Cooperative Interactions Between Androgen Receptor (AR) and Heat

Research Article

Cooperative Interactions between Androgen Receptor (AR) and Heat-Shock Protein 27 Facilitate AR Transcriptional Activity Amina Zoubeidi, Anousheh Zardan, Eliana Beraldi, Ladan Fazli, Richard Sowery, Paul Rennie, Colleen Nelson, and Martin Gleave

The Prostate Centre, Vancouver General Hospital, and Department of Urological Sciences, University of British Columbia, Vancouver, British Columbia, Canada

Abstract (ARE) in the transcriptional regulatory regions of target genes (1, 2). In addition to this transcriptional genomic action, androgens Androgen receptor (AR) transactivation is known to enhance prostate cancer cell survival. However, the precise effectors and other steroid hormones like progesterone and estrogen can by which the prosurvival effects of androgen and AR drive exert rapid nongenomic effects that are not mediated through prostate cancer progression are poorly defined. Here, we nuclear receptors, but rather initiated at the plasma membrane, identify a novel feed-forward loop involving cooperative presumably through surface receptors (3, 4). interactions between ligand-activated AR and heat-shock Like progesterone (PR) and estrogen (ER) receptors, AR can interact with the intracellular tyrosine kinase c-Src to activate protein 27 (Hsp27) phospho-activation that enhance AR the mitogen-activated protein kinase (MAPK) pathway (5, 6). In stability, shuttling, and transcriptional activity, thereby androgen-sensitive LNCaP prostate cancer cells, AR interacts with increasing prostate cancer cell survival. Androgen-bound AR 78 82 the SH3 domain of Src within minutes of androgen treatment to induces rapid Hsp27 phosphorylation on Ser and Ser promote cell survival, proliferation, and differentiation (5, 6). residues in an AR- and p38 kinase–dependent manner. After Androgens also rapidly stimulate Raf-1 and Erk-2, both compo- this androgen-induced, non-nuclear phospho-activation, nents of the MAPK signaling cascade (5), suggesting that androgen- Hsp27 displaces Hsp90 from a complex with AR to chaperone stimulated MAPK activation occurs via nongenomic mechanisms. AR into the nucleus and interact with its response elements to This nongenomic action of androgen can also influence classic enhance its genomic activity. Inhibition of Hsp27 phosphor- genomic AR activity, including modulation of AR by coactivators ylation, or knockdown using the antisense drug OGX-427, (7). Phosphoinositide-3-kinase (PI3K)/Akt is another pathway shifted the association of AR with Hsp90 to MDM2, increased involved in nongenomic activity of ER and PR (8, 9), as well as proteasome-mediated AR degradation, decreased AR tran- AR (3, 10). In fact, androgen-activated PI3K and Akt enhance cell scriptional activity, and increased prostate cancer LNCaP cell growth and survival in AR-positive cells, which can be inhibited by apoptotic rates. OGX-427 treatment of mice bearing LNCaP PI3K inhibitors or dominant-negative Akt. AR is found in a large xenografts transfected with an androgen-regulated, probasin- protein complex with p85a-PI3K and Src, both required for luciferase reporter construct resulted in decreased biolumi- androgen-stimulated PI3K/Akt activation (3, 10). nescence and serum PSA levels as pharmacodynamic readouts Molecular chaperones are involved in processes of folding, of AR activity, as well as AR, Hsp27, and Hsp90 protein levels activation, trafficking, and transcriptional activity of most steroid in LNCaP tumor tissue. These data identify novel nongenomic receptors, including AR. In the absence of ligand, AR is predomi- mechanisms involving androgen, AR, and Hsp27 activation nantly cytoplasmic, maintained in an inactive but highly responsive that cooperatively interact to regulate the genomic activity of state by a large dynamic heterocomplex composed of heat-shock AR and justify further investigation of Hsp27 knockdown as an proteins (Hsp), co-chaperones, and tetratricopeptide repeat (TPR)– AR disrupting therapeutic strategy in prostate cancer. [Cancer containing proteins. Ligand binding leads to a conformational Res 2007;67(21):10455–65] change in the AR and dissociation from the large Hsp complex. Subsequently, the AR translocates to the nucleus, interacts with Introduction coactivators, dimerizes, and binds to its ARE to transactivate target The androgen receptor (AR), a ligand-dependent transcription gene expression (11). Dissociation of the AR-chaperone complex factor and member of the class I subgroup of the nuclear receptor after ligand binding is viewed as a general regulatory mechanism of superfamily, plays a key role in prostate carcinogenesis and AR signaling (11). Molecular chaperones remain important players progression. The classic model of androgen-regulated AR tran- in the events downstream of receptor activation and throughout the scriptional activity has not fully defined the many diverse effects of life cycle of the AR. For example, the Hsp90 inhibitor, geldanamycin, androgens on prostate cancer cell survival and growth. In response destabilizes AR and increases its proteasomal degradation, thereby to androgen, cytoplasmic AR rapidly translocates to the nucleus decreasing the expression of AR-regulated genes (12). Recent reports and interacts with sequence-specific androgen response elements further highlight the important roles of other co-chaperones on AR activation. Bag-1L is overexpressed in hormone refractory prostate cancer (13) and enhances the transactivation of the AR by using its NH2 and COOH-terminal domains to bind to COOH- and NH2- Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). terminal sequences of AR (14). Another co-chaperone, FKBP52, also Requests for reprints: Martin E. Gleave, Department of Urologic Sciences, increases AR transactivation activity (15, 16), and FKBP52 knock-out University of British Columbia, 2775 Laurel Street, Level 6, Vancouver, BC, Canada, mice exhibit defects in male reproductive tissues, including V6H 3Z6. Phone: 604-875-4818; Fax: 604-875-5654; E-mail: [email protected]. I2007 American Association for Cancer Research. ambiguous external genitalia and defects in prostate and seminal doi:10.1158/0008-5472.CAN-07-2057 vesicle development. www.aacrjournals.org 10455 Cancer Res 2007; 67: (21). November 1, 2007

