Targeting Androgen Receptor Leads to Suppression of Prostate Cancer via Induction of Autophagy

Targeting Androgen Receptor Leads to Suppression of Prostate Cancer via Induction of Autophagy Qi Jiang, Shuyuan Yeh, Xiaohai Wang, Defeng Xu, Qiaoxia Zhang, Xinquin Wen, Shujie Xia* and Chawnshang Chang* From the George Whipple Laboratory for Cancer Research, Departments of Pathology and Urology, University of Rochester Medical Center (QJ, SY, XW, CC), Rochester, New York, Department of Urology, Shanghai First People’s Hospital, Shanghai Jiaotong University (QJ, XW, SX), Shanghai, Sex Hormone Research Center, Shenzhen Hematology Institute, First Affiliated Hospital of Shenzhen University (QZ), Shenzhen and Department of Urology, Third Affiliated Hospital, Sun Yat-Sen University (XW), Guangzhou, People’s Republic of China, and Sex Hormone Research Center, China Medical University/Hospital (CC), Taichung, Taiwan, Republic of China

Purpose: Androgen receptor has a critical role in prostate cancer development and progression. Cell death via autophagy may also contribute to prostate cancer progression. We determined the role and regulatory effects of androgen receptor on the autophagy process of prostate cancer cells. Materials and Methods: Using a series of morphological approaches, such as transmission electron microscopy, monodansylcadaverine (Sigma®) and GFPLC3 fluorescence microscopy assay, and Western blot we monitored the autophagic process in 3 pairs of prostate cancer cell lines to study the relationship to androgen receptor signals. Results: Androgen receptor knockdown in androgen receptor positive cells, such as LNCaP or CWRrv1 human prostate cancer cells, led to increased autophagy. Adding functional androgen receptor to androgen receptor negative cells, such as PC3 human prostate cancer cells, resulted in decreased autophagy. This suggests that androgen receptor could have a negative role in regulating autophagy. Mechanism dissection indicated that androgen receptor might repress autophagy via modulation of p62 expression. A therapeutic approach of targeting androgen receptor to increase autophagy using the androgen receptor degradation enhancer ASC-J9® suppressed prostate cancer growth. Conclusions: Findings provide evidence that the androgen receptor might promote prostate cancer cell growth via autophagy down-regulation. Targeting the androgen receptor via ASC-J9 might lead to tumor suppression via the induction of autophagy. This may represent a new, potential therapeutic approach and mechanism for prostate cancer.

Abbreviations and Acronyms AR ⫽ androgen receptor CHIP ⫽ chromatin immunoprecipitation DHT ⫽ dihydrotestosterone FM ⫽ fluorescence microscopy MDC ⫽ monodansylcadaverine PCa ⫽ prostate cancer PCR ⫽ polymerase chain reaction RAPA ⫽ rapamycin RT ⫽ reverse transcriptase Submitted for publication January 4, 2012. Supported by National Institutes of Health Grant CA156700, the George Whipple Professorship Endowment, and Taiwan Department of Health Clinical Trial and Research Center of Excellence grant DOH99-TD-B-111-004 (China Medical University, Taichung, Taiwan). * Correspondence: E-mail: xsjurologist@163. com and [email protected]).

Key Words: prostate; prostatic neoplasms; autophagy; receptors, androgen; 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,4,6-heptatrien-3-one AUTOPHAGY is a catabolic process involving physiological turnover of longlived proteins and damaged organelles in the autophagosomes, which are double membrane structures1,2 that perform this turnover of cytosolic proteins and remove unwanted organelles. Autophagy was proposed as an

important process for cell death, in addition to apoptosis and necrosis.3 However, the role of autophagy in cancer cell death is in dispute.4 Some studies show that autophagy has a role in preventing apoptosis in cancer cells due to nutritional stress.5 In contrast, others demonstrate that autophagy has an

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anticancer effect.4,6 Early reports indicated that autophagy might induce PCa cell death7,8 but detailed mechanisms remain unclear. AR has a critical role in PCa development and progression.9 However, the regulatory role of AR in the autophagic process remains poorly understood. Parikh et al used statin as an inducer, which induced autophagy in PCa PC3 AR negative cells but not in LNCaP AR positive cells.7 In contrast, Li et al found that androgen (DHT) treatment inhibited autophagy in LNCaP cells but not in PC3 cells.10 Due to these contrasting results the roles of androgen/AR signals in altering PCa cell autophagy remains unclear. We determined the role and regulatory effects of AR on the autophagy process of PCa cells and speculated about new, potential therapeutic approaches to PCa treatment.

