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. Author manuscript; available in PMC: 2017 Jun 1.
Published in final edited form as: Clin Cancer Res. 2015 Dec 28;22(11):2744–2754. doi: 10.1158/1078-0432.CCR-15-2119

Co-targeting Androgen Receptor Splice Variants and mTOR signaling pathway for the Treatment of Castration-Resistant Prostate Cancer

Minoru Kato 1, Carmen A Banuelos 1, Yusuke Imamura 1, Jacky K Leung 1, Daniel P Caley 1, Jun Wang 1, Nasrin R Mawji 1, Marianne D Sadar 1,*
PMCID: PMC4891302  NIHMSID: NIHMS748235  PMID: 26712685

Abstract

Purpose

The PI3K/Akt/mTOR pathway is activated in most castration-resistant prostate cancer (CRPC). Transcriptionally active androgen receptor (AR) plays a role in the majority of CRPC. Therefore, co-targeting full-length (FL) AR and PI3K/Akt/mTOR signaling has been proposed as a possible more effective therapeutic approach for CRPC. However, truncated AR-splice variants (AR-Vs) that are constitutively active and dominant over FL-AR are associated with tumor progression and resistance mechanisms in CRPC. It is currently unknown how blocking the PI3K/Akt/mTOR pathway impacts prostate cancer driven by AR-Vs. Here we evaluated the efficacy and mechanism of combination therapy to block mTOR activity together with EPI-002, an AR N-terminal domain (NTD) antagonist that blocks the transcriptional activities of FL-AR and AR-Vs in models of CRPC.

Experimental design

To determine the functional roles of FL-AR, AR-Vs and PI3K/Akt/mTOR pathways, we employed EPI-002 or enzalutamide and BEZ235 (low dose) or everolimus in human prostate cancer cells that express FL-AR or FL-AR and AR-Vs (LNCaP95). Gene expression and efficacy were examined in vitro and in vivo.

Results

EPI-002 had antitumor activity in enzalutamide-resistant LNCaP95 cells that was associated with decreased expression of AR-V target genes (e.g., UBE2C). Inhibition of mTOR provided additional blockade of UBE2C expression. A combination of EPI-002 and BEZ235 decreased the growth of LNCaP95 cells in vitro and in vivo.

Conclusion

Co-targeting mTOR and AR NTD to block transcriptional activities of FL-AR and AR-Vs provided maximum antitumor efficacy in PTEN-null, enzalutamide resistant CRPC.

Keywords: prostate cancer, androgen receptor splice variant, EPI, PI3K/Akt/mTOR

Introduction

Androgen deprivation therapy (ADT) is initially effective for most recurring prostate cancer (PC). Unfortunately, the malignancy will eventually begin to grow again to form castration resistant prostate cancer (CRPC). Transcriptionally active androgen receptor (AR) plays a major role in CRPC in spite of reduced blood levels of androgen (1, 2). AR mechanisms of resistance to ADT include: overexpression of AR (3, 4); gain-of-function mutations in AR LBD (5); intratumoral androgen synthesis (6); altered expression and function of AR coactivators (7, 8); aberrant post-translational modifications of AR (9, 10); and expression of AR splice variants (AR-Vs) which lack ligand-binding domain (LBD) (1, 11–13). Anti-androgens such as bicalutamide and enzalutamide target AR LBD, but have no effect on truncated constitutively active AR-Vs such as AR-V7 (14). Expression of AR-V7 is associated with resistance to current hormone therapies (14, 15). EPI compounds were developed to specifically target the AR amino-terminal domain (NTD) to block the transcriptional activities of FL-AR and AR-Vs, which results in antitumor activity in CRPC xenografts (11, 12, 16). EPI-506, an analogue of EPI-002, is in clinical trials for CRPC patients that are resistant to enzalutamide or abiraterone.

The PI3K/Akt/mTOR pathway is a key oncogenic pathway in various cancers (17), and is linked to resistance to ADT in PCs (18). Alterations of components in PI3K/Akt/mTOR pathway occur in 42% of primary prostate tumors and 100% of metastatic tumors (19). The PI3K/Akt/mTOR pathway is constitutively active due to loss of PTEN in the majority of advanced prostate cancers (20). Targeting PI3K/Akt/mTOR is therefore considered a promising approach to treat CRPC (21, 22). However, the effects of inhibiting PI3K/Akt/mTOR signaling on AR are controversial (23–26). There are numerous inhibitors that target PI3K/Akt/mTOR signaling such as rapamycin and its analogs, dual TORC1/2 inhibitors, pan-PI3K inhibitors, isoform-specific PI3K inhibitors, Akt inhibitors, and dual PI3K/TORC1/2 inhibitors. Here, we used BEZ235, a dual PI3K/TORC1/2 inhibitor, to achieve better blockade of PI3K/akt/mTOR signaling by inhibiting possible re-activation of PI3K and Akt through mTOR dependent feedback loops (27, 28). Reciprocal feedback regulation of PI3K and FL-AR signaling in PTEN-deficient prostate cancer has been reported (23). From those studies, concomitant blockade of PI3K/Akt/mTOR and FL-AR signaling pathways were proposed to achieve better antitumoral activity in CRPC (29). To date there are no reports that examine the impact of such a combination on human prostate cancer cells that are resistant to antiandrogens and express AR-Vs. Here we evaluate the therapeutic efficacy of a combination of EPI-002 and BEZ235 using CRPC models both in vitro and in vivo.

Materials and Methods

Cells, reporter assays and reagents

LNCaP, COS-1 and DU145 cells, plasmids (PSA-luciferase, PB-luciferase, ARR3-luciferase, 5xGal4UAS-TATA- luciferase, AR1-558Gal4DBD) and transfection protocols are described (7, 11, 12). Cell lines were obtained from the following: LNCaP cells from Dr. Leland Chung (Cedars-Sinai Medical Center, Los Angeles, CA) in September 1993; COS-1 cells from Dr. Rob Kay (BC Cancer Agency, Vancouver, BC) in April 2000; DU145 cells from Dr. Victor Ling (BC Cancer Agency, Vancouver, BC) in October 1998; LNCaP95 cells from Dr. Stephen Plymate (University of Washington, Seattle, WA) in February in 2012. LNCaP and COS-1 cells were not authenticated in our laboratory, but were regularly tested to ensure mycoplasma-free (VenorTMGeM Mycoplasma Detection kit, Sigma-Aldrich, St. Louis, MO). LNCaP95 and DU145cells were authenticated by short tandem repeat (STR) analysis and tested to ensure mycoplasma-free by DDC Medical (Fairfield, OH) in September 2013. All cells used were passaged in our laboratory for fewer than 3 months after resurrection. EPI-002 was provided by NAEJA (Edmonton, Alberta), enzalutamide from Omega Chem (St-Romuald, Quebec), NVP-BEZ235 from SelleckChem (Boston, MA), synthetic androgen, R1881, from Perkin-Elmer (Woodbridge, ON), Interleukin-6 from R&D Systems (Minneapolis, MN), forskolin from EMD Millipore (Billerica, MA). Silencer select siRNA for p110 beta (# s10523, 10524 and 10525), p110 gamma (# s10529, 10530 and 10531), and lipofectamine RNAiMAX were from Life Technologies (Carlsbad, CA). All cells were maintained in culture no more than 10 passages and regularly tested to ensure they were mycoplasma-free.