Another class of Hsps that complex with ER and glucocorticoid were replaced by CSS F R1881 24 h after transfection for another 24 h. receptors (GR) are ATP independent and include Hsp27 (17). Hsp27 Luciferase activities measured using the Dual-Luciferase Reporter Assay is a cytoprotective chaperone expressed in response to many stress System (Promega) with the aid of a microplate luminometer (EG&G signals to regulate key effectors of the apoptotic machinery, Berthold). All experiments were carried out in triplicate wells and repeated five times using different preparations of plasmids. including the apoptosome, the caspase activation complex (18, 19), Northern blot analysis. Total RNA was isolated from LNCaP cells treated and proteasome-mediated degradation of apoptosis-regulatory with OGX-427 or mismatch control, transfected with empty vector, Hsp27 wt, proteins (20, 21). Antisense knockdown of Hsp27 delays prostate or Hsp27 TM, and treated with SB203580 before R1881 (10 nmol/L) cancer xenograft growth and androgen-independent progression treatment for 24 h using TRIzol/chloroform extraction (Invitrogen Life (22, 23). Although Hsp27 is induced by estrogens and glucocorti- Technologies, Inc.). A 10-Ag aliquot from each sample was subjected coids (24, 25), its relationship with AR and androgens is undefined. to horizontal electrophoresis and followed by hybridization with Hsp27 Here, we identify a feed-forward loop whereby androgen-bound AR (700 pb) or prostate-specific antigen (PSA; 2 kb) cDNA probe as described induces rapid Hsp27 phosphorylation that, in turn, cooperatively (23) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) facilitates genomic activity of the AR, thereby enhancing prostate cDNA probe for normalization of loading levels. cancer cell survival. Electromobility shift assay. LNCaP cells were treated with indicated concentrations of OGX-427 or mismatch control. Forty-eight hours after the second treatment, nucleoplasmic proteins were extracted using CeLytic Materials and Methods NuCLEAR Extraction Kit (Sigma). The nuclear extract (10 Ag) was incubated Cell culture and antisense oligonucleotide transfection. LNCaP cells in a final volume of 20 AL containing DNA-binding buffer [10 mmol/L Tris- are maintained in RPMI with 5% fetal bovine serum (FBS; ref. 26) and HCl (pH, 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L treated with Hsp27 antisense oligonucleotide (ASO) designated OGX-427 phenylmethylsulfonyl fluoride, 20% v/v glycerol] and 1.5 Ag of poly(deox- (OncoGenex Technology, Inc.) or mismatch control oligonucleotide (ODN) yiosinic-deoxycytidic acid) (Roche) for 10 min at room temperature with OligofectAMINE in serum-free OPTI-MEM (Invitrogen Life Technol- and then incubated for 20 min at room temperature with double-stranded 32 ogies, Inc.) for 20 min. Four hours later, 5% FBS was added. Cells were P-labeled (with specific activity of about 300,000 cpm) PSA-ARE treated once daily for 2 successive days and harvested 48 h after the second oligonucleotide (27). treatment. Chromatin immunoprecipitation. LNCaP cells were treated with Plasmids, reagents, and antibodies. An Hsp27 wild type (wt) was 10 nmol/L of R1881 for 4 h and parafolmaldehyde cross-linked and subcloned into pcDNA3.1-GFP (Invitrogen-Life Technologies). The Hsp27 sonicated. Chromatin immunoprecipitation (ChIP) assay was done using EZ triple mutant (TM) was generated by introducing direct mutagenesis, ChIP kit according to the manufacturer (Upstate) on the PSA gene regions replacing Ser15, Ser78, and Ser82 with alanine using QuikChangeR II XL, site- ARE I and ARE III as described in ref. 28. directed mutagenesis kit according to the manufacturer’s instructions Reverse transcription-PCR. About 4 Ag of total RNA were reverse (Stratagene). R1881 (Perkin-Elmer) was dissolved in 100% ethanol. Cyclo- transcribed in cDNA using 100 pmol of random hexamer primers hexamide, MG-132, SB 203580, and LY 294002 were purchased from (Pharmacia) and Moloney murine leukemia virus reverse transcriptase Calbiochem; Hsp27, pHsp27, and Hsp90 were from StressGen; MDM2, (Life Technologies). The cDNA was successively amplified with two pairs AR N-20, and AR-441 were from Santa Cruz Biotechnology, Inc.; and of AR-specific primers. About 5 AL of cDNA were used as the starting pAkt/Akt, pp38/p38, and poly(ADP ribose)polymerase (PARP) were from DNA template in the PCR assay. To verify RNA quality, each sample was Cell Signaling Technology. amplified with a set of primers specific to h-actin (5¶-TGATCCA- Cell proliferation and apoptosis assays. LNCaP cells were plated in CATCTGCTGGAAGGTGG-3¶ sense and 5¶-GGACCTGACTGACTACCTCAT- RPMI with 5% FBS and switched to charcoal-stripped serum (CSS) the next GAA-3¶ antisense). Analysis of PCR products was done by electrophoresis in day with or without 10 nmol/L R1881. After a time course exposure, LNCaP 2% agarose gel and visualized by ethidium bromide staining. cell growth was measured using the crystal violet assay as previously Protein stability. LNCaP cells were plated and treated with OGX-427 described (23). Detection and quantitation of apoptotic cells were done by or mismatch control or transfected with Hsp27 wt or empty vector as flow-cytometric analysis as previously described (23). Each assay was done described above. Media were changed 48 h later to RPMI + 5% serum six times. containing 10 Amol/L of cyclohexamide incubated at 37jC for 2 to 6 or 16 h. Western blot analysis and immunoprecipitation. Total proteins Western blot was done using AR, Hsp27, and vinculin antibodies. (500 Ag) were pre-cleared with protein-G sepharose (Invitrogen Life In vivo imaging of luciferase activity. Bioluminescence imaging was Technologies) for 1 h at 4jC and immunoprecipitated with 2 Ag of anti- done with a highly sensitive, cooled CCD camera mounted in a light-tight Hsp27, anti-AR, or immunoglobulin G (IgG) as a control overnight at 4jC. The specimen box (Xenogen Corporation) using the protocol as previously immune complexes were recovered with protein-G sepharose for 2 h and then described (29). Before imaging, mice were injected i.p. with 100 mg/kg of the washed with radioimmunoprecipitation assay buffer (RIPA) at least thrice, substrate D-luciferin and anesthetized with 1% of isoflurane. At 15 min centrifuged, and submitted to SDS-PAGE, followed by Western blotting. postinjection, mice were imaged by IVIS 200, and bioluminescence was Immunofluorescence. LNCaP cells were grown on coverslips and monitored by Living Image software. treated FR1881 for 15 min. For Hsp27 knockdown, cells were treated with Animal treatment. Male athymic nude mice (Harlan Sprague-Dawley, 50 nmol/L Hsp27 ASO as described above. After treatment, cells were fixed Inc.) were injected s.c. with 1 Â 106 LNCaP-Probasin–driven luciferase cells. in ice-cold methanol completed with 3% acetone for 10 min at À20jC. Cells Once tumors were palpable with serum PSA levels f25 ng/mL and were then washed thrice with PBS and incubated with 0.2% Triton/PBS for bioluminescence detectable using the IVIS-Imaging System, mice were 10 min, followed by washing and 30 min blocking in 3% nonfat milk before treated with 20 mg/kg of OGX-427 or mismatch control i.p. once daily for the addition of antibodies overnight to detect Hsp27 (1:500) and AR (1:250). 7 days. Tumor volume, serum PSA, and bioluminescence measurements Antigens were visualized using anti-rabbit or anti-mouse antibodies were done at baseline and on days 4 and 8. coupled to FITC or rhodamine (1:500; 30 min). Photomicrographs were Statistical analysis. All data were analyzed by Student’s t test. Levels of taken at 20Â magnification using Zeiss Axioplan II fluorescence statistical significance were set at P < 0.05. microscope, followed by analysis with imaging software (Northern Eclipse, Empix Imaging, Inc.). Results Transfection and luciferase assay. LNCaP cells (2.5 Â 105) were plated on six-well plates and transfected using lipofectin (6 AL per well; Invitrogen Androgens and Hsp27 protect LNCaP cells from apoptotic Life Technologies, Inc.). The total amount of plasmid DNA used was stress. Androgen is assumed to have an important role as a normalized to 2 Ag per well by the addition of a control plasmid. Media survival factor in prostate epithelial cells. The synthetic androgen

