CooperativeInteractionsbetweenAndrogenReceptor(AR)and Heat ... · Res 2007;67(21):10455–65]...

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

Androgen receptor (AR) transactivation is known to enhanceprostate cancer cell survival. However, the precise effectorsby which the prosurvival effects of androgen and AR driveprostate cancer progression are poorly defined. Here, weidentify a novel feed-forward loop involving cooperativeinteractions between ligand-activated AR and heat-shockprotein 27 (Hsp27) phospho-activation that enhance ARstability, shuttling, and transcriptional activity, therebyincreasing prostate cancer cell survival. Androgen-bound ARinduces rapid Hsp27 phosphorylation on Ser78 and Ser82

residues in an AR- and p38 kinase–dependent manner. Afterthis androgen-induced, non-nuclear phospho-activation,Hsp27 displaces Hsp90 from a complex with AR to chaperoneAR into the nucleus and interact with its response elements toenhance its genomic activity. Inhibition of Hsp27 phosphor-ylation, or knockdown using the antisense drug OGX-427,shifted the association of AR with Hsp90 to MDM2, increasedproteasome-mediated AR degradation, decreased AR tran-scriptional activity, and increased prostate cancer LNCaP cellapoptotic rates. OGX-427 treatment of mice bearing LNCaPxenografts transfected with an androgen-regulated, probasin-luciferase reporter construct resulted in decreased biolumi-nescence and serum PSA levels as pharmacodynamic readoutsof AR activity, as well as AR, Hsp27, and Hsp90 protein levelsin LNCaP tumor tissue. These data identify novel nongenomicmechanisms involving androgen, AR, and Hsp27 activationthat cooperatively interact to regulate the genomic activity ofAR and justify further investigation of Hsp27 knockdown as anAR disrupting therapeutic strategy in prostate cancer. [CancerRes 2007;67(21):10455–65]

Introduction

The androgen receptor (AR), a ligand-dependent transcriptionfactor and member of the class I subgroup of the nuclear receptorsuperfamily, plays a key role in prostate carcinogenesis andprogression. The classic model of androgen-regulated AR tran-scriptional activity has not fully defined the many diverse effects ofandrogens on prostate cancer cell survival and growth. In responseto androgen, cytoplasmic AR rapidly translocates to the nucleusand interacts with sequence-specific androgen response elements

(ARE) in the transcriptional regulatory regions of target genes(1, 2). In addition to this transcriptional genomic action, androgensand other steroid hormones like progesterone and estrogen canexert rapid nongenomic effects that are not mediated throughnuclear receptors, but rather initiated at the plasma membrane,presumably through surface receptors (3, 4).

Like progesterone (PR) and estrogen (ER) receptors, AR caninteract with the intracellular tyrosine kinase c-Src to activatethe mitogen-activated protein kinase (MAPK) pathway (5, 6). Inandrogen-sensitive LNCaP prostate cancer cells, AR interacts withthe SH3 domain of Src within minutes of androgen treatment topromote cell survival, proliferation, and differentiation (5, 6).Androgens also rapidly stimulate Raf-1 and Erk-2, both compo-nents of the MAPK signaling cascade (5), suggesting that androgen-stimulated MAPK activation occurs via nongenomic mechanisms.This nongenomic action of androgen can also influence classicgenomic AR activity, including modulation of AR by coactivators(7). Phosphoinositide-3-kinase (PI3K)/Akt is another pathwayinvolved in nongenomic activity of ER and PR (8, 9), as well asAR (3, 10). In fact, androgen-activated PI3K and Akt enhance cellgrowth and survival in AR-positive cells, which can be inhibited byPI3K inhibitors or dominant-negative Akt. AR is found in a largeprotein complex with p85a-PI3K and Src, both required forandrogen-stimulated PI3K/Akt activation (3, 10).

Molecular chaperones are involved in processes of folding,activation, trafficking, and transcriptional activity of most steroidreceptors, including AR. In the absence of ligand, AR is predomi-nantly cytoplasmic, maintained in an inactive but highly responsivestate by a large dynamic heterocomplex composed of heat-shockproteins (Hsp), co-chaperones, and tetratricopeptide repeat (TPR)–containing proteins. Ligand binding leads to a conformationalchange in the AR and dissociation from the large Hsp complex.Subsequently, the AR translocates to the nucleus, interacts withcoactivators, dimerizes, and binds to its ARE to transactivate targetgene expression (11). Dissociation of the AR-chaperone complexafter ligand binding is viewed as a general regulatory mechanism ofAR signaling (11). Molecular chaperones remain important playersin the events downstream of receptor activation and throughout thelife cycle of the AR. For example, the Hsp90 inhibitor, geldanamycin,destabilizes AR and increases its proteasomal degradation, therebydecreasing the expression of AR-regulated genes (12). Recent reportsfurther highlight the important roles of other co-chaperones on ARactivation. Bag-1L is overexpressed in hormone refractory prostatecancer (13) and enhances the transactivation of the AR by using itsNH2 and COOH-terminal domains to bind to COOH- and NH2-terminal sequences of AR (14). Another co-chaperone, FKBP52, alsoincreases AR transactivation activity (15, 16), and FKBP52 knock-outmice exhibit defects in male reproductive tissues, includingambiguous external genitalia and defects in prostate and seminalvesicle development.