MATERIALS AND METHODS Cell Culture and Treatments In the current study 3 pairs of PCa cell lines were used, including PC3 and PC3AR9, CWR22R-kdAR and CWR22R, and LNCaP-siAR and LNCaP-sc. PC3 and PC3AR9, and CWR22R-kdAR and CWR22R cells were maintained at 37C in 5% CO2 in complete RPMI 1640 medium supplemented with 10% fetal bovine serum. CWR22R-kdAR is an AR knockdown cell line from CWR22R.11,12 PC3AR9 is a PCa PC3 cell line with the addition of functional AR.13 Prepared LNCaP cells with silenced AR and scrambled AR (LNCaP-siAR and LNCaP-sc, respectively) were maintained according to the protocol.14 After plating in RPMI 1640 medium supplemented with 10% charcoal stripped fetal bovine serum and antibiotics at 37C at 5% CO2 overnight, cells were treated with 100 nM RAPA (Calbiochem®), and 1 and 10 nM DHT (Sigma). At 24 hours cells were harvested for experiments. For ASC-J9 experiments, cells were treated with 5 ␮M ASC-J9 24 hours before RAPA and DHT treatments.

Transmission Electron Microscopy After 24-hour 100 nM RAPA treatment, cells were trypsinized, fixed for 24 hours with 2.5% glutaraldehyde in 0.1 M sodium cacodylate, rinsed twice in the same buffer and post fixed for 30 minutes using buffered 1.0% osmium tetroxide. Cells were rinsed in buffer, trapped in agarose, treated with 0.5% uranyl acetate for 1 hour in the dark at room temperature and dehydrated in a graded series of ethanol to 100%. They were transitioned to propylene oxide, infiltrated in Epon®/Araldite® resin for 24 hours, embedded in molds and polymerized for 48 hours at 70C. Blocks were cut at 1 ␮ on glass slides using an ultramicrotome to determine area into 70 nm sections with a diamond knife. The thin sections were collected on 200 mesh nickel grids and stained with aqueous uranyl acetate and lead citrate. Grids were examined with a 7650 transmission electron microscope (Hitachi, Tokyo, Japan). Cells were photographed using an attached 11 megapixel

Erlangshen digital camera (Gatan, Pleasanton, California).

Assays Monodansylcadaverine. Cells growing on coverslips were incubated with 0.05 mM MDC added directly to culture medium. After incubation at 37C for 10 minutes cells were fixed in 4% paraformaldehyde for 15 minutes and immediately analyzed using a fluorescence microscope (Nikon, Tokyo, Japan) with 380 and 525 nm wavelength excitation and emission filters, respectively. GFP-LC3 FM. For GFP-LC3 FM assay cells were plated in 6-well plates and transduced with GFP-LC3 according to the reagent protocol (Invitrogen™). At 24 hours cells were subjected to various treatments. They were subsequently fixed in 4% paraformaldehyde for 5 minutes and analyzed using a fluorescence microscope with 485 and 520 nm wavelength excitation and emission filters, respectively. MDC and GFP-LC3 assay results were ranked, including 1— 0 to 4 punctates per cell, 2—5 to 9, 3—10 to 14, 4 —15 to 19 and 5—more than 19. Cell viability. MTT solution (5 mg/ml in phosphate buffered saline) was added to each well of the 24-well plate and incubated for 4 hours at 37C. Supernatant was discarded and 0.5 ml dimethyl sulfoxide per well were added to dissolve the formazan. Absorbance was immediately measured at 570 nm using an absorbance reader (Bio-Rad®). Western blot. Cells (5 ⫻ 105) were plated in 10 cm2 plates for various treatments. At 24 hours cells were harvested. Total cellular protein was extracted from cell pellets by homogenization in RIPA buffer containing 10 mM TrisHCl (pH 8.0), 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.8% Triton X-100 and protease inhibitor cocktail (Roche, Mannheim, Germany) at 4C for 30 minutes. Protein samples with 20 to 40 ␮g protein per sample were subjected to electrophoretic analysis in l5%/5% sodium dodecyl sulfate stacked polyacrylamide gels and then electrotransferred to polyvinylidene fluoride membranes. Membranes were blocked for 1 hour in Tris-buffered saline-Tween-20, composed of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl and 0.1% Tween 20, with 5% nonfat milk powder. Membranes were washed and probed with primary antibody at 4C overnight. After washing membranes were incubated for 1 hour at room temperature with horseradish peroxidase linked secondary antibody. All bands were normalized to actin.