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R1881 enhances LNCaP cell survival in the presence of cytotoxic induced by OGX-427, a second-generation ASO that potently stress, including etoposide (30), cyclohexamide (10), and PI3K decreases Hsp27 levels (23). OGX-427–induced knockdown of inhibitors (31). We recently reported that Hsp27 conferred Hsp27 was associated with decreased apoptotic rates in R1881- resistance to androgen ablation in LNCaP (23) and next set out treated cells, as measured by fluorescence-activated cell sorting to explore whether androgen- and Hsp27-induced prosurvival (FACS) and PARP cleavage (Fig. 1B and C). Collectively, these activities were interrelated. Before beginning to study relationships results indicate that androgens suppress apoptosis induced by between androgen and Hsp27, we first confirmed that R1881 added Hsp27 knockdown or paclitaxel. to androgen-depleted CSS increased LNCaP cell growth (Fig. 1A, Androgens lead to rapid Hsp27 phosphorylation via p38 left) and decreased apoptotic rates (Fig. 1A, right) compared with MAPK pathway. Androgens stimulate growth and survival via CSS alone. R1881 protected LNCaP cells to paclitaxel treatment, both genomic and nongenomic pathways. Recently identified lowering apoptotic rates and increasing cell growth (Supplemen- nongenomic effects include activation of Src, PI3K, and Akt tary Data S1). R1881 also protected LNCaP cells from apoptosis (3, 10). Because Akt has been reported to phosphorylate Hsp27 in

Figure 1. Androgen enhances LNCaP cell survival. A, left, R1881 enhances LNCaP cell growth. Cells were treated with 1 nmol/L R1881 for 2, 4, and 6d, and cell growth rates were determined by MTS assay and compared with control (day of treatment defined as 100%). Right, R1881 protects LNCaP cells from apoptosis. Cells were treated with 1 nmol/L R1881 for 4 d, and proportion of cells in sub-G0,G0-G1,S,G2-M was determined by propidium iodide staining. B and C, R1881 protects cells from apoptosis induced by OGX-427. B, left, cell growth rates were compared with control (1 d after transfection) using MTS assay 2 and 5 d post-transfection. B, right, apoptotic rates were determined 5 d post-transfection. C, LNCaP cells were pretreated with 10 nmol/L R1881 or CSS for 48 h before treatment with OGX-427 or mismatch control. PARP cleavage and Hsp27 expression levels were measured by Western blot. All experiments were repeated at least thrice. www.aacrjournals.org 10457 Cancer Res 2007; 67: (21). November 1, 2007