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Requests for reprints: Martin E. Gleave, Department of Urologic Sciences,University of British Columbia, 2775 Laurel Street, Level 6, Vancouver, BC, Canada,V6H 3Z6. Phone: 604-875-4818; Fax: 604-875-5654; E-mail: [email protected].

I2007 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-07-2057

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Another class of Hsps that complex with ER and glucocorticoidreceptors (GR) are ATP independent and include Hsp27 (17). Hsp27is a cytoprotective chaperone expressed in response to many stresssignals to regulate key effectors of the apoptotic machinery,including the apoptosome, the caspase activation complex (18, 19),and proteasome-mediated degradation of apoptosis-regulatoryproteins (20, 21). Antisense knockdown of Hsp27 delays prostatecancer xenograft growth and androgen-independent progression(22, 23). Although Hsp27 is induced by estrogens and glucocorti-coids (24, 25), its relationship with AR and androgens is undefined.Here, we identify a feed-forward loop whereby androgen-bound ARinduces rapid Hsp27 phosphorylation that, in turn, cooperativelyfacilitates genomic activity of the AR, thereby enhancing prostatecancer cell survival.

Materials and Methods

Cell culture and antisense oligonucleotide transfection. LNCaP cellsare maintained in RPMI with 5% fetal bovine serum (FBS; ref. 26) and

treated with Hsp27 antisense oligonucleotide (ASO) designated OGX-427

(OncoGenex Technology, Inc.) or mismatch control oligonucleotide (ODN)

with OligofectAMINE in serum-free OPTI-MEM (Invitrogen Life Technol-ogies, Inc.) for 20 min. Four hours later, 5% FBS was added. Cells were

treated once daily for 2 successive days and harvested 48 h after the second

treatment.Plasmids, reagents, and antibodies. An Hsp27 wild type (wt) was

subcloned into pcDNA3.1-GFP (Invitrogen-Life Technologies). The Hsp27

triple mutant (TM) was generated by introducing direct mutagenesis,replacing Ser15, Ser78, and Ser82 with alanine using QuikChangeR II XL, site-directed mutagenesis kit according to the manufacturer’s instructions(Stratagene). R1881 (Perkin-Elmer) was dissolved in 100% ethanol. Cyclo-hexamide, MG-132, SB 203580, and LY 294002 were purchased fromCalbiochem; Hsp27, pHsp27, and Hsp90 were from StressGen; MDM2,AR N-20, and AR-441 were from Santa Cruz Biotechnology, Inc.; andpAkt/Akt, pp38/p38, and poly(ADP ribose)polymerase (PARP) were fromCell Signaling Technology.

Cell proliferation and apoptosis assays. LNCaP cells were plated in

RPMI with 5% FBS and switched to charcoal-stripped serum (CSS) the next

day with or without 10 nmol/L R1881. After a time course exposure, LNCaP

cell growth was measured using the crystal violet assay as previouslydescribed (23). Detection and quantitation of apoptotic cells were done by

flow-cytometric analysis as previously described (23). Each assay was done

six times.

Western blot analysis and immunoprecipitation. Total proteins(500 Ag) were pre-cleared with protein-G sepharose (Invitrogen Life

Technologies) for 1 h at 4jC and immunoprecipitated with 2 Ag of anti-

Hsp27, anti-AR, or immunoglobulin G (IgG) as a control overnight at 4jC. Theimmune complexeswere recoveredwith protein-G sepharose for 2 h and then

washed with radioimmunoprecipitation assay buffer (RIPA) at least thrice,

centrifuged, and submitted to SDS-PAGE, followed by Western blotting.

Immunofluorescence. LNCaP cells were grown on coverslips andtreated FR1881 for 15 min. For Hsp27 knockdown, cells were treated with

50 nmol/L Hsp27 ASO as described above. After treatment, cells were fixed

in ice-cold methanol completed with 3% acetone for 10 min at �20jC. Cells

were then washed thrice with PBS and incubated with 0.2% Triton/PBS for10 min, followed by washing and 30 min blocking in 3% nonfat milk before

the addition of antibodies overnight to detect Hsp27 (1:500) and AR (1:250).

Antigens were visualized using anti-rabbit or anti-mouse antibodiescoupled to FITC or rhodamine (1:500; 30 min). Photomicrographs were

taken at 20� magnification using Zeiss Axioplan II fluorescence

microscope, followed by analysis with imaging software (Northern Eclipse,

Empix Imaging, Inc.).Transfection and luciferase assay. LNCaP cells (2.5 � 105) were plated

on six-well plates and transfected using lipofectin (6 AL per well; Invitrogen

Life Technologies, Inc.). The total amount of plasmid DNA used was

normalized to 2 Ag per well by the addition of a control plasmid. Media

were replaced by CSS F R1881 24 h after transfection for another 24 h.Luciferase activities measured using the Dual-Luciferase Reporter Assay

System (Promega) with the aid of a microplate luminometer (EG&G

Berthold). All experiments were carried out in triplicate wells and repeated

five times using different preparations of plasmids.Northern blot analysis. Total RNA was isolated from LNCaP cells treated

with OGX-427 or mismatch control, transfected with empty vector, Hsp27 wt,

or Hsp27 TM, and treated with SB203580 before R1881 (10 nmol/L)

treatment for 24 h using TRIzol/chloroform extraction (Invitrogen LifeTechnologies, Inc.). A 10-Ag aliquot from each sample was subjected

to horizontal electrophoresis and followed by hybridization with Hsp27

(700 pb) or prostate-specific antigen (PSA; 2 kb) cDNA probe as described

(23) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)cDNA probe for normalization of loading levels.