Real-Time RT-PCR After 24-hour treatment, total cellular RNA was extracted using TRIzol® reagent according to the manufacturer recommended protocol. Total RNA (1 ␮g) was used to generate cDNA. The RT reaction was done using an RT system procedure. RT product (1 ␮l) served as the template in a 20 ␮l PCR reaction using SYBR® Premix Ex Taq™ PCR Mastermix. After initial denaturation at 95C for 10 minutes 45 cycles were performed with a denaturation step at 95C for 30 seconds, annealing at 60C for 30 seconds and extension at 72C for 30 seconds. Primers were designed using Primer3 (http://frodo.wi.mit.edu/). As an internal control, cDNA for GAPDH was amplified. Primer se-

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Figure 1. Altered AR expression led to autophagy modulation in PCa cells. Autophagosome formation was detected in PC3 (A to D) and PC3AR9 (E to H) cells by TEM after 24 hours of 100 nM RAPA treatment. Sham treatment served as control. Arrows indicate autophagosome structures. Scale bars 1 ␮ (A, C, E and G), 0.5 ␮ (B, F and H) and 0.2 ␮ (D).

quences were 5=-CTC CTC CAC CTT TGA CGC TG-3= (forward) and 5=-CAT ACC AGG AAA TGA GCT TGA CAA-3= (reverse) for GAPDH, 5=-TGT CCA TCT TGT CGT CTT C-3= (forward) and 5=-CCT CTC CTT CCT CCT GTA G-3= (reverse) for AR, and 5=-CGG CTT CCA GGC GCA CTA CC-3= (forward) and 5=-GGT CCC GCC GGC ACT CTT TT-3= (reverse) for p62. Each sample was run in triplicate. Target gene relative expression was estimated using the 2(–␦␦ threshold count) method.

Chromatin Immunoprecipitation CHIP was performed in LNCaP cells, as previously reported.15 immunoprecipitation was done at 4C overnight with 3 ␮g rabbit polyclonal antibody AR (NH27) and normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, California). The primer sequences used for the p62 promoter 5= upstream region were p62 (sense) 5=-CAT GGC AGG GTC GGA ACA G-3= and (antisense) 5=-TGA CTC AGC AAT ATC CTC AGT TGG-3=.

p62 siRNA Transfection Cells were seeded at 50% confluence per well in 6-well plates overnight. They were transfected with p62 siRNA or Non-Targeting siRNA (Dharmacon, Denver, Colorado) using Lipofectamine™ 2000. Transfected cells were used for experiments at 24 hours.

Statistical Analysis Statistically significant differences were identified with SPSS®, version 14. Statistical analysis was done by ANOVA and, when only 2 values were compared, by the paired Student t test. Variations are represented by the SD. Significance was considered at p ⬍0.05.

RESULTS Functional AR Led to Decreased AR Negative PCa Cell Autophagy The autophagic process can be analyzed using autophagic inducers, such as RAPA, tamoxifen, etc. RAPA directly inhibits mammalian target of RAPA and represents the most commonly used, specific autophagy inducer.16,17 To study the potential roles of AR during changes in autophagosome morphology we first stably transfected PC3 cells with functional AR-cDNA, whose expression is regulated by a 5.6 kb promoter of human AR (PC3-AR9). We then used the transmission electron microscopy assay to compare the AR effect on autophagy.18 After 24 hours RAPA treatment, human PCa PC3-AR9 cells had less autophagosome formation than PC3 cells with transfected vector (fig. 1), suggesting that AR negatively modulates autophagy in PC3 cells. PCa AR Negative Role in Autophagy in Androgen Dependent and Castration Resistant PCa Cells To further verify that AR modulated autophagy in more than 1 PCa cell type and eliminate the possible artificial effect due to AR over expression in PC3 cells we applied a further strategy to modulate AR expression. We used siRNA to knock down AR in another 2 human PCa cell lines, CWR22R (named CWR22R-kdAR) and LNCaP (named LNCaP-siAR). Results revealed relative AR expression in these cells (fig. 2).

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Figure 2. AR had negative role in regulating autophagy after 24-hour RAPA treatment. AR and LC3 protein expression was detected by Western blot in prostate cancer cell line pairs PC3 and PC3AR9, CWR22R-kdAR and CWR22R, and LNCaP-siAR and LNCaP-sc (A). Actin served as loading control. Using MDC assay (B) and GFP-LC3 FM (C) autophagosomes were detected in AR negative/knockdown cell lines PC3, CWR22R-kdAR and LNCaP-siAR, and AR positive cell lines PC3AR9, CWR22R and LNCaP-sc. Cell scores were nonnormally distributed and are shown as mean of at least 20 per group. Single asterisk indicates p ⬍0.05. Double asterisks indicate p ⬍0.01.