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Research intact neutrophils (32), we tested whether androgen leads to Hsp27 Androgen-induced Hsp27 phosphorylation is AR dependent. phosphorylation. Interestingly, androgen induced rapid phosphor- To determine whether AR is required for androgen-induced ylation of Hsp27 on both Ser78 and Ser82 residues in a dose- and phosphorylation of Hsp27, changes in Ser78 and Ser82 phosphory- time-dependent manner (Fig. 2A). Within 15 min after incubation lation levels were analyzed after treatment with R1881 F the anti- with 10 nmol/L R1881, Ser78 and Ser82 phosphorylation levels androgen bicalutimide. Bicalutimide inhibited R1881-induced increased 3.2- and 5.3-fold, respectively (Fig. 2A, right). Hsp27 phosphorylation at both sites (Fig. 3A), suggesting that To identify upstream effectors of androgen-induced Hsp27 ligand binding to AR is required for Hsp27 phosphorylation by phosphorylation, we analyzed R1881-induced changes in p38 R1881. Next, PC3 cells, which do not express endogenous AR, were kinase and Akt phosphorylation levels, both previously associated transfected with increasing amounts of AR or empty vector with or with Hsp27 activation (32, 33). R1881 enhances p38 kinase and Akt without R1881. As shown in Fig. 3A, Hsp27 phosphorylation levels at phosphorylation in a time-dependent manner, with maximum both Ser78 and Ser82 sites increased with increasing AR levels after stimulation at 15 and 5 min, respectively (Fig. 2B, left and right). R1881 treatment. In AR-negative PC3 cells, Hsp27 phosphorylation These results suggest that Hsp27 may be phosphorylated via p38 is insensitive to androgens, but is enhanced when AR is transiently and/or Akt pathways. Preincubation of LNCaP cells with the p38 overexpressed in PC3 cells. These results confirm that AR is kinase inhibitor, SB 203580, abolished R1881-induced Hsp27 required for androgen-induced Hsp27 phosphorylation. phosphorylation (Fig. 2C, left), whereas the Akt inhibitor, LY To further define mechanisms of androgen-induced Hsp27 294002, did not alter androgen-induced Hsp27 phosphorylation phosphorylation, we determined whether Hsp27 and AR interact (Fig. 2C, right), indicating that androgen-induced Hsp27 phosphor- using coimmunoprecipitation for Hsp27 and AR in LNCaP cells. ylation occurs via the p38 kinase pathway. Hsp27 is detected in AR immunoprecipitated complexes, and

Figure 2. Androgen phosphorylation of Hsp27 requires p38 MAPK pathway. A, R1881 leads to rapid phosphorylation of Hsp27 on both Ser78 and Ser82 residues in a dose- (left) and time-dependent (right) manner: LNCaP cells were maintained in CSS for 16h before R1881 treatment. Total proteins were analyzed by Western blotting with anti–phospho-Hsp27 Ser78 or Ser82 or anti–total Hsp27 for control loading. B, R1881 leads to phosphorylation of p38 kinase (left) and Akt (right) in a time-dependent manner as shown using anti–phospho-Akt or anti–phospho-p38 antibodies. Anti–total Akt or anti–total p38 was used for control loading. C, the p38 inhibitor SB203580 (10 Amol/L; left), but not the Akt inhibitor LY98059 (right), inhibits R1881-induced Hsp27 phosphorylation. After 5 or 15 min R1881 (for AKT or p38, respectively), total lysates were analyzed by Western blotting Hsp27 anti–phospho-Hsp27 Ser78 or Ser82 or anti–total Hsp27 for control loading.

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Figure 3. Androgen-induced Hsp27 phosphorylation requires AR. A, bicalutimide inhibits R1881-induced Hsp27 phosphorylation. LNCaP cells were starved for 16h before adding 10 nmol/L R1881 for 15 min F 1 Amol/L bicalutamide, and total protein analyzed by Western blotting with anti–phospho-Hsp27 Ser78 or Ser82, or anti–total Hsp27 for control loading. B, androgen increases phospho-Hsp27 levels in AR-transfected PC-3 cells. PC3 cells were transiently transfected with 100 to 500 ng of pcDNA-hAR, followed by 10 nmol/L R1881 for 15 min, and total protein was then analyzed by Western blotting with anti-AR, anti–phospho-Hsp27 Ser78 or Ser82, or anti–total Hsp27 for control loading. C, AR interacts with Hsp27. 500 Ag of total extract were immunoprecipitated with 2 Ag of anti-Hsp27, anti-AR, or IgG as a control overnight at 4jC. The immune complexes were recovered with protein-G sepharose for 2 h and submitted to Western blotting with anti-AR or anti-Hsp27 (left). AR and Hsp27 colocalize and translocate into the nucleus after R1881: LNCaP cells were treated FR1881 for 15 min and fixed in methanol/acetone for immunofluorescence staining with anti-Hsp27 and anti-AR antibodies (right). D, androgen increases Hsp27 levels in complex with AR as Hsp90 levels decrease. LNCaP cells were maintained in CSS for 16h and treated for indicated times with 10 nmol/L R1881. 500 Ag of total extract were immunoprecipitated using AR, and Western blot was done using Hsp90, Hsp27, and AR antibodies. conversely, AR is detected in Hsp27 immunoprecipitated com- gated the effects of phospho-Hsp27 levels on AR transcriptional plexes (Fig. 3C, left). The domain of AR required for the interaction activity. This was done by gain-of-function strategies using over- with Hsp27 was next analyzed using glutathione S-transferase expressing wt Hsp27, as well as loss-of-function strategies using an (GST)–pulldown assays. GST pulldown after initial equimolar Hsp27 ASO (OGX-427), dominant-negative Hsp27 phosphorylation normalization of GST-AR domain fusion as previously described TM, or an Hsp27 phosphorylation inhibitor (p38 kinase inhibitor, (34) indicates that Hsp27 binds with NH2-terminal and ligand SB 203580). PSA transactivation assays were done using LNCaP cells binding (COOH-terminal) domains, but not the DNA-binding transiently transfected with the luciferase reporter plasmid domain of AR (Supplementary Data S2). Furthermore, immunoflu- regulated by the PSA enhancer-promoter region (6.1 kb) in the orescence illustrates that Hsp27 and AR colocalize in the cytoplasm presence or absence of an increasing amount of exogenous wt of LNCaP cells cultured in the absence of androgens. Importantly, Hsp27 (0, 0.25, 0.5, and 1 Ag). R1881 treatment increased AR and of potential functional relevance, both proteins translocate and reporter gene expression 34-fold, whereas wt Hsp27 overexpression colocalize in the nucleus after R1881 treatment (Fig. 3C, right). increases androgen-stimulated transcriptional activity of PSA a Before ligand binding, AR exists in a complex with Hsp90 and further 3-fold (Fig. 4A, left). In contrast, Hsp27 knockdown using other co-chaperones. The AR-Hsp90 interaction maintains AR in a OGX-427 decreased the transactivation of the androgen-regulated ligand-binding conformation necessary for efficient response to PSA reporter in a dose-dependent manner, further supporting a role hormone (35). Upon ligand binding, AR is released from Hsp90 and for Hsp27 in AR transactivation (Fig. 4A, right). is translocated into the nucleus. Interestingly, AR immunoprecip- To determine whether Hsp27 phosphorylation is required for itation blots show that shortly after androgen treatment, Hsp27 R1881-induced AR-mediated gene activation, we overexpressed an levels increase in complex with AR as Hsp90 levels decrease Hsp27 TM (Ser15, Ser78, and Ser82 substituted by alanine) in a dose- (Fig. 3D), suggesting that upon ligand binding, phospho-activated dependent manner and tested its ability to affect R1881-stimulated Hsp27 replaces Hsp90 to chaperone AR into the nucleus. PSA transactivation. As shown in Fig. 4B (left), Hsp27 TM Effect of Hsp27 on genomic activity of AR. Many proteins transfection inhibited luciferase transactivation from the PSA participate in the activation of AR, either by direct binding or as enhancer in response to R1881. Similarly, inhibition of androgen- part of a tertiary complex regulating the activity of other induced, p38 kinase–mediated Hsp27 phosphorylation using the transactivators. Because Hsp27 colocalizes with and shuttles p38 kinase inhibitor, SB 203580, suppressed R1881-mediated trans- ligand-activated AR during nuclear translocation, we next investi- activation of PSA enhancer–driven luciferase activity (Fig. 4B, www.aacrjournals.org 10459 Cancer Res 2007; 67: (21). November 1, 2007