Cell proliferation BrdU immunoassay

LNCaP95 cells (8,000 cells/well) were seeded in a 96-well plate and incubated for 48hr in RPMI with 10% charcoal stripped serum before pre-treating for 1hr with DMSO, EPI-002 (25 uM), enzalutamide (10 uM), BEZ235 (15nM) and combination of EPI-002 (25 uM) and BEZ235 (15 nM) in serum-free conditions prior to addition of 0.1 nM R1881or EtOH. BrdU incorporation was measured after 2 days using BrdU ELISA kit (Roche Diagnostics).

Transfection and luciferase assay

LNCaP95 cells (150,000 cells/well) and LNCaP cells (100,000 cells/well) in 12-well plates were transfected with PSA-luciferase, ARR3-luciferase or PB-luciferase reporters and pre-treated the next day with DMSO, EPI-002 (25 uM), enzalutamide (10 uM), BEZ235 (15 nM) or its combination for 1 hr before addition of R1881 or EtOH under serum-free and phenol red-free conditions. After 48 hr of incubation, cells were harvested and luciferase activities measured and normalized to protein concentration. Transactivation of AR NTD was measured in LNCaP and LNCaP95 cells as described (7). Transcriptional activity of AR-Vs was performed using AR-negative Cos-1 cells as reported (30). Transient knockdown of p110 β or p110 δ was performed in LNCaP95 cells using lipofectamine RNAiMAX.

Western blot analysis

LNCaP95 cells were serum-starved for 24hr, followed by treatment with DMSO, EPI-002 (25uM), enzalutamide (10 uM), BEZ235 (15nM) or a combination for 1hr prior to addition of R1881 or EtOH for 48hr. Antibodies used were: AR (1:1000; Santa Cruz), AR-V7 (1:400; Precision), p110α (1:500; BD Bioscience), p110β (1:1000; abcam), p100γ (1:1000; abcam), p110δ (1:1000; abcam), UBE2C (Boston Biochem; 1:1000), PTEN (1:1000), pS6 (1:2000), pAktThr308 (1:1000), pAktSer473 (1:2000), p4EBP1 (1:1000), total-Akt (1:1000), total-S6 (1:1000), total-4EBP1 (1:1000), pERK/MAPK (1:1000), total-ERK/MAPK (1:1000) from Cell Signaling technology (Danvers, MA). β-actin (1:10,000, Abcam) was used as a loading control.

Gene expression analysis

LNCaP95 cells (180,000 cells/well) in 6-well plates were serum-starved for 24hr before treating with vehicle, EPI-002 (35 uM), enzalutamide (10 uM), BEZ235 (15 nM) or its combination for 1 hr prior to the addition of R1881 (1nM) or EtOH for 48hr. Total RNA was isolated using PureLink RNA Mini Kit (Life technologies) and reverse transcribed to cDNA with High Capacity RNA-to-DNA Kit (Life Technologies). Quantitative real-time RT-PCR was performed in triplicates for each biological sample. Expression levels were normalized to RPL13A housekeeping gene. Primers are as described (11, 31).

LNCaP95 FL-AR sequence

Total RNA (3ug) from LNCaP95 cells was reverse transcribed using Superscript III First Strand Synthesis Kit (Invitrogen) with 5uM Oligo (dT)20. The coding region of FL-AR was PCR amplified with Platinum DNA Taq Polymerase High Fidelity in 50ul with 1U Taq polymerase, 1mmol/L MgSO4, 0.2mmol/L deoxynucleotide mix, and 2%DMSO with the AR forward primer 5′-AGGGGAGGCGGGGTAAGGGAAGTA-3′ and AR reverse primer 5′-CATGAGCTGGGGTGGGGAAATAGG-3′. The PCR fragment was gel purified and cloned into PCR 2.1 TOPO cloning vector using TOPO TA cloning kit from Invitrogen and transformed into chemically competent TOP10 cells. FL-AR plasmids were sequenced at the NAPS core Unit at UBC (http://naps.msl.ubc.ca).

Animal studies

Six to eight weeks old male NOD-SCID mice were maintained in the Animal Care Facility in the British Columbia Cancer Research Centre. All animal experiments were approved by the University of British Columbia Animal Care Committee. Mice were castrated two weeks before inoculating LNCaP95 cells (5 million cells/tumor) subcutaneously, and divided into 4 groups: vehicle control (N-Methyl-2-pyrrolidone : polyethylene glycol 400 (10 / 90, v / v), n = 10), EPI-002 (100 mg / kg bodyweight, n = 8), BEZ235 (5 mg / kg body weight, n = 10) and combination of EPI-002 (100 mg/kg body weight) and BEZ235 (5 mg / kg body weight) (n = 8). Solutions were prepared fresh daily and application volume was 5 ml / kg body weight / dose. Animals were treated by oral gavage, qd, for 2 weeks when tumors reached approximately 80 mm3. Body weight was measured everyday and tumor volumes were measured twice a week using a caliper by the formula length x width x height x 0.52. Tumors were harvested 1 hr after the last treatment and prepared for western blot analyses, gene expression assays and immunohistochemistry.

Immunohistochemistry

Sections (5 um thick) were processed and endogenous peroxidase blocked. Incubations with anti-pS6 (1:200; cell signaling), anti-UBE2C (1:200; Boston Biochem) and anti-Ki-67 (1:50; Dako) were at 4°C overnight. Antigen was detected with 3,3-diaminobenzidine and counterstaining with hematoxylin. For TUNEL staining, ApopTag® Fluorescein In Situ Apoptosis Detection Kit (MILLIPORE) was used.