TARGETING ANDROGEN RECEPTOR LEADS TO SUPPRESSION OF PROSTATE CANCER

After RAPA treatment we examined the expression of LC3II, an essential marker recruited to the autophagosome membrane.19 As reported,20 increased LC3II expression (LC3II-to-internal control protein ratio) correlated well with the autophagosome accumulation. Consistently PC3 cells without AR, and CWR22R-kdAR and LNCaP-siAR cells with AR knockdown had more LC3II protein expression than cells with higher AR expression, including PC3AR9, CWR22R and LNCaP-sc cells (fig. 2, A). The groups with 10 and 1 nM DHT (representing the prostate DHT concentration before and after androgen deprivation therapy, respectively), and ethanol showed little difference in LC3II expression (fig. 2, A). This suggests that AR may function as a negative regulator of autophagy in the presence of different androgen concentrations. We then examined autophagosome formation after RAPA treatment in the 3 pairs of human PCa cells in the presence of 10 and 1 nM DHT using different assays. On MDC staining assay we first detected punctate structures, indicating autophagosome formation,21 in cells after RAPA treatment. The number of MDC stained, punctate structures was greater in PC3, CWR22R-kdAR and LNCaPsiAR cells than in PC3AR9, CWR22R and LNCaP-sc cells (fig. 2, B). We stably transfected these cells with GFP-LC3 fusion protein, which is a specific marker of autophagosomes, and analyzed them by FM and found similar results. The number of punctates marked with GFP-LC3 remained less in PC3AR9, CWR22R and LNCaP-sc cells compared to PC3, CWR22RkdAR and LNCaP-siAR cells after incubation with RAPA (fig. 2, C). AR Repressed Autophagy via p62 Modulation To determine potential molecular mechanisms and better understand how AR represses autophagy we examined the expression of p62, which has important roles in autophagy. AR positively regulated p62 protein and mRNA expression in the 3 pairs of PCa cell lines tested (fig. 3, A and B). To further determine the mechanism by which AR regulates p62 we performed CHIP assay in LNCaP cells. AR could bind to the p62 promoter and regulate p62 transcription in the presence of different androgen concentrations (fig. 3, C). We then detected p62 expression after inducing autophagy in the 3 pairs of PCa cells with RAPA. There was no significant difference in p62 expression when cells were vs were not treated with DHT. Results revealed that adding AR to PC3-AR9 cells led to higher p62 expression while AR knockdown in CWR22R-kdAR and LNCaP-siAR cells resulted in decreased p62 expression (fig. 3, D).

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To further validate that AR up-regulation of p62 is essential for AR to suppress autophagy we knocked down p62 using siRNA in PC3AR9, CWR22R and LNCaP-sc AR positive cells (fig. 3, E). Expression of the autophagic marker LC3II increased after p62 knockdown in AR positive cells (fig. 3, F), suggesting that p62 is essential for mediating AR down-regulated autophagy. AR Targeting by ASC-J9 Induced Autophagy and Suppressed PCa Cell Growth Early studies documented that autophagy has important roles in PCa progression.7,8,10 Thus, we tested the impact on PCa cell growth by targeting AR mediated autophagy. Results demonstrated that AR could function as a negative regulator to modulate autophagy regardless of the androgen concentration in various PCa cells (fig. 1 to 3). This suggests that targeting androgen via classic antiandrogens to suppress/ decrease androgen binding to AR may have little effect on this AR mediated autophagy. Thus, we applied the AR degradation enhancer ASC-J9, which selectively degrades AR in PCa cells and suppresses PCa cell growth,22 to target AR mediated autophagy. ASC-J9 (5 ␮M) degraded AR in PC3-AR9 and CWR22R cells at the human physiological concentration of 10 nM DHT (fig. 4, A). The consequences of degraded AR led to induced autophagy, which was accompanied by suppressed PCa cell growth (fig. 4, B and C).