Figure 4. Effect of Hsp27 on AR transactivation. A, Hsp27 overexpression increases AR transactivation. LNCaP cells were transiently cotransfected with 1 Ag of PSA-luciferase and indicated concentrations of wt Hsp27, followed by R1881 or vehicle for 24 h. Total amount of plasmid DNA transfected was normalized to 2 Ag per well by the addition of an empty vector (left). Hsp27 knockdown decreases AR transactivation. LNCaP cells were treated with the indicated dose of OGX-427 or mismatch control (right). Cells were harvested, and luciferase activity was determined. Columns, means of at least three independent experiments done in triplicate. Fold is measured relative to PSA activation with no treatment. B, effect of Hsp27 phosphorylation on AR transactivation. LNCaP cells were transiently cotransfected with 1 Ag of PSA-luciferase with indicated concentrations of Hsp27 TM (Ser15,Ser78, and Ser82 were mutated to alanine) followed by R1881 or vehicle for 24 h. The total amount of plasmid DNA transfected was normalized to 2 Agperwellbytheadditionofanemptyvector(left). LNCaP cells were transfected with 1 Ag per well of PSA-luciferase before the indicated concentration of SB 203580 for 1 h before R1881 treatment (right). Columns, means of at least three independent experiments done in triplicate. Fold is measured relative to PSA activation with no treatment. C, Hsp27 knockdown decreases PSA mRNA expression (left). LNCaP cells were treated as indicated and RNA extracted for Northern blot analysis of PSA and Hsp27, using GAPDH as a loading control. Hsp27 TM decreases PSA mRNA expression (middle). LNCaP cells were transfected with Hsp27 TM; 24 h after transfection, cells were treated with R1881 for 16h, and RNA was extracted for Northern blot analysis. p38 kinase inhibition decreases PSA mRNA expression ( right). LNCaP cells were treated for 2 h with SB203580 (10 Amol/L) before R1881, and RNA were extracted for Northern blot analysis. D, Hsp27 knockdown decreases AR binding to its response elements. EMSA was done using radiolabeled PSA oligonucleotides with nuclear cell extracts isolated from LNCaP treated with different concentrations of Hsp27 ASO. For controls, 10 Ag of nuclear protein were incubated 20 min with cold PSA oligonucleotide. E, Hsp27 recruitment to the promoter of PSA gene. LNCaP cells were treated with 10 nmol/L R1881 for 4 h. Soluble chromatin was prepared from formaldehyde cross-linked and sonicated cell culture. Immunoprecipitation was done using AR or Hsp27 antibodies, along with IgG as control. The final DNA extractions were amplified using the primers for ARE I and ARE III.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Androgen Receptor Interactions with Hsp27 right). Collectively, these results indicate that androgen-induced a heterodimer complex with Hsp90 to provide stability for ligand- p38 kinase-mediated phosphorylation of Hsp27 is important for unbound AR. Indeed, without Hsp90 binding, the unfolded protein AR-mediated PSA expression. will be recognized and degraded by the ubiquitin-proteasome The preceding data identified a role for phospho-Hsp27 in the system (12, 35). Hsp27 knockdown by OGX-427 significantly transactivation of PSA. To provide further evidence to substantiate disrupts the association between AR and Hsp90 (Fig. 5D, left). this hypothesis, Northern blot analyses were done (Fig. 4C) and This indicates that Hsp27 knockdown–induced dissociation indicated that levels of AR-regulated endogenous PSA mRNA between AR and Hsp90 may render the AR-Hsp90 heterocomplex decreases significantly after Hsp27 knockdown (OGX-427; Fig. 4C, vulnerable to degradation by the proteasome-mediated pathway. left), p38 kinase inhibition (SB 203580; Fig. 4C, middle), or Hsp27 Because AR has been reported to be ubiquitinated by the E3 ligase, phosphorylation inhibition (Hsp27 TM; Fig. 4C, right). These results MDM2 (36, 37), we next set out to determine whether interaction confirm that Hsp27 levels and phosphorylation status enhance between MDM2 and AR increased after OGX-427–induced Hsp27 AR-mediated PSA mRNA expression. knockdown. As predicted, Hsp27 knockdown increased the As a transcription factor, AR translocates to the nucleus after association between endogenous MDM2 and AR as shown by co- androgen binding, where it interacts with ARE to transactivate its immunoprecipitation experiments in Fig. 5D (middle). Further- target genes. Suppression of Hsp27 levels or phosphorylation more, Hsp27 knockdown increased levels of ubiquitinated AR (Fig. negatively regulates androgen-stimulated transcriptional activity of 5D, right). These results indicate that OGX-427–induced Hsp27 PSA. To determine whether AR interaction with its response knockdown dissociates the Hsp90-AR heterocomplex and increases elements is affected by Hsp27 levels, we next analyzed the effects of MDM2-mediated AR ubiquitination and degradation. Hsp27 knockdown on AR binding to PSA-ARE using electropho- In vivo Hsp27 knockdown by OGX-427 decreases LNCaP retic mobility shift assay (EMSA) with nuclear lysates of LNCaP proliferation rates, serum PSA levels, and AR client protein cells treated with OGX-427. As shown in Fig. 4D, 10 nmol/L R1881 expression levels. Our results establish a novel mechanism increased AR binding to ARE, and this effect was specifically whereby ligand-activated AR phospho-activates Hsp27 to cooper- blocked by excess cold PSA oligonucleotide. Hsp27 knockdown atively enhance AR nuclear translocation and transcriptional using OGX-427 decreased AR binding to its ARE, confirming that activity. Hsp27 knockdown leads to AR degradation and reduced Hsp27 knockdown decreases AR binding to its ARE. Collectively, PSA transcription and LNCaP cell growth in vitro. We sought to the preceding data indicate that phospho-activated Hsp27 plays a determine whether in vivo Hsp27 knockdown with OGX-427 critical role in androgen-induced nuclear translocation and decreased AR activity in LNCaP xenografts. Male nude mice were transcriptional activity of the AR. injected s.c. with 1 Â 106 LNCaP-Probasin–driven luciferase- We next explored whether Hsp27 forms a complex with AR on transfected cells (Supplementary Data S3), and once tumors were the ARE of the PSA promoter using ChIP assay in LNCaP cells palpable with serum PSA levels f25 ng/mL and bioluminescence FR1881. h-Actin fragment was amplified as control. As shown in was detectable using the IVIS Xenogen monitor, mice were Fig. 4E, Hsp27 is present, along with AR (which serves as a positive treated with 20 mg/kg of OGX-427 or mismatch control. A bio- control) on the AREs. In the presence of R1881, higher levels of AR luminescent signal first became detectable when serum PSA levels and Hsp27 were recruited to the promoter proximal region (ARE I) reached f5 ng/mL and was easily detected at serum PSA levels and also to the enhancer region (ARE III) of the PSA promoter. above 20 ng/mL. Mice were subjected to three different types of These data indicate that Hsp27 continues to interact with the AR measurements: bioluminescence of probasin-promoter–driven transcriptional complex at the level of the ARE and seems to be luciferase activity (a measure of in vivo AR activity in LNCaP regulated by androgen. xenografts), serum PSA levels, and tumor volume. Measurements Hsp27 knockdown induces AR degradation via the protea- were done at baseline before treatment (day 0), during treat- some-mediated pathway. Hsp27 knockdown or inhibition of ment (day 4), and at the end of treatment (day 8). Beginning day 4, phosphorylation inhibits androgen-stimulated nuclear transloca- OGX-427 decreased bioluminescence and reduced circulating PSA tion of the AR with subsequent suppression of AR-regulated gene levels by 60% (P < 0.01), with a slight 15% decrease in tumor expression. To investigate the fate of AR after Hsp27 knockdown, volume (Fig. 6A and B); individual data are presented in changes in AR mRNA and protein levels after treatment with Supplementary Data S4. In contrast, mice treated with mismatch OGX-427 were evaluated. AR mRNA levels did not change after control ODN showed an anticipated increasing trend on bio- Hsp27 knockdown (Fig. 5A, left). In contrast, Hsp27 knockdown luminescence and PSA levels, coincident with increasing tumor decreased AR (Fig. 5A, middle) and Hsp90 (Fig. 5A, right) protein volume during the 8-day treatment period. Western blot of snap- levels in a dose-dependent manner. The effect of Hsp27 knockdown frozen xenograft samples (Fig. 6C) and immunostaining data on AR protein stability was next evaluated using cyclohexamide, (Supplementary Data S5) indicate that OGX-427 decreased LNCaP which inhibits protein synthesis. As shown in Fig. 5C, AR protein xenograft levels of AR, Hsp27, and Hsp90. In addition, OGX-427 levels decrease significantly with rapid degradation after OGX-427– also decreased Ki67 staining (Supplementary Data). Collectively, induced knockdown of Hsp27. In contrast, Hsp27 overexpression these data suggest that the in vivo anticancer activity of OGX-427 prolonged AR half-life compared with empty vector-transfected results, in part, from AR disruption, and that serum PSA may controls (Fig. 5C). AR degradation after Hsp27 knockdown occurs prove a useful surrogate of pharmacodynamic activity as OGX-427 via the proteasome pathway because treatment with the protea- moves into human trials. some inhibitor MG-132 suppressed Hsp27 knockdown-induced AR degradation (Fig. 5C). Taken together, these findings indicate that Hsp27 knockdown induces AR degradation via a proteasome- Discussion mediated pathway. Hsp27 is an ATP-independent chaperone that is phospho- Hsp27 knockdown disrupts AR-Hsp90 association and activated by cell stress to prevent aggregation and/or regulate increases AR-MDM2 association and ubiquitination. AR forms activity/degradation of certain client proteins. The chaperone activity www.aacrjournals.org 10461 Cancer Res 2007; 67: (21). November 1, 2007