Electromobility shift assay. LNCaP cells were treated with indicated

concentrations of OGX-427 or mismatch control. Forty-eight hours after thesecond treatment, nucleoplasmic proteins were extracted using CeLytic

NuCLEAR Extraction Kit (Sigma). The nuclear extract (10 Ag) was incubated

in a final volume of 20 AL containing DNA-binding buffer [10 mmol/L Tris-

HCl (pH, 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/Lphenylmethylsulfonyl fluoride, 20% v/v glycerol] and 1.5 Ag of poly(deox-

yiosinic-deoxycytidic acid) (Roche) for 10 min at room temperature

and then incubated for 20 min at room temperature with double-stranded32P-labeled (with specific activity of about 300,000 cpm) PSA-AREoligonucleotide (27).

Chromatin immunoprecipitation. LNCaP cells were treated with

10 nmol/L of R1881 for 4 h and parafolmaldehyde cross-linked andsonicated. Chromatin immunoprecipitation (ChIP) assay was done using EZ

ChIP kit according to the manufacturer (Upstate) on the PSA gene regions

ARE I and ARE III as described in ref. 28.

Reverse transcription-PCR. About 4 Ag of total RNA were reverse

transcribed in cDNA using 100 pmol of random hexamer primers

(Pharmacia) and Moloney murine leukemia virus reverse transcriptase

(Life Technologies). The cDNA was successively amplified with two pairs

of AR-specific primers. About 5 AL of cDNA were used as the starting

DNA template in the PCR assay. To verify RNA quality, each sample was

amplified with a set of primers specific to h-actin (5¶-TGATCCA-

CATCTGCTGGAAGGTGG-3¶ sense and 5¶-GGACCTGACTGACTACCTCAT-

GAA-3¶ antisense). Analysis of PCR products was done by electrophoresis in

2% agarose gel and visualized by ethidium bromide staining.

Protein stability. LNCaP cells were plated and treated with OGX-427

or mismatch control or transfected with Hsp27 wt or empty vector as

described above. Media were changed 48 h later to RPMI + 5% serumcontaining 10 Amol/L of cyclohexamide incubated at 37jC for 2 to 6 or 16 h.

Western blot was done using AR, Hsp27, and vinculin antibodies.

In vivo imaging of luciferase activity. Bioluminescence imaging was

done with a highly sensitive, cooled CCD camera mounted in a light-tight

specimen box (Xenogen Corporation) using the protocol as previously

described (29). Before imaging, mice were injected i.p. with 100 mg/kg of the

substrate D-luciferin and anesthetized with 1% of isoflurane. At 15 min

postinjection, mice were imaged by IVIS 200, and bioluminescence was

monitored by Living Image software.

Animal treatment. Male athymic nude mice (Harlan Sprague-Dawley,

Inc.) were injected s.c. with 1 � 106 LNCaP-Probasin–driven luciferase cells.

Once tumors were palpable with serum PSA levels f25 ng/mL and

bioluminescence detectable using the IVIS-Imaging System, mice were

treated with 20 mg/kg of OGX-427 or mismatch control i.p. once daily for

7 days. Tumor volume, serum PSA, and bioluminescence measurements

were done at baseline and on days 4 and 8.Statistical analysis. All data were analyzed by Student’s t test. Levels of

statistical significance were set at P < 0.05.

Results

Androgens and Hsp27 protect LNCaP cells from apoptoticstress. Androgen is assumed to have an important role as asurvival factor in prostate epithelial cells. The synthetic androgen

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Cancer Res 2007; 67: (21). November 1, 2007 10456 www.aacrjournals.org



R1881 enhances LNCaP cell survival in the presence of cytotoxicstress, including etoposide (30), cyclohexamide (10), and PI3Kinhibitors (31). We recently reported that Hsp27 conferredresistance to androgen ablation in LNCaP (23) and next set outto explore whether androgen- and Hsp27-induced prosurvivalactivities were interrelated. Before beginning to study relationshipsbetween androgen and Hsp27, we first confirmed that R1881 addedto androgen-depleted CSS increased LNCaP cell growth (Fig. 1A,left) and decreased apoptotic rates (Fig. 1A, right) compared withCSS alone. R1881 protected LNCaP cells to paclitaxel treatment,lowering apoptotic rates and increasing cell growth (Supplemen-tary Data S1). R1881 also protected LNCaP cells from apoptosis

induced by OGX-427, a second-generation ASO that potentlydecreases Hsp27 levels (23). OGX-427–induced knockdown ofHsp27 was associated with decreased apoptotic rates in R1881-treated cells, as measured by fluorescence-activated cell sorting(FACS) and PARP cleavage (Fig. 1B and C). Collectively, theseresults indicate that androgens suppress apoptosis induced byHsp27 knockdown or paclitaxel.Androgens lead to rapid Hsp27 phosphorylation via p38

MAPK pathway. Androgens stimulate growth and survival viaboth genomic and nongenomic pathways. Recently identifiednongenomic effects include activation of Src, PI3K, and Akt(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 6 d,and cell growth rates were determined by MTS assay and compared with control (day of treatment defined as 100%). Right, R1881 protects LNCaP cells fromapoptosis. 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 and5 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 beforetreatment with OGX-427 or mismatch control. PARP cleavage and Hsp27 expression levels were measured by Western blot. All experiments were repeated at leastthrice.