Results

EPI-002 inhibited AR-V7, mTOR, and blocked BEZ235-induced FL-AR transcriptional activity

A combination of PI3K inhibitor with antiandrogen is suggested to provide potential therapeutic advantage for the treatment of CRPC (23, 29). Constitutively active AR-Vs lack LBD to which antiandrogens bind. Thus cells that express these variants may be resistant to antiandrogens. No studies are reported that examine combination of PI3K/Akt/mTOR inhibitor plus antiandrogen, or plus an AR NTD antagonist, on prostate cancer cells that express constitutively active AR-Vs.

LNCaP95 human prostate cancer cells are androgen-independent and enzalutamide-resistant (32, 33). Proliferation of LNCaP95 cells is driven by truncated AR-Vs in spite of endogenous expression of functional FL-AR (32, 33). FL-AR from LNCaP95 cells was sequenced and no unique mutations that would confer resistance to enzalutamide were detected (data not shown) relative to the parental LNCaP FL-AR sequence (Accession J03180, 34). The only differences detected between parental LNCaP FL-AR and LNCaP95 FL-AR were in polymorphic regions in the NTD with LNCaP95 FL-AR having 6 extra glutamines and 2 less glycines. The T877A mutation originally detected in parental LNCaP FL-AR was maintained in the LNCaP95 FL-AR. EPI-002 (EPI) is an antagonist of AR activation function 1 (AF-1) that blocks the activity of both full-length and truncated AR species (11, 12, 33).

LNCaP and LNCaP95 cells are PTEN null and express p110δ and p110β isoforms albeit LNCaP95 have lower levels of p110β compared to LNCaP (Fig 1A, left). Using siRNA to p110δ or p110β, revealed that phosphorylation of Akt (pAkt) in LNCaP95 cells depends predominantly upon p110δ (Fig 1A, right). BEZ235 (BEZ) is a dual PI3K/mTOR inhibitor and in cell-free assays has the following IC50s: p110α, 4 nM; mTOR (p70S6K), 6 nM; p110δ, 7 nM; ATR, 21 nM; and p110β, 75 nM (35, 36). At higher concentrations BEZ inhibits EGFR/Erb1 >8.5 uM and many more kinases at >10uM including Akt1, IGF1R, and CDK1 (35). However, the previously reported concentration of 500 nM BEZ that was used to inhibit pAkt in LNCaP cells (23) also inhibited pAkt here in LNCaP95 cells but was associated with enormous cytotoxicity making it difficult to interpret the data. Titration experiments revealed a non-toxic concentration of 15nM BEZ that was subsequently used in all experiments, but at this concentration it did not impact pAkt (Fig 1B, left). In LNCaP95 cells, BEZ (15 nM) inhibited phosphorylation of S6 (pS6) ribosomal protein, an mTOR-regulated protein but not p4EBP1 levels (Fig. 1B, left). Consistent with previous reports, BEZ increased protein levels of FL-AR but unexpected were the decreased levels of UBE2C at 48 hrs which is an AR-V7 target gene (Fig 1B, left). Everolimus, an mTOR inhibitor, reduced pS6 at 10 nM and in the absence of androgen also reduced levels of UBE2C (Fig. 1B, left).

Figure 1. Co-targeting AR-NTD and mTOR with EPI and low dose BEZ or everolimus.

Figure 1

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A, Comparative expression levels of p110 isoforms, pAkt, and pS6 were evaluated in cell lines (left). Knockdown of p110 β (siB1,2,3) or p110 δ (siD1,2,3) in LNCaP95 cells for 48 hrs followed by analyses of levels of pAkt (right). B, Titration experiments of BEZ (left) and everolimus (right). LNCaP95 cells were exposed for 24 or 48 hr to BEZ or 24 hrs to everolimus at various concentrations. Effects of enzalutamide (ENZ), EPI and BEZ on mTOR and AR pathways in LNCaP95 (C) and its parental LNCaP (D) cells. Cells were treated with DMSO, EPI (25 uM), ENZ (10 uM), BEZ (15 nM) or combination for 1 hr prior to the addition of R1881 (1nM) or EtOH for 48 hr. These concentrations were used throughout the manuscript unless stated otherwise.

Combination experiments with BEZ (15nM) were examined in LNCaP95 cells compared to parental LNCaP cells. EPI reduced pS6 levels regardless of androgen status (Fig 1C, left). In the absence of androgen, BEZ increased levels of FL-AR and AR-V7. EPI, but not enzalutamide (ENZ), also reduced expression of UBE2C, consistent with previous reports (12, 33). In the presence of androgen, EPI had no effect on levels of FKBP5, a gene transcriptionally regulated by FL-AR (Fig 1C, right). Protein levels of PSA were undetectable in LNCaP95 cells.

LNCaP cells do not express constitutively active AR-Vs and are androgen sensitive with proliferation dependent on AR. No studies have been reported using a concentration of 15nM BEZ in LNCaP cells. BEZ had no effect on pAKT at this concentration. Combinations of BEZ with ENZ or EPI were substantially better than BEZ monotherapy in blocking pS6. (Fig 1D, left). In the absence of androgen, BEZ increased levels of FL-AR (Fig 1D, right). Consistent with results obtained with LNCaP95 cells, EPI was a poor inhibitor of androgen-induced FKBP5 in spite of being comparable to ENZ in blocking androgen-induced levels of PSA (Fig 1D, right). PSA was increased with BEZ regardless of androgen status. Thus, although BEZ increased protein levels of FL-AR, AR-V7, and possibly downstream target genes of FL-AR (PSA), these increases were partially attenuated by EPI or ENZ.