Figure 5. Effect of Hsp27 knockdown on AR expression and stability. A, AR mRNA levels are not affected by Hsp27 knockdown (left). LNCaP cells were treated in a dose-dependent manner with OGX-427 or mismatch control oligos, and AR Hsp27 mRNA levels in LNCaP cells were analyzed by reverse transcription-PCR, and actin was monitored as a control. AR and Hsp90 protein levels are decreased by Hsp27 knockdown. LNCaP cells were treated with the indicated concentration of OGX-427 or mismatch controls, and protein levels of AR (middle), Hsp90 (right), and Hsp27 were determined by Western blot. Vinculin was used as a loading control. B, Hsp27 levels affect AR stability. LNCaP cells were treated with 70 nmol/L OGX-427 or mismatch control (left) or transfected with empty vector or Hsp27 wt and then treated with 10 Amol/L cycloheximide for the indicated time period. DMSO was used as control. AR and Hsp27 protein levels were measured by Western blot analysis. C, Hsp27 knockdown accelerates proteasomal degradation of AR. LNCaP cells were treated with OGX-427 or mismatch control and 10 Amol/L MG-132 for 6h. DMSO was used as control. AR protein level was measured by Western blot analysis. D, effect of Hsp27 knockdown on AR/Hsp90 association: LNCaP cells were treated with 70 nmol/L OGX-427 or mismatch control in the presence of FBS. Immunoprecipitation was done using AR antibody, and Western blot analysis was done using Hsp90 antibodies (left). Effect of Hsp27 knockdown on AR/MDM2 association: LNCaP cells were treated with 70 nmol/L OGX-427 in the presence of MG-132 (10 Amol/L) for 6h. AR immunoprecipitation and Western blot was done with anti-MDM2 ( middle). Effect of Hsp27 knockdown on AR/ubiquitin association: LNCaP cells were treated with 70 nmol/L OGX-427 in the presence of MG-132 (10 Amol/L) for 6h. AR immunoprecipitation and Western blot was done with anti-ubiquitin antibody (right). Membrane was stripped and blotted with anti-AR as control immunoprecipitation. Input was blotted with Hsp27 antibody. of Hsp27 is regulated by stress-induced changes in phosphorylation radiation (41, 42). We recently reported that overexpression of and oligomerization (38). As a cytoprotective chaperone situated as a Hsp27 in LNCaP cells suppressed castration-induced apoptosis and Hub at the center of many pathways regulating cellular response to confers androgen resistance (23), whereas Hsp27 knockdown using therapeutic stress, targeted inhibition of Hsp27 would inhibit many ASO potently decreases Hsp27 levels, increases caspase-3 cleavage pathways implicated in cancer progression and resistance. The and apoptosis, enhances paclitaxel chemosensitivity, and delays cytoprotective effects of Hsp27 result from its ubiquitin binding and tumor progression in vivo (22, 23). degradation of InB (20), direct interference of caspase activation, Human Hsp27 is phosphorylated on three serine residues, Ser15, modulation of oxidative stress, and regulation of the cytoskeleton Ser78, and Ser82. p38 kinase and Akt were reported to be the Hsp27 (19, 39). Higher levels of Hsp27 are commonly detected in many kinases (32, 33), although protein kinase C (PKC) a and y and cyclic cancers including prostate (22, 23, 40) and is associated with AMP–dependent kinase can also phosphorylate Hsp27 (43). Hsp27 metastasis, poor prognosis, and resistance to chemotherapy or becomes phosphorylated when exposed to angiotensin (43),