Androgen Receptor Interactions with Hsp27




intact neutrophils (32), we tested whether androgen leads to Hsp27phosphorylation. Interestingly, androgen induced rapid phosphor-ylation of Hsp27 on both Ser78 and Ser82 residues in a dose- andtime-dependent manner (Fig. 2A). Within 15 min after incubationwith 10 nmol/L R1881, Ser78 and Ser82 phosphorylation levelsincreased 3.2- and 5.3-fold, respectively (Fig. 2A, right).

To identify upstream effectors of androgen-induced Hsp27phosphorylation, we analyzed R1881-induced changes in p38kinase and Akt phosphorylation levels, both previously associatedwith Hsp27 activation (32, 33). R1881 enhances p38 kinase and Aktphosphorylation in a time-dependent manner, with maximumstimulation at 15 and 5 min, respectively (Fig. 2B, left and right).These results suggest that Hsp27 may be phosphorylated via p38and/or Akt pathways. Preincubation of LNCaP cells with the p38kinase inhibitor, SB 203580, abolished R1881-induced Hsp27phosphorylation (Fig. 2C, left), whereas the Akt inhibitor, LY294002, did not alter androgen-induced Hsp27 phosphorylation(Fig. 2C, right), indicating that androgen-induced Hsp27 phosphor-ylation occurs via the p38 kinase pathway.

Androgen-induced Hsp27 phosphorylation is AR dependent.To determine whether AR is required for androgen-inducedphosphorylation of Hsp27, changes in Ser78 and Ser82 phosphory-lation levels were analyzed after treatment with R1881 F the anti-androgen bicalutimide. Bicalutimide inhibited R1881-inducedHsp27 phosphorylation at both sites (Fig. 3A), suggesting thatligand binding to AR is required for Hsp27 phosphorylation byR1881. Next, PC3 cells, which do not express endogenous AR, weretransfected with increasing amounts of AR or empty vector with orwithout R1881. As shown in Fig. 3A , Hsp27 phosphorylation levels atboth Ser78 and Ser82 sites increased with increasing AR levels afterR1881 treatment. In AR-negative PC3 cells, Hsp27 phosphorylationis insensitive to androgens, but is enhanced when AR is transientlyoverexpressed in PC3 cells. These results confirm that AR isrequired for androgen-induced Hsp27 phosphorylation.

To further define mechanisms of androgen-induced Hsp27phosphorylation, we determined whether Hsp27 and AR interactusing coimmunoprecipitation for Hsp27 and AR in LNCaP cells.Hsp27 is detected in AR immunoprecipitated complexes, and

Figure 2. Androgen phosphorylation ofHsp27 requires p38 MAPK pathway.A, R1881 leads to rapid phosphorylationof Hsp27 on both Ser78 and Ser82 residuesin a dose- (left ) and time-dependent(right ) manner: LNCaP cells weremaintained in CSS for 16 h before R1881treatment. Total proteins were analyzed byWestern blotting with anti–phospho-Hsp27Ser78 or Ser82 or anti–total Hsp27 forcontrol loading. B, R1881 leads tophosphorylation of p38 kinase (left ) andAkt (right ) in a time-dependent manneras shown using anti–phospho-Akt oranti–phospho-p38 antibodies. Anti–totalAkt or anti–total p38 was used for controlloading. C, the p38 inhibitor SB203580(10 Amol/L; left ), but not the Akt inhibitorLY98059 (right ), inhibits R1881-inducedHsp27 phosphorylation. After 5 or 15 minR1881 (for AKT or p38, respectively),total lysates were analyzed by Westernblotting Hsp27 anti–phospho-Hsp27 Ser78

or Ser82 or anti–total Hsp27 for controlloading.

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conversely, AR is detected in Hsp27 immunoprecipitated com-plexes (Fig. 3C, left). The domain of AR required for the interactionwith Hsp27 was next analyzed using glutathione S-transferase(GST)–pulldown assays. GST pulldown after initial equimolarnormalization of GST-AR domain fusion as previously described(34) indicates that Hsp27 binds with NH2-terminal and ligandbinding (COOH-terminal) domains, but not the DNA-bindingdomain of AR (Supplementary Data S2). Furthermore, immunoflu-orescence illustrates that Hsp27 and AR colocalize in the cytoplasmof LNCaP cells cultured in the absence of androgens. Importantly,and of potential functional relevance, both proteins translocate andcolocalize in the nucleus after R1881 treatment (Fig. 3C, right).