BEZ increased FL-AR transcriptional activity in LNCaP95 and inhibited transactivation of AR NTD in LNCaP cells

PSA-, ARR3- and PB-luciferase are three well-characterized androgen-induced AR-driven reporter gene constructs. BEZ (15 nM) significantly increased PSA-, ARR3- and PB-luciferase activities in LNCaP95 cells treated with androgen which were blocked by both ENZ and EPI (Fig. 2A). To confirm this change was through inhibition of mTOR, LNCaP95 cells were treated with everolimus (EVE, 10 nM) which yielded a similar increase in PSA-luciferase activity (Fig. 2A, right panel). Importantly, in LNCaP cells BEZ did not enhance the activity of FL-AR in response to androgen (Fig.2B). To address if BEZ affected AR-Vs transcriptional activities, Cos-1 cells that do not express endogenous AR were co-transfected with PB-luciferase and expression vectors for AR-V567es or AR-V7. BEZ had no effect on the transcriptional activities of either AR-V567es or AR-V7 in Cos-1 cells (Fig 2C). Protein levels of AR-V7 and AR-567es were comparable to endogenous levels in LNCaP cells (Fig 2C, lower). To determine if BEZ directly enhanced AR transactivation, we employed the AR NTD transactivation assay using both LNCaP and LNCaP95 cells. The AR NTD is essential for full transcriptional activities (37). In LNCaP cells, transactivation of the AR NTD can be induced with IL-6 or by stimulation of the PKA pathway with forskolin (FSK). In LNCaP95 cells, there is high intrinsic activity of AR NTD that cannot be further induced by stimulation of these pathways. In LNCaP cells, BEZ as well as EPI (positive control) significantly inhibited AR-NTD transactivation induced by IL-6 (Fig 2D). BEZ had no effect on AR-NTD transactivation induced by FSK in LNCaP cells or on the intrinsic activity of AR NTD in LNCaP95. Taken together, BEZ has differential effects on AR transcriptional activities that possibly involve cell-specific differences in signal transduction pathways.

Figure 2. Effect of inhibition of mTOR on AR transcriptional activity.

Figure 2

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LNCaP95 (A) and LNCaP (B) cells transfected with PSA-, ARR3- or PB-luciferase reporters were treated with DMSO, EPI, ENZ, BEZ or combination for 1hr prior to the addition of R1881 for 48hr. LNCaP95 cells transfected with PSA-luciferase reporter were also treated with everolimus (10 nM) or combination with ENZ or EPI (A, right panel). C, Cos-1 cells transfected with PB-luciferase and expression vectors for AR-V567es or AR-V7 were treated with DMSO, EPI, BEZ or combination of EPI and BEZ (left). Ectopic protein levels of AR-V7 and AR-V567es in Cos-1 cells are shown relative to endogenous levels of FL-AR in LNCaP cells (right). D, Transactivation assays of the AR NTD were performed in LNCaP (Left and middle) and LNCaP95 cells (right) cotransfected with p5xGal4UAS-TATA-luciferase and AR NTD-Gal4DBD. EPI, BEZ, or combination of EPI and BEZ were added 1hr before addition of IL-6 (50 ng/ml) or FSK (50 uM) in LNCaP cells and harvested after 24hr. LNCaP95 cells were harvested 24hr after the treatment of indicated compound (right). Luciferase activities are shown as percentage of vehicle control. Data is presented as the mean ± SEM from three independent experiments. One-Way ANOVA, post-hoc Turkey’s multiple comparisons test. * indicate vs DMSO control. # indicate vs BEZ treatment group. n.s.; not statistically significant; *p < 0.05; ** p < 0.01; *** p < 0.001; ****p < 0.0001; ## p < 0.01; ### p < 0.001; #### p < 0.0001.

EPI inhibited FL-AR and AR-V7 regulated genes and BEZ inhibited AR-V7 regulated genes

LNCaP95 cells were used to examine the effects of BEZ and combination therapies on endogenous gene expression regulated by FL-AR and AR-Vs. EPI and ENZ inhibited expression of KLK3, TMPRSS2 and FKBP5, which are genes regulated by FL-AR in response to androgen. BEZ significantly increased androgen-induced levels of PSA transcripts compared to levels induced by androgen alone (Fig 3A, compare column 4 to column 1). In the absence of androgen, BEZ also induced levels of PSA transcript which could be blocked by EPI but not ENZ. No similar effects were observed for TMPRSS2 or FKBP5 in response to BEZ.

Figure 3. EPI and BEZ effects on endogenous genes regulated by FL-AR and AR-V7.

Figure 3

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LNCaP95 cells were treated with DMSO, EPI (35 uM), ENZ, BEZ or combination of ENZ and BEZ or EPI and BEZ for 1 hr prior to the addition of R1881 or EtOH for 48 hr. A, Transcript levels of FL-AR regulated genes KLK3, FKBP5, and TMPRRSS2. B, Transcript levels of AR-V7 regulated genes UBE2C, CDC20, and Akt1. C, Transcript levels of FL-AR and AR-V7. All transcripts were normalized to levels of RPL13A. Error bars represent the mean ± SEM from three independent experiments. One-Way ANOVA, post-hoc Turkey’s multiple comparisons test. * indicate vs DMSO control. † indicate vs EPI treatment group. # indicate vs BEZ treatment group. n.s.; not statistically significant; *p < 0.05; ** p < 0.01; *** p < 0.001; ****p < 0.0001; †† p < 0.01; # p < 0.05; ### p < 0.001; #### p < 0.0001.

AR-V7 regulates a subset of genes that are unique from FL-AR. ENZ increased levels of UBE2C transcripts in cells treated with androgen, while monotherapy with EPI or BEZ attenuated UBE2C levels regardless of androgen (Fig 3B). In the absence of androgen, ENZ had no effect on transcript levels of any of the AR-V7 target genes, contrary to monotherapies with EPI or BEZ that consistently reduced levels of expression of these AR-V7 target genes. Combination of EPI and BEZ were significantly more effective than monotherapies. BEZ did not increase levels of FL-AR transcript (Fig 3C, left). Surprising was the greater than 2-fold increase in transcript levels of AR-V7 induced with BEZ in the absence of androgen which was blocked by EPI (Fig 3C).

Combination therapy with EPI and BEZ significantly reduced CRPC tumor growth

Proliferation of LNCaP95 cells is androgen-independent and driven by AR-V7 (32, 38). As expected, ENZ had no effect on proliferation of these cells (Fig 4A). Thus LNCaP95 cells are ENZ-resistant. EPI or BEZ monotherapies inhibited proliferation with the combination being significantly better than each monotherapy. Everolimus (EVE) also inhibited the proliferation of LNCaP95 cells, indicating that this additional inhibition was by blocking mTOR (Fig 4B). EVE in combination with EPI was significantly better than the monotherapies.

Figure 4. Combination therapy with EPI and BEZ significantly decreased tumor growth both in vitro and in vivo compared to vehicle and each monotherapy treated group.