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Androgen Receptor Interactions with Hsp27 interleukin-6 (IL-6), IL-1, heat shock and tumor necrosis factor-a dependent manner. This rapid androgen/AR-mediated Hsp27 (44). Using LNCaP cells, which express AR and are sensitive to phosphorylation identifies a nongenomic mechanism for AR in androgens, we show that androgens increase phospho-Hsp27 levels LNCaP cells. These findings are in agreement with previous studies within minutes in a dose-, time-, and p38 kinase pathway– indicating that steroids can act via classic steroid receptors or

Figure 6. OGX-427 suppresses probasin luciferase (Pb-Luc) bioluminescence as well as AR, Hsp90, and Hsp27 levels in vivo. A, in vivo imaging of LNCaP–Pb-Luc xenografts after OGX-427 treatment by IVIS imaging system. Intact male mice were injected s.c. with 1 Â 106 LNCaP-Pb-Luc cells and, once tumors formed with serum PSA levels f25 ng/mL, were treated with 20 mg/kg of OGX-427 or mismatch for 7 d. Bioluminescence indicates probasin luciferase activity before (day 0), during (day 4), and after (day 8) treatment. B, effect of OGX-427 treatment on serum PSA levels. Changes in serum PSA levels from mice treated with OGX-427 or mismatch control were measured using IMX immunoassays (left). Effect of OGX-427 treatment on LNCaP tumor volume. Xenograft volume was measured by caliper at indicated time (right). Points, average of five mice per group; bars, SE. C, total LNCaP xenograft proteins were extracted in RIPA buffer after mismatch or OGX-427 treatment (five mice per group) and Western blots done with AR, Hsp90, and Hsp27 antibodies; vinculin was used as a loading control. D, schema illustrating cooperative interactions between ligand-activated AR and Hsp27 phosphorylation: Androgen binding to AR leads to rapid Hsp27 phosphorylation via p38 kinase pathway, which displaces Hsp90 and chaperones AR to the nucleus to enhance activation of AR-regulated genes (left). OGX-427 induced Hsp27 knockdown destabilizes AR/Hsp90 heterocomplex and leads to MDM2-mediated ubiquitination and AR proteasomal degradation (right).

