Fig. 3E, Supplemental Fig. 1b), suggesting a protective role of Hsp27 in protein homeostasis. 3.3.

Cotargeting heat shock protein 90 and heat shock protein

27 amplifies endoplasmic reticulum stress and treatment-induced apoptosis

Since Hsp90 inhibitors induce Hsp27, and Hsp27 functions as a mediator of ER stress regulation and treatment resistance [22], we next evaluated if Hsp27 knockdown potentiated the effect of Hsp90 inhibition. Combined Hsp27 (using OGX-427) and Hsp90 (with PF-04928473 or 17-AAG) inhibition induced Grp78 and higher levels of CHOP and XBP1 splicing compared with control or each drug alone (Fig. 4A and 4B, Supplemental Fig. 2a). To assess synergy, combination index (CI) values were calculated according to the Chou and Talalay median effect principle [23]. Dose-response curves show that OGX-427 plus PF-04929113 or OGX-427 plus 17-AAG treatments more potently inhibited tumor cell growth than monotherapy (Fig. 4C). The CI curves and plots (lower than one) indicate that OGX-427 synergistically enhanced the effect of both Hsp90 inhibitors on tumor cell growth (Fig. 4C) and induction of apoptosis. Flow cytometric analysis showed that apoptotic rates (subG1 fraction) increased significantly when OGX-427 was combined with PF-04929113 (38%) compared with control ScrB (4.5%), OGX-427 (8.7%), or control ScrB plus PF-04928473 (25.8%) (Fig. 4D). Enhanced apoptosis was confirmed by the caspase-3 activity assay and cleaved-PARP expression (Fig. 4E, 4f, Supplemental Fig. 2b). In addition, OGX-427 blocked PF-094928473-induced Hsp27 expression and decreased

Please cite this article in press as: Lamoureux F, et al. 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. Eur Urol (2014), http://dx.doi.org/10.1016/j.eururo.2013.12.019

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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 mM 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 mM carbobenzoxy-Leu-Leu-leucinal (MG132) for 48 h. Western blotting analyses were performed for Hsp27, Grp78, P-eiF2a, 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.

Akt, AR, and PSA expression when combined with Hsp90 inhibition in LNCaP cells (Fig. 4F). Collectively, these data confirm that cotargeting Hsp27 and Hsp90 amplified ER stress, overwhelming adaptive responses to increase stressinduced apoptosis.

3.4.

OGX-427 potentiates PF-04929113 activity in LNCaP

castrate-resistant prostate cancer xenografts

The effects of combined treatment with OGX-427 and PF04929113 were evaluated in LNCaP CRPC xenografts. All

Please cite this article in press as: Lamoureux F, et al. 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. Eur Urol (2014), http://dx.doi.org/10.1016/j.eururo.2013.12.019

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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 mM 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) Dosedependent 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 (

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.

Although prostate cancer responds initially to androgen ablation therapies, progression to castration-resistant prostate cancer (CRPC) frequently occu...
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