Before ligand binding, AR exists in a complex with Hsp90 andother co-chaperones. The AR-Hsp90 interaction maintains AR in aligand-binding conformation necessary for efficient response tohormone (35). Upon ligand binding, AR is released from Hsp90 andis translocated into the nucleus. Interestingly, AR immunoprecip-itation blots show that shortly after androgen treatment, Hsp27levels increase in complex with AR as Hsp90 levels decrease(Fig. 3D), suggesting that upon ligand binding, phospho-activatedHsp27 replaces Hsp90 to chaperone AR into the nucleus.Effect of Hsp27 on genomic activity of AR. Many proteins

participate in the activation of AR, either by direct binding or aspart of a tertiary complex regulating the activity of othertransactivators. Because Hsp27 colocalizes with and shuttlesligand-activated AR during nuclear translocation, we next investi-

gated the effects of phospho-Hsp27 levels on AR transcriptionalactivity. This was done by gain-of-function strategies using over-expressing wt Hsp27, as well as loss-of-function strategies using anHsp27 ASO (OGX-427), dominant-negative Hsp27 phosphorylationTM, or an Hsp27 phosphorylation inhibitor (p38 kinase inhibitor,SB 203580). PSA transactivation assays were done using LNCaP cellstransiently transfected with the luciferase reporter plasmidregulated by the PSA enhancer-promoter region (6.1 kb) in thepresence or absence of an increasing amount of exogenous wtHsp27 (0, 0.25, 0.5, and 1 Ag). R1881 treatment increased ARreporter gene expression 34-fold, whereas wt Hsp27 overexpressionincreases androgen-stimulated transcriptional activity of PSA afurther 3-fold (Fig. 4A, left). In contrast, Hsp27 knockdown usingOGX-427 decreased the transactivation of the androgen-regulatedPSA reporter in a dose-dependent manner, further supporting a rolefor Hsp27 in AR transactivation (Fig. 4A, right).

To determine whether Hsp27 phosphorylation is required forR1881-induced AR-mediated gene activation, we overexpressed anHsp27 TM (Ser15, Ser78, and Ser82 substituted by alanine) in a dose-dependent manner and tested its ability to affect R1881-stimulatedPSA transactivation. As shown in Fig. 4B (left), Hsp27 TMtransfection inhibited luciferase transactivation from the PSAenhancer in response to R1881. Similarly, inhibition of androgen-induced, p38 kinase–mediated Hsp27 phosphorylation using thep38 kinase inhibitor, SB 203580, suppressed R1881-mediated trans-activation of PSA enhancer–driven luciferase activity (Fig. 4B,

Figure 3. Androgen-induced Hsp27 phosphorylation requires AR. A, bicalutimide inhibits R1881-induced Hsp27 phosphorylation. LNCaP cells were starved for16 h 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 with100 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 oranti-Hsp27 (left ). AR and Hsp27 colocalize and translocate into the nucleus after R1881: LNCaP cells were treatedFR1881 for 15 min and fixed in methanol/acetone forimmunofluorescence 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 16 h and treated for indicated times with 10 nmol/L R1881. 500 Ag of total extract were immunoprecipitated using AR, andWestern blot was done using Hsp90, Hsp27, and AR antibodies.





Figure 4. Effect of Hsp27 on AR transactivation. A, Hsp27 overexpression increases AR transactivation. LNCaP cells were transiently cotransfected with1 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 normalizedto 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 ofOGX-427 or mismatch control (right ). Cells were harvested, and luciferase activity was determined. Columns, means of at least three independent experimentsdone in triplicate. Fold is measured relative to PSA activation with no treatment. B, effect of Hsp27 phosphorylation on AR transactivation. LNCaP cells weretransiently cotransfected with 1 Ag of PSA-luciferase with indicated concentrations of Hsp27 TM (Ser15, Ser78, and Ser82 were mutated to alanine) followedby R1881 or vehicle for 24 h. The total amount of plasmid DNA transfected was normalized to 2 Ag per well by the addition of an empty vector (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 knockdowndecreases PSA mRNA expression (left ). LNCaP cells were treated as indicated and RNA extracted for Northern blot analysis of PSA and Hsp27, using GAPDHas a loading control. Hsp27 TM decreases PSA mRNA expression (middle ). LNCaP cells were transfected with Hsp27 TM; 24 h after transfection, cells weretreated with R1881 for 16 h, and RNA was extracted for Northern blot analysis. p38 kinase inhibition decreases PSA mRNA expression (right ). LNCaP cells weretreated for 2 h with SB203580 (10 Amol/L) before R1881, and RNA were extracted for Northern blot analysis. D, Hsp27 knockdown decreases AR bindingto its response elements. EMSA was done using radiolabeled PSA oligonucleotides with nuclear cell extracts isolated from LNCaP treated with differentconcentrations of Hsp27 ASO. For controls, 10 Ag of nuclear protein were incubated 20 min with cold PSA oligonucleotide. E, Hsp27 recruitment to the promoterof 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 forARE I and ARE III.

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right). Collectively, these results indicate that androgen-inducedp38 kinase-mediated phosphorylation of Hsp27 is important forAR-mediated PSA expression.

