hsp27 regulates epithelial mesenchymal transition ... · hsp27 regulates epithelial mesenchymal...

Therapeutics, Targets, and Chemical Biology

Hsp27 Regulates Epithelial Mesenchymal Transition,Metastasis, and Circulating Tumor Cells in Prostate Cancer

Masaki Shiota1, Jennifer L. Bishop1, Ka Mun Nip1, Anousheh Zardan1, Ario Takeuchi1,Thomas Cordonnier1, Eliana Beraldi1, Jenny Bazov1, Ladan Fazli1, Kim Chi1,3,Martin Gleave1,2, and Amina Zoubeidi1,2

AbstractDefining the mechanisms underlying metastatic progression of prostate cancer may lead to insights into how

to decrease morbidity and mortality in this disease. An important determinant of metastasis is epithelial-to-mesenchymal transition (EMT), and the mechanisms that control the process of EMT in cancer cells are stillemerging. Here, we report that the molecular chaperone Hsp27 (HSPB1) drives EMT in prostate cancer,whereas its attenuation reverses EMT and decreases cell migration, invasion, and matrix metalloproteinaseactivity. Mechanistically, silencing Hsp27 decreased IL-6–dependent STAT3 phosphorylation, nuclear trans-location, and STAT3 binding to the Twist promoter, suggesting that Hsp27 is required for IL-6–mediated EMTvia modulation of STAT3/Twist signaling. We observed a correlation between Hsp27 and Twist in patients withprostate cancer, with Hsp27 and Twist expression each elevated in high-grade prostate cancer tumors. Hsp27inhibition by OGX-427, an antisense therapy currently in phase II trials, reduced tumor metastasis in a murinemodel of prostate cancer. More importantly, OGX-427 treatment decreased the number of circulating tumorcells in patients with metastatic castration-resistant prostate cancer in a phase I clinical trial. Overall, this studydefines Hsp27 as a critical regulator of IL-6–dependent and IL-6–independent EMT, validating this chaperoneas a therapeutic target to treat metastatic prostate cancer. Cancer Res; 73(10); 3109–19. �2013 AACR.

IntroductionProstate cancer is the most common cancer and the

second leading cause of cancer-related death in males inNorth America. Although early detection and treatment oflocalized Prostate cancer has improved outcomes, manymen still die of metastatic disease that occurs when patientsfail antiandrogen therapies and progress to castration-resis-tant prostate cancer (CRPC; ref. 1). CRPC progression is acomplex process whereby cancer cells acquire the ability tosurvive and proliferate in the absence of testicular andro-gens. Metastasis of CRPC cells requires a process calledepithelial-to-mesenchymal transition (EMT), which endowsmalignant cells with enhanced migratory and survival attri-butes that facilitate cancer establishment in new sites (2).Understanding molecular mechanisms that enable prostatecancer cell dissemination, in particular characterizing EMTeffectors, will yield new insights into mechanisms of metas-

tasis and may provide novel therapeutic targets that canprevent lethal disease.

During EMT, malignant epithelial cells acquire mesenchy-mal characteristics with defined morphology, protein expres-sion, and gene signatures. The loss of epithelial cell markers,including E-cadherin, concomitant with gain of mesenchymalmarkers like N-cadherin, vimentin, and fibronectin, are hall-marks of EMT. EMT can be initiated by a wide variety of signalsin the tumor environment, activating transcription factorsTwist and Snail and leading to repression of E-cadherin (2).In particular, the interleukin (IL)-6/STAT3 signaling pathway isa well-characterized inducer of EMT in many cancers, includ-ing head and neck (3), non–small lung (4), and breast (5).Importantly, IL-6 overexpression in patients with prostatecancer has been implicated in the development of CRPC andhigh levels of IL-6 correlate with tumor burden and metastasis(6), however, whether IL-6 signaling plays a role in EMTinduction in prostate cancer remains unknown.