Figure 4

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A, LNCaP95 cells were treated with DMSO, EPI, ENZ, BEZ or combination of EPI and BEZ for 1hr prior to the addition of R1881 (0.1 nM) for 48 hr. Proliferation was measured by BrdU incorporation. B, LNCaP95 cells were also treated with everolimus (EVE, 10 nM) instead with BEZ235. Proliferation was assessed by BrdU incorporation. Data for “A” and “B” are shown as ratio to vehicle control. C, LNCaP95 tumor growth in castrated mice administered vehicle, a half-dose of EPI (100mg/kg body weight), BEZ (5mg/kg body weight) or combination daily by oral gavage for two weeks. D, Body weight change over the duration of the experiment. E, Western blot analyses of protein lysates from xenografts harvested 1hr after the last treatment (top). Three xenografts from each treatment group are shown. β-actin is a loading control. The ratios of phosphoprotein to total protein are shown for pAkt ser473, pS6 and p4EBP1 (bottom). Error bars represent the mean ± SEM from at least three independent experiments. One-Way ANOVA, post-hoc Turkey’s multiple comparisons test. * indicate vs DMSO control. † indicate vs EPI-002 treatment group. # indicate vs BEZ235 treatment group. n.s.; not statistically significant; *p < 0.05; ** p < 0.01; *** p < 0.001; ****p < 0.0001; † p < 0.05; †† p < 0.01; †††† p < 0.0001; # p < 0.05; ## p < 0.01; #### p < 0.0001.

A small pilot in vivo study was completed to determine the non-toxic oral dose of BEZ that could be administered daily. Doses of BEZ at 45mg/kg body weight resulted in the mortality of 66% of the animals (data not shown). BEZ orally administered daily at 5mg/kg body weight was non-toxic and sufficient to block mTOR but not pAkt in tumors. Therefore a dose of BEZ at 5 mg/kg body weight was used in the following in vivo studies. Castrated mice were daily treated orally either with vehicle (NMP:PEG400, 1:9, v/v), a half-dose of EPI (100mg/kg), BEZ (5mg/kg) or a combination (EPI 100mg/kg + BEZ 5 mg/kg). Final tumor volume in the EPI+BEZ combination group was significantly reduced compared to those in vehicle (DMSO), EPI and BEZ groups (Fig. 4C). There was no significant difference in body weight among the treatment groups (Fig. 4D). Protein levels of FL-AR and AR-V7 in response to BEZ were reduced (Fig 4E) contrary to in vitro results which may be the result of differences in time course, exposure levels and contributions from other signaling pathways in the tumor microenvironment which are not present in vitro. Consistent with in vitro data, protein levels of UBE2C and pS6 were reduced in harvested tumors treated with EPI, BEZ and combination treatment. No significant change observed in levels of pAkt and p4EBP1 when normalized to total Akt or 4EBP1, respectively (Fig. 4E, bottom). In vivo, EPI and BEZ monotherapies reduced protein levels of FKBP5, a FL-AR target gene, and UBE2C, an AR-V7 target gene thereby supporting that the transcriptional activities of FL-AR and AR-Vs were blocked. Immunohistochemical analysis of these same harvested xenografts revealed that EPI and BEZ reduced levels of UBE2C and pS6 staining (Fig. 5A), which were consistent with western blot data. EPI significantly decreased proliferation (Fig. 5B) and increased apoptosis (Fig. 5C) as indicated with staining of Ki67 and TUNEL, respectively.

Figure 5. EPI decreased proliferation, UBE2C and pS6 immunostaining, and increased apoptosis in CRPC LNCaP95 xenografts.

Figure 5

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A, Immunohistochemistry of representative xenografts stained for hematoxylin and eosin (H-E), UBE2C, pS6, Ki-67 and TUNEL. B, % Ki67 and C, % TUNEL positive cells were counted in sections from xenografts for each treatment. At least 3000 cells per xenograft were counted. Cells that were positive for Ki67 or TUNEL staining were counted in sections from 3 xenografts per treatment. The total number of cells counted was as follows: 4,712 (vehicle, Ki67), 4,833 (EPI, Ki67), 5,167 (BEZ, Ki67), 4123 (combination, Ki67), 4502 (vehicle, TUNEL), 3733 (EPI, TUNEL), 4109 (BEZ, TUNEL) and 3715 (combination, TUNEL). Error bar represent the mean ± SEM. One-Way ANOVA post hoc Bonferroni’s multiple comparison test, *p < 0.05; ***p < 0.001; **** p < 0.0001.

Discussion

AR-Vs are a potential mechanism of resistance to abiraterone and enzalutamide in CRPC (14, 39, 40). AR-NTD targeting drugs have benefits over drugs targeting the AR-LBD because the NTD is essential for the transcriptional activities of both FL-AR and AR-Vs. Antagonists of AR-NTD, such as EPI, could therefore provide therapeutic responses for CRPC patients with malignancies that express constitutively active AR-Vs and are resistant to abiraterone or antiandrogens. In addition to AR, the PI3K/Akt/mTOR pathway is implicated as a potential driver of CRPC (21, 26, 41). Previous reports have shown therapeutic benefits for the treatment of CRPC by a combination of antiandrogen with an inhibitor of PI3K/Akt/mTOR (23, 26, 29). However, those studies focused on cross-talk with FL-AR and PI3K/Akt/mTOR pathways. Since CRPC that is resistant to antiandrogens and abiraterone has been shown to be correlated to expression of constitutively active AR-Vs, it is of interest to investigate therapeutic effects and mechanisms by combination treatments using an inhibitor of both FL-AR and AR-Vs, such as EPI, with an inhibitor of PI3K/Akt/mTOR. Here such studies showed the following: 1) A low, non-toxic concentration of BEZ (15 nM) that did not inhibit pAkt was a potent inhibitor of mTOR; 2) Inhibition of mTOR caused an increase in levels of FL-AR (protein) and its target gene PSA (protein and transcript); 3) Inhibition of mTOR also increased levels of AR-Vs, but decreased endogenous expression of its target genes such as UBE2C, CDC20, and Akt1; 4) Inhibition of mTOR decreased the proliferation of enzalutamide-resistant human prostate cancer cells which are considered to be driven by AR-V7. Combination therapy to block mTOR and the AR-NTD provided significantly better suppression of proliferation than individual monotherapies; and 5) Co-targeting PI3K/Akt/mTOR and AR-NTD in vivo was superior to monotherapies and sufficient to suppress FL-AR and AR-Vs transcriptional activities, and decrease the growth of enzalutamide-resistant CRPC xenografts. Together, these findings support the rationale for co-targeting mTOR and AR-NTD (blocks both FL-AR and AR-Vs) signaling pathways for the treatment of CRPC.