The preceding data identified a role for phospho-Hsp27 in thetransactivation of PSA. To provide further evidence to substantiatethis hypothesis, Northern blot analyses were done (Fig. 4C) andindicated that levels of AR-regulated endogenous PSA mRNAdecreases significantly after Hsp27 knockdown (OGX-427; Fig. 4C,left), p38 kinase inhibition (SB 203580; Fig. 4C, middle), or Hsp27phosphorylation inhibition (Hsp27 TM; Fig. 4C, right). These resultsconfirm that Hsp27 levels and phosphorylation status enhanceAR-mediated PSA mRNA expression.

As a transcription factor, AR translocates to the nucleus afterandrogen binding, where it interacts with ARE to transactivate itstarget genes. Suppression of Hsp27 levels or phosphorylationnegatively regulates androgen-stimulated transcriptional activity ofPSA. To determine whether AR interaction with its responseelements is affected by Hsp27 levels, we next analyzed the effects ofHsp27 knockdown on AR binding to PSA-ARE using electropho-retic mobility shift assay (EMSA) with nuclear lysates of LNCaPcells treated with OGX-427. As shown in Fig. 4D , 10 nmol/L R1881increased AR binding to ARE, and this effect was specificallyblocked by excess cold PSA oligonucleotide. Hsp27 knockdownusing OGX-427 decreased AR binding to its ARE, confirming thatHsp27 knockdown decreases AR binding to its ARE. Collectively,the preceding data indicate that phospho-activated Hsp27 plays acritical role in androgen-induced nuclear translocation andtranscriptional activity of the AR.

We next explored whether Hsp27 forms a complex with AR onthe ARE of the PSA promoter using ChIP assay in LNCaP cellsFR1881. h-Actin fragment was amplified as control. As shown inFig. 4E , Hsp27 is present, along with AR (which serves as a positivecontrol) on the AREs. In the presence of R1881, higher levels of ARand Hsp27 were recruited to the promoter proximal region (ARE I)and also to the enhancer region (ARE III) of the PSA promoter.These data indicate that Hsp27 continues to interact with the ARtranscriptional complex at the level of the ARE and seems to beregulated by androgen.Hsp27 knockdown induces AR degradation via the protea-

some-mediated pathway. Hsp27 knockdown or inhibition ofphosphorylation inhibits androgen-stimulated nuclear transloca-tion of the AR with subsequent suppression of AR-regulated geneexpression. To investigate the fate of AR after Hsp27 knockdown,changes in AR mRNA and protein levels after treatment withOGX-427 were evaluated. AR mRNA levels did not change afterHsp27 knockdown (Fig. 5A, left). In contrast, Hsp27 knockdowndecreased AR (Fig. 5A, middle) and Hsp90 (Fig. 5A, right) proteinlevels in a dose-dependent manner. The effect of Hsp27 knockdownon AR protein stability was next evaluated using cyclohexamide,which inhibits protein synthesis. As shown in Fig. 5C , AR proteinlevels decrease significantly with rapid degradation after OGX-427–induced knockdown of Hsp27. In contrast, Hsp27 overexpressionprolonged AR half-life compared with empty vector-transfectedcontrols (Fig. 5C). AR degradation after Hsp27 knockdown occursvia the proteasome pathway because treatment with the protea-some inhibitor MG-132 suppressed Hsp27 knockdown-induced ARdegradation (Fig. 5C). Taken together, these findings indicate thatHsp27 knockdown induces AR degradation via a proteasome-mediated pathway.Hsp27 knockdown disrupts AR-Hsp90 association and

increases AR-MDM2 association and ubiquitination. AR forms

a heterodimer complex with Hsp90 to provide stability for ligand-unbound AR. Indeed, without Hsp90 binding, the unfolded proteinwill be recognized and degraded by the ubiquitin-proteasomesystem (12, 35). Hsp27 knockdown by OGX-427 significantlydisrupts the association between AR and Hsp90 (Fig. 5D, left).This indicates that Hsp27 knockdown–induced dissociationbetween AR and Hsp90 may render the AR-Hsp90 heterocomplexvulnerable to degradation by the proteasome-mediated pathway.Because AR has been reported to be ubiquitinated by the E3 ligase,MDM2 (36, 37), we next set out to determine whether interactionbetween MDM2 and AR increased after OGX-427–induced Hsp27knockdown. As predicted, Hsp27 knockdown increased theassociation between endogenous MDM2 and AR as shown by co-immunoprecipitation experiments in Fig. 5D (middle). Further-more, Hsp27 knockdown increased levels of ubiquitinated AR (Fig.5D, right). These results indicate that OGX-427–induced Hsp27knockdown dissociates the Hsp90-AR heterocomplex and increasesMDM2-mediated AR ubiquitination and degradation.In vivo Hsp27 knockdown by OGX-427 decreases LNCaP