In addition to IL-6, elevated expression of the heat shockprotein Hsp27 occurs in prostate cancer and is associated withCRPCprogression (7). Hsp27 is anATP-independentmolecularchaperone that is highly induced in response to cellularstresses, including exposure to mitogens, inflammatory cyto-kines, growth factors, hormones, oxidative stress, and antican-cer agents (8–10). Not surprisingly, therefore, Hsp27 has beenidentified as a critical mediator in cancer progression, pre-venting apoptosis in transformed cells (7, 11, 12). In addition,Hsp27 enhances migration and invasion in breast cancer cells(13), mediates EMT in lung cancer cells (14), and has been

Authors' Affiliations: 1The Vancouver Prostate Centre; 2Department ofUrologic Sciences, University of British Columbia; and 3Vancouver CancerCentre, BC Cancer Agency, Vancouver, British Columbia, Canada

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

Corresponding Author: Amina Zoubeidi, The Vancouver ProstateCentre, 2660 Oak Street, Vancouver, British Columbia, V6H 3Z6,Canada. Phone: 604-875-4818; Fax: 604-875-5654; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-3979

�2013 American Association for Cancer Research.

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Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3979

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implicated as an inducer of EMT during kidney fibrosis (15).Like IL-6, Hsp27 interacts closely with STAT3; for example,there is a direct interaction between Hsp27 and STAT3 inbreast and prostate cancer cells (7, 16). Importantly, theinteraction between Hsp27 and STAT3 in prostate cancer cellsis required for Hsp27-mediated protection from apoptosisduring androgen ablation (7).

Epithelial-to-mesenchymal transition is a definitive featureof aggressive metastatic prostate cancer. Increased expressionof IL-6 and Hsp27, which are known to induce EMT in othercancer models, strongly correlate with CRPC, prostate cancertumor burden, and poor patient prognosis. However, it isunknown whether Hsp27 regulates EMT in prostate cancercells downstream of IL-6, or if targeting Hsp27 is a viablestrategy to control metastasis in CRPC. Herein, we show thatHsp27 overexpression drives EMT in prostate cancer cells andis required for STAT3-mediated IL-6 induction of EMT. More-over, inhibition of Hsp27 reverses EMT in vitro, preventstumor metastasis in a murine prostate cancer model and,importantly, reduces numbers of circulating tumor cells (CTC)in patients with CRPC.

Materials and MethodsAntibodies and reagents

OligofectAMINE, lipofectin, FBS, and ZO-1 antibody werefrom Invitrogen-Life Technologies, Inc.; IL-6 was fromResearch Diagnostic Inc.; and Dual-Luciferase Reporter AssaySystem was from Promega. Total Hsp27/pHsp27 antibodieswere from Assay Designs; Total STAT3, pSTAT3Y705, andpSTAT3S727 and pGSK3b antibodies were from Cell SignalingTechnology; protein-G sepharose, Twist antibody was fromSanta Cruz Biotechnology Inc.; E-cadherin, vimentin, andfibronectin antibodies were from BD Biosciences; and LaminB1 and antiactin were from Abcam and Sigma, respectively.

Cell culture and transfectionLNCaP were provided by Dr. Leland W.K. Chung (1992,

MDACC, Houston, TX), tested and authenticated by whole-genome and whole-transcriptome sequencing (IlluminaGenome Analyzer IIx, July 2009). ARCaPE and ARCaPM [ref. 17;derived from ARCaP cells and developed in the laboratory ofDr. Chung (18)] and LNCaP cells weremaintained in RPMIþ5%FBS. LNCaP were stably transfected with shRNA Hsp27 (SantaCruz), selected using puromycin (Invitrogen-Life Technolo-gies, Inc.) and maintained in Dulbecco's Modified Eagle Medi-um (DMEM)þ5% FBS. For siRNA transfection, cells weretreated with Hsp27 siRNA or Scramble siRNA using Oligofec-tAMINE as described previously (12). Twenty-four hours afterthe second transfection, the cells were serum starved overnightand stimulated with IL-6 as indicated.

Western blot analysis and immunoprecipitationTotal proteins were extracted as described previously (12) or

fractionated using the CelLytic NuCLEAR Extraction Kit (Sig-ma) according to manufacturer's protocol. For immunopre-cipitation, proteins were extracted with immunoprecipitationlysis/wash buffer [25 mmol/L Tris-HCl (pH 7.4), 150 mmol/L

NaCl, 1 mmol/L EDTA, and 1% NP-40, pH 7.4], precleared withprotein-G sepharose for 1 hour, and 500 mg protein wasimmunoprecipitated overnight at 4�Cwith 2 mg of anti-STAT3,or immunoglobulin G (IgG) as a control. Immune complexeswere recovered with protein-G sepharose for 2 hours andwashed with IP lysis/wash buffer 3 times after Westernblotting.