Previous work has implicated that inhibition of FL-AR activates Akt through reducing levels of PHLPP (23). Here, ENZ and EPI both decreased mTOR-regulated pS6 while androgen increased pS6. These data suggest that FL-AR regulates mTOR activity, which is consistent with recent studies (42, 43). Importantly, here levels of AR were increased by both BEZ and also EVE without decreasing pAkt. This suggests that mTOR plays an important role in regulating AR protein levels and that Akt was not directly involved. PI3K/Akt inhibitors can have various effects on AR protein levels in prostate cancer cell lines through Akt-independent mechanisms (25). Also surgical and chemical castration is reported to have no effect on activation of Akt and mTOR (26). Here BEZ increased protein levels of FL-AR and AR-V7 without concomitant increases in levels of their respective transcripts. Thus, inhibition of mTOR may regulate AR protein levels through post-translational modifications (44). Taken together, our results suggest the possibility of an alternative mechanism of cross-talk between these pathways apart from a reciprocal feedback regulation of Akt and AR signaling. A model of possible cross-talk mechanisms among FL-AR, AR-V7 and mTOR signaling pathway is shown in Fig. 6.

Fig. 6. Hypothetical model of cross-talk mechanisms between FL-AR, AR-V and mTOR signaling pathways.

Fig. 6

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A, EPI-002 inhibits transcriptional activity of FL-AR and AR-Vs which results in reduced expression of target genes such as PSA and UBE2C respectively. EPI reduces mTOR-regulated pS6. B, mTOR inhibitors such as everolimus (EVE) and low dose of BEZ blocks mTOR with concomitant increases in levels of FL-AR and AR-V. Increased levels of FL-AR lead to increased levels of expression of its target gene, PSA. Levels of AR-V7 target genes such as UBE2C are inhibited possibly by decreased transactivation of AR NTD or other unidentified mechanisms. C, Increased transcriptional activity of FL-AR due to increased levels in response to mTOR inhibition is blocked by AR-NTD antagonist. Combination of AR-NTD antagonist (EPI) and mTOR inhibitor blocks mTOR-regulated pS6 and transcriptional activity of AR-V7 to reduce levels of its target gene, UBE2C. Lines represent effects on activity. Thick arrows represent changes in levels of expression.

Here an important observation was that in spite of increased levels of AR-V7 protein by inhibition of mTOR there was decreased expression of AR-V7 target genes, such as UBE2C, CDC20, and Akt1. Reduced levels of AR-V7 target genes by inhibition of mTOR appeared specific because there was no decrease in expression of genes regulated by FL-AR or other non-AR-V7 mechanisms (e.g. transcript levels of FL-AR or AR-V7). This suggests that inhibition of mTOR might attenuate transactivation of AR-V7 possibly by mechanisms involving posttranslational modifications of AR-Vs or coregulators unique to AR-Vs, but this remains to be established. Paradoxically, increased expression of FL-AR target genes and enhanced reporter activities in response to BEZ seem likely due to induced levels of FL-AR protein rather than increased transactivation because BEZ did not increase the transcriptional activities of ectopic AR-V567es or AR-V7 in Cos-1 cells, or transactivation of AR-NTD. However, cell-specific responses to inhibition of mTOR would not be unexpected due to variation in signaling pathways in different cell lines.

Up-regulation of the FL-AR pathway was, at least in part, blocked here by ENZ and EPI, which supports a rational that co-targeting both PI3K/Akt/mTOR and FL-AR should achieve better efficacy. However, a clinical trial using a combination of everolimus and bicalutamide to block those pathways failed to achieve a better response when compared to bicalutamide monotherapy (45). A possible explanation of the lack of efficacy could be that the FL-AR signaling pathway was not directly related to the CRPC growth observed and perhaps those tumors were driven by AR-Vs which would not be impacted by an inhibitor of the AR-LBD which is consistent with clinical evidence that neither ENZ nor abiraterone can reverse resistance mediated by AR-Vs (15). In accordance with this notion, ENZ did not inhibit the growth of LNCaP95 cells, despite that it effectively blocked FL-AR transcriptional activity. Most importantly, EPI and BEZ, but not ENZ, reduced levels of UBE2C, an AR-V7 target gene.

In conclusion, our findings demonstrate that co-targeting mTOR and AR-NTD to block both FL-AR and AR-Vs showed maximum antitumor efficacy in PTEN-negative ENZ-resistant CRPC with acceptable tolerability. Since AR-LBD targeting drugs may have limited or no effect on AR-Vs, this novel approach may provide a therapeutic advantage for CRPC patients that are resistant to abiraterone or antiandrogens by a mechanism involving expression of AR-Vs.

Translation Relevance.

Truncated constitutively active AR-Vs are correlated to resistance to drugs targeting AR-LBD either directly with antiandrogens, or indirectly with approaches that reduce levels of androgen. EPI-002 is an AR NTD antagonist that inhibits transcriptional activities of FL-AR and AR-Vs. PI3K/Akt/mTOR is activated in advanced prostate cancer and accounts in part for resistance to androgen deprivation therapy. Here we characterised a functional role of combination treatment with EPI-002 and mTOR inhibitors in a model of enzalutamide-resistant CRPC that is driven by AR-Vs. Our findings suggest that inhibition of mTOR activates FL-AR transcriptional activity independent of Akt which has been previously reported to increase expression of AR. Most importantly, inhibition of mTOR decreased levels of expression of AR-V7 target genes, such as UBE2C which is correlated to CRPC. These results support a rational for co-targeting the AR NTD and mTOR for the treatment of CRPC patients.

Acknowledgments

Financial support: This research was supported by grants to MDS from US National Cancer Institute (2R01 CA105304) and the USA Department of Defence (W81XWH-11-1-0551).

Footnotes

Disclosure of Potential Conflicts of Interest: CAB, NRM, JW and MDS are shareholders of ESSA Pharma Inc. MDS receives compensation as a consultant and is a Director and Officer of ESSA Pharma Inc. No potential conflicts of interest were disclosed for the other authors.