DISCUSSION Much evidence supports a role for autophagy in sustaining cell survival but some data support the notion that cell death resulting from progressive cellular consumption may be attributable to unrestrained autophagy.23 Also, early studies suggested that several commonly activated oncogenes, such as PI3K, PKB, TOR and Bcl-2, inhibit autophagy while some commonly mutated or epigenetically silenced tumor suppressor genes, such as p53, PTEN, DAPk and TSC1/ TSC2, stimulate autophagy.24 These findings indicate that autophagy is positively linked to the tumor suppressor pathway. Autophagy can suppress cancer progression via several pathways.25 The most obvious one is by suppressing tumorigenesis through its death promoting effects. Autophagy can also affect tumor progression by preventing DNA damage and negatively regulating cell growth. What role does autophagy have in PCa? Parikh7 and Suh8 et al found that autophagy could induce cell death rather than have cytoprotective effects in PCa cells. These observations suggest that autophagy has a protective role against PCa. In our study we detected more autophagy signals induced by RAPA treatment in AR negative PC3 cells than in

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Figure 3. AR repressed autophagy via p62. AR and p62 protein (A) and mRNA (B) expressions were detected in AR negative/knockdown cell lines PC3, CWR22R-kdAR and LNCaP-siAR, and AR positive cell lines PC3AR9, CWR22R and LNCaP-sc. KD, kDa. CHIP using 10 nM DHT as AR antibody revealed that AR bound to promoter regions of p62 in androgen independent way (C). Quantitative PCR values were normalized to input and plotted as relative enrichment in 3 preparations. Single asterisk indicates p ⬍0.05. Double asterisks indicate t test p ⬍0.01. p62 expression was detected in 3 pairs of PCa cells when autophagy was induced by RAPA in presence of 0, 1 and 10 nM DHT (D). p62 was knocked down by p62 siRNA in PC3AR9, CWR22R and LNCaP-sc cells (E). Autophagic marker LC3II was examined by Western blot (F).

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Figure 4. ASC-J9 induced autophagy via AR degradation after 5 ␮M ASC-J9 treatment with 100 nM RAPA and 10 nM DHT or vehicle in PC3AR9 and CWR22R cells. AR, p62 and LC3II protein expression on Western blot (A). MTT assay showed viability of PC3AR9 (B) and CWR22R (C) cells.

AR positive PC3-AR9 cells. AR is a critical transcription factor for regulating prostate development as well as carcinogenesis. Our findings indicate that AR is a negative regulator of autophagy in androgen dependent and castration resistant PCa cells. To further verify this finding we identified more autophagosome formation in AR negative and AR knockdown PCa cells than in AR positive cells. The same LC3II protein expression was observed. These findings consistently verify that AR has a negative role in regulating PCa autophagy. To mimic the human androgen concentration before and after castration we treated cells with 10 and 1 nM DHT. There were no significant differences among the 10 and 1 nM, and ethanol treatment groups. p62 is a scaffold protein involved in multiple signaling pathways.26 It binds ubiquitin and LC3, and regulates the formation of protein aggregates.27 An early report indicated that p62 accumulation was sufficient to activate the DNA damage response and enhance tumor growth while tumorigenesis could be suppressed by eliminating p62.28 Over expression of p62 is linked to several human disorders, especially neurodegenerative disease and hepatocellular carcinoma.29 However, the molecular mechanisms of p62 in autophagy deficient conditions and its pathophysiological roles in PCa remain unclear. Mathew et al reported that sustained p62 expression resulting from autophagy defects was sufficient to promote tumorigenesis.28 We found that an overabundance of p62 could suppress PCa cell autophagy. Also, AR could bind to the p62 promoter to up-regulate p62 protein and mRNA levels under the different condi-

tions with different androgen concentrations to exert a suppression effect on autophagy. p62 knockdown impaired AR regulated autophagy, an indication that AR can function via p62 to modulate autophagic signals. Since the AR regulated autophagy pathway was found under different conditions at different androgen concentrations, traditional androgen deprivation therapy to decrease the androgen concentration and treat PCa may not reverse this pathway. Thus, we applied the AR degradation enhancer ASC-J9 to degrade AR in AR positive cells. Results revealed increased autophagy and decreased cell growth compared to those of sham treated AR positive cells. Accordingly targeting AR with ASC-J9 leads to more autophagy, which may result in further suppression of PCa growth.

CONCLUSIONS To our knowledge we identified a new functional mechanism of AR in PCa. Targeting AR to promote autophagy, for example by applying ASC-J9 to degrade AR, may represent a new, potential therapeutic approach to PCa in the future.

ACKNOWLEDGMENTS Karen Bentley, Electron Microscope Research Core, University of Rochester, performed electron microscope studies. Dr. Rennie, University of British Columbia, provided LNCaP-siAR and LNCaP-sc cells. ASC-J9 was provided by AndroScience Corp.

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