proliferation rates, serum PSA levels, and AR client proteinexpression levels. Our results establish a novel mechanismwhereby ligand-activated AR phospho-activates Hsp27 to cooper-atively enhance AR nuclear translocation and transcriptionalactivity. Hsp27 knockdown leads to AR degradation and reducedPSA transcription and LNCaP cell growth in vitro . We sought todetermine whether in vivo Hsp27 knockdown with OGX-427decreased AR activity in LNCaP xenografts. Male nude mice wereinjected s.c. with 1 � 106 LNCaP-Probasin–driven luciferase-transfected cells (Supplementary Data S3), and once tumors werepalpable with serum PSA levels f25 ng/mL and bioluminescencewas detectable using the IVIS Xenogen monitor, mice weretreated with 20 mg/kg of OGX-427 or mismatch control. A bio-luminescent signal first became detectable when serum PSA levelsreached f5 ng/mL and was easily detected at serum PSA levelsabove 20 ng/mL. Mice were subjected to three different types ofmeasurements: bioluminescence of probasin-promoter–drivenluciferase activity (a measure of in vivo AR activity in LNCaPxenografts), serum PSA levels, and tumor volume. Measurementswere done at baseline before treatment (day 0), during treat-ment (day 4), and at the end of treatment (day 8). Beginning day 4,OGX-427 decreased bioluminescence and reduced circulating PSAlevels by 60% (P < 0.01), with a slight 15% decrease in tumorvolume (Fig. 6A and B); individual data are presented inSupplementary Data S4. In contrast, mice treated with mismatchcontrol ODN showed an anticipated increasing trend on bio-luminescence and PSA levels, coincident with increasing tumorvolume during the 8-day treatment period. Western blot of snap-frozen xenograft samples (Fig. 6C) and immunostaining data(Supplementary Data S5) indicate that OGX-427 decreased LNCaPxenograft levels of AR, Hsp27, and Hsp90. In addition, OGX-427also decreased Ki67 staining (Supplementary Data). Collectively,these data suggest that the in vivo anticancer activity of OGX-427results, in part, from AR disruption, and that serum PSA mayprove a useful surrogate of pharmacodynamic activity as OGX-427moves into human trials.

Discussion

Hsp27 is an ATP-independent chaperone that is phospho-activated by cell stress to prevent aggregation and/or regulateactivity/degradation of certain client proteins. The chaperone activity





of Hsp27 is regulated by stress-induced changes in phosphorylationand oligomerization (38). As a cytoprotective chaperone situated as aHub at the center of many pathways regulating cellular response totherapeutic stress, targeted inhibition of Hsp27 would inhibit manypathways implicated in cancer progression and resistance. Thecytoprotective effects of Hsp27 result from its ubiquitin binding anddegradation of InB (20), direct interference of caspase activation,modulation of oxidative stress, and regulation of the cytoskeleton(19, 39). Higher levels of Hsp27 are commonly detected in manycancers including prostate (22, 23, 40) and is associated withmetastasis, poor prognosis, and resistance to chemotherapy or

radiation (41, 42). We recently reported that overexpression ofHsp27 in LNCaP cells suppressed castration-induced apoptosis andconfers androgen resistance (23), whereas Hsp27 knockdown usingASO potently decreases Hsp27 levels, increases caspase-3 cleavageand apoptosis, enhances paclitaxel chemosensitivity, and delaystumor progression in vivo (22, 23).

Human Hsp27 is phosphorylated on three serine residues, Ser15,Ser78, and Ser82. p38 kinase and Akt were reported to be the Hsp27kinases (32, 33), although protein kinase C (PKC) a and y and cyclicAMP–dependent kinase can also phosphorylate Hsp27 (43). Hsp27becomes phosphorylated when exposed to angiotensin (43),

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 treatedin 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 ofOGX-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 andthen 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 blotanalysis. C, Hsp27 knockdown accelerates proteasomal degradation of AR. LNCaP cells were treated with OGX-427 or mismatch control and 10 Amol/L MG-132for 6 h. DMSO was used as control. AR protein level was measured by Western blot analysis. D, effect of Hsp27 knockdown on AR/Hsp90 association: LNCaP cellswere 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 wasdone 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 ofMG-132 (10 Amol/L) for 6 h. 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 6 h. AR immunoprecipitation and Western blot was done withanti-ubiquitin antibody (right ). Membrane was stripped and blotted with anti-AR as control immunoprecipitation. Input was blotted with Hsp27 antibody.

Cancer Research




interleukin-6 (IL-6), IL-1, heat shock and tumor necrosis factor-a(44). Using LNCaP cells, which express AR and are sensitive toandrogens, we show that androgens increase phospho-Hsp27 levelswithin minutes in a dose-, time-, and p38 kinase pathway–

dependent manner. This rapid androgen/AR-mediated Hsp27phosphorylation identifies a nongenomic mechanism for AR inLNCaP cells. These findings are in agreement with previous studiesindicating 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-Lucxenografts after OGX-427 treatment by IVIS imaging system. Intact male mice were injected s.c. with 1 � 106 LNCaP-Pb-Luc cells and, once tumors formed withserum 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-427or mismatch control were measured using IMX immunoassays (left ). Effect of OGX-427 treatment on LNCaP tumor volume. Xenograft volume was measured bycaliper at indicated time (right ). Points, average of five mice per group; bars, SE. C, total LNCaP xenograft proteins were extracted in RIPA buffer after mismatchor 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, schemaillustrating cooperative interactions between ligand-activated AR and Hsp27 phosphorylation: Androgen binding to AR leads to rapid Hsp27 phosphorylation via p38kinase pathway, which displaces Hsp90 and chaperones AR to the nucleus to enhance activation of AR-regulated genes (left). OGX-427 induced Hsp27 knockdowndestabilizes AR/Hsp90 heterocomplex and leads to MDM2-mediated ubiquitination and AR proteasomal degradation (right ).