Authors’ Contributions

Conception and design: M. Kato, M.D. Sadar

Development of methodology: M. Kato, N. Mawji, M.D. Sadar

Acquisition of data: M. Kato, C. Banuelos, Y. Imamura, J. Leung, D. Caley

Analysis and interpretation of data: M. Kato, N. Mawji, M.D. Sadar

Writing, review and/or revision of the manuscript: M. Kato, M.D. Sadar

Administrative, technical, or material support: N. Mawji, J. Wang, M.D. Sadar

Study supervision: M.D. Sadar

References

  • 1.Karantanos T, Corn PG, Thompson TC. Prostate cancer progression after androgen deprivation therapy: mechanisms of castrate resistance and novel therapeutic approaches. Oncogene. 2013;32:5501–11. doi: 10.1038/onc.2013.206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Harris WP, Mostaghel EA, Nelson PS, Montgomery B. Androgen deprivation therapy: progress in understanding mechanisms of resistance and optimizing androgen depletion. Nature clinical practice Urology. 2009;6:76–85. doi: 10.1038/ncpuro1296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Visakorpi T, Hyytinen E, Koivisto P, Tanner M, Keinanen R, Palmberg C, et al. In vivo amplification of the androgen receptor gene and progression of human prostate cancer. Nature genetics. 1995;9:401–6. doi: 10.1038/ng0495-401. [DOI] [PubMed] [Google Scholar]
  • 4.Koivisto P, Visakorpi T, Kallioniemi OP. Androgen receptor gene amplification: a novel molecular mechanism for endocrine therapy resistance in human prostate cancer. Scandinavian journal of clinical and laboratory investigation Supplementum. 1996;226:57–63. [PubMed] [Google Scholar]
  • 5.Culig Z, Hobisch A, Cronauer MV, Cato AC, Hittmair A, Radmayr C, et al. Mutant androgen receptor detected in an advanced-stage prostatic carcinoma is activated by adrenal androgens and progesterone. Molecular endocrinology. 1993;7:1541–50. doi: 10.1210/mend.7.12.8145761. [DOI] [PubMed] [Google Scholar]
  • 6.Cai C, Chen S, Ng P, Bubley GJ, Nelson PS, Mostaghel EA, et al. Intratumoral de novo steroid synthesis activates androgen receptor in castration-resistant prostate cancer and is upregulated by treatment with CYP17A1 inhibitors. Cancer research. 2011;71:6503–13. doi: 10.1158/0008-5472.CAN-11-0532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Ueda T, Mawji NR, Bruchovsky N, Sadar MD. Ligand-independent activation of the androgen receptor by interleukin-6 and the role of steroid receptor coactivator-1 in prostate cancer cells. The Journal of biological chemistry. 2002;277:38087–94. doi: 10.1074/jbc.M203313200. [DOI] [PubMed] [Google Scholar]
  • 8.Xu J, Wu RC, O’Malley BW. Normal and cancer-related functions of the p160 steroid receptor co-activator (SRC) family. Nature reviews Cancer. 2009;9:615–30. doi: 10.1038/nrc2695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Gioeli D, Paschal BM. Post-translational modification of the androgen receptor. Molecular and cellular endocrinology. 2012;352:70–8. doi: 10.1016/j.mce.2011.07.004. [DOI] [PubMed] [Google Scholar]
  • 10.van der Steen T, Tindall DJ, Huang H. Posttranslational modification of the androgen receptor in prostate cancer. International journal of molecular sciences. 2013;14:14833–59. doi: 10.3390/ijms140714833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Andersen RJ, Mawji NR, Wang J, Wang G, Haile S, Myung JK, et al. Regression of castrate-recurrent prostate cancer by a small-molecule inhibitor of the amino-terminus domain of the androgen receptor. Cancer cell. 2010;17:535–46. doi: 10.1016/j.ccr.2010.04.027. [DOI] [PubMed] [Google Scholar]
  • 12.Myung JK, Banuelos CA, Fernandez JG, Mawji NR, Wang J, Tien AH, et al. An androgen receptor N-terminal domain antagonist for treating prostate cancer. The Journal of clinical investigation. 2013;123:2948–60. doi: 10.1172/JCI66398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Sun S, Sprenger CC, Vessella RL, Haugk K, Soriano K, Mostaghel EA, et al. Castration resistance in human prostate cancer is conferred by a frequently occurring androgen receptor splice variant. The Journal of clinical investigation. 2010;120:2715–30. doi: 10.1172/JCI41824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Li Y, Chan SC, Brand LJ, Hwang TH, Silverstein KA, Dehm SM. Androgen receptor splice variants mediate enzalutamide resistance in castration-resistant prostate cancer cell lines. Cancer research. 2013;73:483–9. doi: 10.1158/0008-5472.CAN-12-3630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Antonarakis ES, Lu C, Wang H, Luber B, Nakazawa M, Roeser JC, et al. AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. The New England journal of medicine. 2014;371:1028–38. doi: 10.1056/NEJMoa1315815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Martin SK, Banuelos CA, Sadar MD, Kyprianou N. N-terminal targeting of androgen receptor variant enhances response of castration resistant prostate cancer to taxane chemotherapy. Molecular oncology. 2014 doi: 10.1016/j.molonc.2014.10.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Fruman DA, Rommel C. PI3K and cancer: lessons, challenges and opportunities. Nature reviews Drug discovery. 2014;13:140–56. doi: 10.1038/nrd4204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Kaarbo M, Mikkelsen OL, Malerod L, Qu S, Lobert VH, Akgul G, et al. PI3K-AKT-mTOR pathway is dominant over androgen receptor signaling in prostate cancer cells. Cellular oncology : the official journal of the International Society for Cellular Oncology. 2010;32:11–27. doi: 10.3233/CLO-2009-0487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, Carver BS, et al. Integrative genomic profiling of human prostate cancer. Cancer cell. 2010;18:11–22. doi: 10.1016/j.ccr.2010.05.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.McMenamin ME, Soung P, Perera S, Kaplan I, Loda M, Sellers WR. Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced stage. Cancer research. 1999;59:4291–6. [PubMed] [Google Scholar]
  • 21.Bitting RL, Armstrong AJ. Targeting the PI3K/Akt/mTOR pathway in castration-resistant prostate cancer. Endocrine-related cancer. 2013;20:R83–99. doi: 10.1530/ERC-12-0394. [DOI] [PubMed] [Google Scholar]
  • 22.Sarker D, Reid AH, Yap TA, de Bono JS. Targeting the PI3K/AKT pathway for the treatment of prostate cancer. Clinical cancer research : an official journal of the American Association for Cancer Research. 2009;15:4799–805. doi: 10.1158/1078-0432.CCR-08-0125. [DOI] [PubMed] [Google Scholar]