Quantitative PCRTotal RNA was extracted from cells using TRIzol reagent

(Invitrogen) and 2 mg was reversed transcribed using randomhexamers (Applied Biosystems) and 20U of Moloney murineleukemia virus reverse transcriptase (Invitrogen). Quantitativereal time PCR (qRT-PCR) amplification of cDNA was carriedout using the following: Twist1 (Hs00361186_m1), E-cadherin(Hs01023894_m1), vimentin (Hs00185584_m1), fibronectin(Hs01549976_m1), Hsp27 (Hs03044127_g1), and glyceralde-hyde 3-phosphate dehydrogenase (GAPDH; Hs03929097_g1)on the ABI PRISM 7900 HT Sequence Detection System withTaqMan Gene Expression Master Mix (Applied Biosystems).Target gene expression was normalized to GAPDH levels.Results are representative of at least 3 independent experi-ments, with each sample being run in triplicate.

Cell migration assaysTranswell assay. Cells (5� 104) in serum-free media were

seeded in the top chamber and 20% FBS was used as thechemoattractant in the bottom chamber (BD Biosciences).Migrated cells were fixed in 100% methanol and stained using0.5% crystal violet. Quantification of the migrated cells wasdone by counting 4 region of the filter under the microscope.Triplicate filters were used and the experiments were repeated3 times.

Wound healing assay. Scratch wounds were made inconfluent cell monolayers using a pipette tip. Monolayers werewashed twice with serum-free media and the cells were cul-tured in serum-free media with or without 50 ng/mL of IL-6.Cell migration was recorded in 6 different microscopic fields.The percentage of wound healing was calculated by theequation; (percent wound healing) ¼ average of [(gap area:0 hour) � (gap area: 24 hours)]/(gap area: 0 hours).

Gelatin Zymography for MMP activityCells were incubated in DMEM overnight and conditioned

media was collected and concentrated 20� using Amiconcentricon-10 concentrators (Amicon). Protein lysates wereresolved on 8% nondenaturing polyacrylamide gel containing1mg/mL gelatin (Bio-Rad). Gels were incubated in 2.5% TritonX-100 for 1 hour, developed at 37�C overnight in activationbuffer (50 mmol/L Tris-HCl, pH 7.6, 50 mmol/L NaCl, and5 mmol/L CaCl2), stained with Coomassie Blue for 1 hour, anddistained to define signal. Imaging and densitometry wasconducted using a LI-COR odyssey imaging system.

Luciferase assayLNCaP cells (2.5 � 105) were plated in 6-well plates and

transfectedwith Twist orwith STAT3-Luc using lipofectin. Thereporter plasmids (Twist1-Luc �969, �745, �451, �186, and

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Cancer Res; 73(10) May 15, 2013 Cancer Research3110




�105) containing various lengths of the promoter of the wild-type Twist gene were gifted from Dr. Wang LH (Mount SinaiSchool of Medicine, New York, NY; ref. 19). Total plasmidDNA was normalized to 0.5 mg per well by the addition of acontrol plasmid. At 24-hour posttransfection, cells were incu-bated with 50 ng/mL of IL-6 for 6 hours and luciferase activ-ities were measured using a Dual-Luciferase Reporter AssaySystem (Promega) and a microplate luminometer (EG&GBerthold). The Firefly luciferase activities were corrected bythe corresponding Renilla luciferase activities. Results arerepresentative of at least 3 independent experiments. Allexperiments were carried out in triplicate.