through atypical membrane receptors, and that AR mediatesnongenomic activation in response to androgens (6, 10). Ligandbinding to AR induces its association with Src via Src SH3 domainand AR proline-rich domain, triggering Src-dependent pathwayactivation (6, 10). In AR-negative COS-1 and PC3 cells, transfectionof AR is necessary for R1881 induction of c-Src/Raf/extracellularsignal-regulated kinase and Akt, respectively. In AR-positive LNCaPcells, the anti-androgen bicalutamide suppresses R1881-stimulatedHsp27 phosphorylation. In AR-negative PC3 cells, Hsp27 phos-phorylation is insensitive to androgens, but is enhanced when AR istransiently overexpressed in PC3 cells. This nongenomic stimula-tion is supported further by the interaction and colocalization ofHsp27 with AR. Hsp27 binds with AR via its NH2-terminal domainand may directly or indirectly involve other AR coregulators,including signal transducers and activators of transcription 3(STAT3) and ARA55, both of which have been reported to associatewith Hsp27 (45, 46).

In the classic model of androgen action, in response toandrogens, AR dissociates from Hsp90 (11). Interestingly, we foundthat following androgen treatment, Hsp27 becomes more abun-dantly associated with AR as AR dissociates from Hsp90, suggestinga dynamic role for molecular chaperones in AR shuttling. Thisnongenomic action of AR ultimately influences its classic genomiceffects. Once in the nucleus, ligand-bound nuclear receptors likeAR are recruited to target gene promoters either through directbinding to hormone response elements or association with otherpromoter-bound transcription factors (11). Many proteins partic-ipate in the activation of AR, some directly binding to AR, andothers via a tertiary complex with other transactivators. Hsp27 hasnow been identified as a chaperone interacting with the ARtranscriptional complex at the level of its ARE. Recent reportsindicate that the AR coactivators STAT3 and ARA55 also interactwith Hsp27 (23, 45). Hence, Hsp27 and AR may complex with eitherARA55 or STAT3 to cooperatively promote AR translocation andtransactivation. Interestingly, PSA transactivation in LNCaP cells isenhanced by Hsp27 overexpression and suppressed by Hsp27knockdown or inhibition of Hsp27 phosphorylation. Similarly, inER+ MCF-7 breast cancer cells, inhibition of p38 kinase signalingalso blocks ER-mediated transcription by inhibiting nucleartranslocation of ERa (47). Furthermore, Hsp27 knockdowndecreases AR nuclear translocation and binding to its ARE.

Previous studies emphasize the importance of Hsps in steroidreceptor stability. For example, Hsp90 inhibitors such as geldana-mycin induce steroid receptor degradation by directly binding tothe ATP-binding pocket of Hsp90 and thereby inhibiting its function(12, 35). Results shown in Figs. 5 and 6 indicate that Hsp27knockdown destabilizes AR by inducing the dissociation of the AR/Hsp90 heterocomplex and increasing AR association with the E3ligase MDM2, with subsequent ubiquitin-proteasome–mediated ARdegradation. Although both OGX-427 and geldanamycin share theability to abrogate the interaction between AR and Hsp90, they doso by different mechanisms. Interestingly, OGX-427 induces thedegradation of Hsp27, AR, and Hsp90, whereas geldanamycinbinding to the ATP-binding pocket of Hsp90 inhibits its chaperoneactivity (48), induces degradation of client proteins (49), and isaccompanied by stress-activated increases in Hsp70 and Hsp27 (50).

The effects of OGX-427 on AR expression and activity in vitrowere recapitulated in vivo . OGX-427 induced rapid decreases inprobasin-luciferase reporter–driven bioluminescence that corre-lated with early decreases in serum PSA and decreased LNCaPxenograft levels of AR and its chaperones, Hsp90 and Hsp27. Inprostate cancer where the AR is critically important, ligand-activated AR leads to rapid p38 kinase–mediated phosphorylationof Hsp27, which, in turn, complexes with and chaperones the ARto enhance its stability, shuttling, and transcriptional activity.OGX-427–induced knockdown of Hsp27 destabilizes the AR andenhances its ubiquitination and degradation. Collectively, thesedata identify a novel mechanism for Hsp27 as an AR chaperone(as summarized in model illustrated in Fig. 6D) and justifies furtherinvestigation targeting Hsp27 as therapeutic for prostate cancer.

Acknowledgments

Received 6/4/2007; revised 7/25/2007; accepted 8/22/2007.Grant support: Terry Fox Foundation of the National Cancer Institute of Canada

and the NIH Pacific Northwest Prostate Specialized Programs of Research Excellence.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

The University of British Columbia has submitted patent applications, listingDr. Gleave as inventor, on the antisense sequence described in this paper. This IP hasbeen licensed to OncoGenex Technologies, a Vancouver-based biotechnology companythat Dr. Gleave has founding shares in.

We thank Virginia Yago and Mary Bowden for their technical assistance in animalexperimentation.

Cancer Research


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2007;67:10455-10465. Cancer Res Amina Zoubeidi, Anousheh Zardan, Eliana Beraldi, et al. Activityand Heat-Shock Protein 27 Facilitate AR Transcriptional Cooperative Interactions between Androgen Receptor (AR)

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