  • 23.Carver BS, Chapinski C, Wongvipat J, Hieronymus H, Chen Y, Chandarlapaty S, et al. Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer. Cancer cell. 2011;19:575–86. doi: 10.1016/j.ccr.2011.04.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Mulholland DJ, Tran LM, Li Y, Cai H, Morim A, Wang S, et al. Cell autonomous role of PTEN in regulating castration-resistant prostate cancer growth. Cancer cell. 2011;19:792–804. doi: 10.1016/j.ccr.2011.05.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Liu L, Dong X. Complex impacts of PI3K/AKT inhibitors to androgen receptor gene expression in prostate cancer cells. PloS one. 2014;9:e108780. doi: 10.1371/journal.pone.0108780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Zhang W, Zhu J, Efferson CL, Ware C, Tammam J, Angagaw M, et al. Inhibition of tumor growth progression by antiandrogens and mTOR inhibitor in a Pten-deficient mouse model of prostate cancer. Cancer research. 2009;69:7466–72. doi: 10.1158/0008-5472.CAN-08-4385. [DOI] [PubMed] [Google Scholar]
  • 27.Schwartz S, Wongvipat J, Trigwell CB, Hancox U, Carver BS, Rodrik-Outmezguine V, et al. Feedback suppression of PI3Kalpha signaling in PTEN-mutated tumors is relieved by selective inhibition of PI3Kbeta. Cancer cell. 2015;27:109–22. doi: 10.1016/j.ccell.2014.11.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.O’Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D, et al. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer research. 2006;66:1500–8. doi: 10.1158/0008-5472.CAN-05-2925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Thomas C, Lamoureux F, Crafter C, Davies BR, Beraldi E, Fazli L, et al. Synergistic targeting of PI3K/AKT pathway and androgen receptor axis significantly delays castration-resistant prostate cancer progression in vivo. Molecular cancer therapeutics. 2013;12:2342–55. doi: 10.1158/1535-7163.MCT-13-0032. [DOI] [PubMed] [Google Scholar]
  • 30.Banuelos CA, Lal A, Tien AH, Shah N, Yang YC, Mawji NR, et al. Characterization of niphatenones that inhibit androgen receptor N-terminal domain. PloS one. 2014;9:e107991. doi: 10.1371/journal.pone.0107991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Zhang X, Morrissey C, Sun S, Ketchandji M, Nelson PS, True LD, et al. Androgen receptor variants occur frequently in castration resistant prostate cancer metastases. PloS one. 2011;6:e27970. doi: 10.1371/journal.pone.0027970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Hu R, Lu C, Mostaghel EA, Yegnasubramanian S, Gurel M, Tannahill C, et al. Distinct transcriptional programs mediated by the ligand-dependent full-length androgen receptor and its splice variants in castration-resistant prostate cancer. Cancer research. 2012;72:3457–62. doi: 10.1158/0008-5472.CAN-11-3892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Yang YC, Meimetis LG, Tien AH, Mawji NR, Carr G, Wang J, et al. Spongian diterpenoids inhibit androgen receptor activity. Molecular cancer therapeutics. 2013;12:621–31. doi: 10.1158/1535-7163.MCT-12-0978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Lubahn DB, Joseph DR, Sar M, Tan J, Higgs HN, Larson RE, et al. The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate. Mol Endocrinol. 1988;2:1265–1275. doi: 10.1210/mend-2-12-1265. [DOI] [PubMed] [Google Scholar]
  • 35.Maira SM, Stauffer F, Brueggen J, Furet P, Schnell C, Fritsch C, et al. Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity. Molecular cancer therapeutics. 2008;7:1851–63. doi: 10.1158/1535-7163.MCT-08-0017. [DOI] [PubMed] [Google Scholar]
  • 36.Chiarini F, Grimaldi C, Ricci F, Tazzari PL, Evangelisti C, Ognibene A, et al. Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against T-cell acute lymphoblastic leukemia. Cancer research. 2010;70:8097–107. doi: 10.1158/0008-5472.CAN-10-1814. [DOI] [PubMed] [Google Scholar]
  • 37.Quayle SN, Mawji NR, Wang J, Sadar MD. Androgen receptor decoy molecules block the growth of prostate cancer. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:1331–6. doi: 10.1073/pnas.0606718104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Hu R, Dunn TA, Wei S, Isharwal S, Veltri RW, Humphreys E, et al. Ligand-independent androgen receptor variants derived from splicing of cryptic exons signify hormone-refractory prostate cancer. Cancer research. 2009;69:16–22. doi: 10.1158/0008-5472.CAN-08-2764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Yu Z, Chen S, Sowalsky AG, Voznesensky OS, Mostaghel EA, Nelson PS, et al. Rapid induction of androgen receptor splice variants by androgen deprivation in prostate cancer. 2014 doi: 10.1158/1078-0432.CCR-13-1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Liu LL, Xie N, Sun S, Plymate S, Mostaghel E, Dong X. Mechanisms of the androgen receptor splicing in prostate cancer cells. Oncogene. 2014;33:3140–50. doi: 10.1038/onc.2013.284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Edlind MP, Hsieh AC. PI3K-AKT-mTOR signaling in prostate cancer progression and androgen deprivation therapy resistance. Asian journal of andrology. 2014;16:378–86. doi: 10.4103/1008-682X.122876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Wu Y, Chhipa RR, Cheng J, Zhang H, Mohler JL, Ip C. Androgen receptor-mTOR crosstalk is regulated by testosterone availability: implication for prostate cancer cell survival. Anticancer research. 2010;30:3895–901. [PMC free article] [PubMed] [Google Scholar]
  • 43.Munkley J, Rajan P, Lafferty NP, Dalgliesh C, Jackson RM, Robson CN, et al. A novel androgen-regulated isoform of the TSC2 tumour suppressor gene increases cell proliferation. Oncotarget. 2014;5:131–9. doi: 10.18632/oncotarget.1405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Cinar B, De Benedetti A, Freeman MR. Post-transcriptional regulation of the androgen receptor by Mammalian target of rapamycin. Cancer research. 2005;65:2547–53. doi: 10.1158/0008-5472.CAN-04-3411. [DOI] [PubMed] [Google Scholar]
  • 45.Nakabayashi M, Werner L, Courtney KD, Buckle G, Oh WK, Bubley GJ, et al. Phase II trial of RAD001 and bicalutamide for castration-resistant prostate cancer. BJU international. 2012;110:1729–35. doi: 10.1111/j.1464-410X.2012.11456.x. [DOI] [PubMed] [Google Scholar]

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