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Research through atypical membrane receptors, and that AR mediates Previous studies emphasize the importance of Hsps in steroid nongenomic activation in response to androgens (6, 10). Ligand receptor stability. For example, Hsp90 inhibitors such as geldana- binding to AR induces its association with Src via Src SH3 domain mycin induce steroid receptor degradation by directly binding to and AR proline-rich domain, triggering Src-dependent pathway the ATP-binding pocket of Hsp90 and thereby inhibiting its function activation (6, 10). In AR-negative COS-1 and PC3 cells, transfection (12, 35). Results shown in Figs. 5 and 6 indicate that Hsp27 of AR is necessary for R1881 induction of c-Src/Raf/extracellular knockdown destabilizes AR by inducing the dissociation of the AR/ signal-regulated kinase and Akt, respectively. In AR-positive LNCaP Hsp90 heterocomplex and increasing AR association with the E3 cells, the anti-androgen bicalutamide suppresses R1881-stimulated ligase MDM2, with subsequent ubiquitin-proteasome–mediated AR Hsp27 phosphorylation. In AR-negative PC3 cells, Hsp27 phos- degradation. Although both OGX-427 and geldanamycin share the phorylation is insensitive to androgens, but is enhanced when AR is ability to abrogate the interaction between AR and Hsp90, they do transiently overexpressed in PC3 cells. This nongenomic stimula- so by different mechanisms. Interestingly, OGX-427 induces the tion is supported further by the interaction and colocalization of degradation of Hsp27, AR, and Hsp90, whereas geldanamycin Hsp27 with AR. Hsp27 binds with AR via its NH2-terminal domain binding to the ATP-binding pocket of Hsp90 inhibits its chaperone and may directly or indirectly involve other AR coregulators, activity (48), induces degradation of client proteins (49), and is including signal transducers and activators of transcription 3 accompanied by stress-activated increases in Hsp70 and Hsp27 (50). (STAT3) and ARA55, both of which have been reported to associate The effects of OGX-427 on AR expression and activity in vitro with Hsp27 (45, 46). were recapitulated in vivo. OGX-427 induced rapid decreases in In the classic model of androgen action, in response to probasin-luciferase reporter–driven bioluminescence that corre- androgens, AR dissociates from Hsp90 (11). Interestingly, we found lated with early decreases in serum PSA and decreased LNCaP that following androgen treatment, Hsp27 becomes more abun- xenograft levels of AR and its chaperones, Hsp90 and Hsp27. In dantly associated with AR as AR dissociates from Hsp90, suggesting prostate cancer where the AR is critically important, ligand- a dynamic role for molecular chaperones in AR shuttling. This activated AR leads to rapid p38 kinase–mediated phosphorylation nongenomic action of AR ultimately influences its classic genomic of Hsp27, which, in turn, complexes with and chaperones the AR effects. Once in the nucleus, ligand-bound nuclear receptors like to enhance its stability, shuttling, and transcriptional activity. AR are recruited to target gene promoters either through direct OGX-427–induced knockdown of Hsp27 destabilizes the AR and binding to hormone response elements or association with other enhances its ubiquitination and degradation. Collectively, these promoter-bound transcription factors (11). Many proteins partic- data identify a novel mechanism for Hsp27 as an AR chaperone ipate in the activation of AR, some directly binding to AR, and (as summarized in model illustrated in Fig. 6D) and justifies further others via a tertiary complex with other transactivators. Hsp27 has investigation targeting Hsp27 as therapeutic for prostate cancer. now been identified as a chaperone interacting with the AR transcriptional complex at the level of its ARE. Recent reports indicate that the AR coactivators STAT3 and ARA55 also interact Acknowledgments with Hsp27 (23, 45). Hence, Hsp27 and AR may complex with either Received 6/4/2007; revised 7/25/2007; accepted 8/22/2007. ARA55 or STAT3 to cooperatively promote AR translocation and Grant support: Terry Fox Foundation of the National Cancer Institute of Canada and the NIH Pacific Northwest Prostate Specialized Programs of Research Excellence. transactivation. Interestingly, PSA transactivation in LNCaP cells is The costs of publication of this article were defrayed in part by the payment of page enhanced by Hsp27 overexpression and suppressed by Hsp27 charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. knockdown or inhibition of Hsp27 phosphorylation. Similarly, in The University of British Columbia has submitted patent applications, listing ER+ MCF-7 breast cancer cells, inhibition of p38 kinase signaling Dr. Gleave as inventor, on the antisense sequence described in this paper. This IP has also blocks ER-mediated transcription by inhibiting nuclear been licensed to OncoGenex Technologies, a Vancouver-based biotechnology company a that Dr. Gleave has founding shares in. translocation of ER (47). Furthermore, Hsp27 knockdown We thank Virginia Yago and Mary Bowden for their technical assistance in animal decreases AR nuclear translocation and binding to its ARE. experimentation.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cooperative Interactions between Androgen Receptor (AR) and Heat-Shock Protein 27 Facilitate AR Transcriptional Activity

Amina Zoubeidi, Anousheh Zardan, Eliana Beraldi, et al.

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