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assay was con-

ducted according to manufacturer's protocol. LNCaPsh-Control and LNCaPsh-Hsp27 cells treated with 50 ng/mLof IL-6 for 30 minutes were cross-linked with paraformal-dehyde and digested with micrococcal nuclease to achieve aDNA smear of 200—1,000 bp. ChIP assay was conductedusing the SimpleChIP Enzymatic Chromatin IP Kit (AgaroseBeads) according to the manufacturer's protocol (Cell Sig-naling Technology). PCR conditions were 30 cycles of 95�C,60�C, and 72�C for 30 seconds. qRT-PCR was conductedwith 2 mL of DNA extraction, with the following primers:Twist promoter 50-GCCAGGTCGTTTTTGAATGG-30 (for-ward) and 50-CGTGCAGGCGGAAAGTTTGG-30 (reverse) andFastStart Universal SYBR Green Master (ROX; Roche AppliedScience) on the ABI PRISM 7900 HT Sequence DetectionSystem (19). Results are representative of at least 3 inde-pendent experiments.

Prostate tumor immunohistochemistryA total of 106 prostate cancer specimens were obtained

from the Vancouver Prostate Centre Tissue Bank. Two tissuemicroarrays (TMA) were manually constructed (BeecherInstruments) by punching duplicate cores of 1 mm for eachsample. All the specimens were from radical prostatectomyexcept 12 CRPC samples obtained from transurethral resec-tion of prostate. Immunohistochemical staining was con-ducted as previously described (12) using the Ventana Dis-cover XT Autostainer (VentanaMedical System) with enzyme-labeled biotin streptavidin system and a solvent-resistantDAB Map kit by using 1/150 concentration of TWIST-1 andpSTAT3 (Cell Signaling Technology) and 1/25 concentrationsof HSP27 (Sigma) antibodies. TMAswere scored as follows: 0¼no staining, 1¼ faint or focal stain, 2¼ convincing intensity ina minority of cells, and 3 ¼ convincing intensity in a majorityof cells. The overall percentage of cancer cells showing staining(0%–100%) was also indicated. Scoring was conducted at200�.

In vivo tumor metastasis modelPC-3M-luc (C6) cells (2 � 106; Caliper Life Sciences) were

injected into tail veins of 6- to 8-week-old male athymic nudemice (Harlan Sprague-Dawley, Inc.). After 24 hours, mice wererandomly divided into 2 groups, n¼ 10, for treatment with ScrASO or OGX-427. Scr ASO or OGX-427 of 20mg/kgwas injected

intraperitoneally once daily for 7 days followed by 3 weeklytreatments thereafter. On weeks 2, 4, 6, 8, and 10 mice wereinjected intraperitoneally with 150 mg/kg D-luciferin (CaliperLife Sciences), anesthetized with isoflurane, and imaged in thesupine position exactly 11 and 15 minutes after injection in anIVIS200 Imaging System (Caliper Life Sciences). Data wereacquired and analyzed using Living Image software version 3.0(Caliper Life Sciences). Animal procedures were done accord-ing to the guidelines of the Canadian Council on Animal Care.

Circulating tumor cell isolationPatients with CRPC enrolled in OGX-427 phase I of OGX-

427 receiving doses from 400 mg to 1,000 mg i.v. on a 21-dayschedule had whole-blood collected for CTC analyses atscreening, after OGX-427 initiation, and on day 1 of everycycle. CTC enumeration was conducted as previouslydescribed (12). Blood samples were drawn into 10 mLevacuated blood draw tubes (CellSave; Veridex LLC), main-tained at room temperature, and processed within 96 hoursof collection on the CellSearch System (Veridex LLC) as permanufacturer's instructions. CTC were defined as nucleatedcells lacking CD45 and expressing cytokeratin as per man-ufacturer's instructions.

ResultsHsp27 promotes epithelial-to-mesenchymal transition

Clinically, Hsp27 is highly expressed in many cancers (20),including breast (21), prostate (22), where it is associatedwith aggressive tumor behavior, metastasis, and poor progno-sis (23, 24) and its overexpression enhances migration andinvasion (25). However, a defining role for Hsp27 in EMT inprostate cancer remained unexplored. To evaluate the rela-tionship between Hsp27 and EMT, Hsp27 was overexpressedin RWPE-2 cells, a normal epithelial prostate cell line trans-formed with K-Ras, and in the epithelial prostate cancer cellline, ARCaPE (17, 18, 26). Hsp27 overexpression in both linescaused decreases in E-cadherin expression (Fig. 1A) andincreases in vimentin and fibronectin (Fig. 1B), whereas itsknockdown using siRNA in the prostate cancer cell lineDU145, lung cancer cell line A549, and breast cancer cell lineMCF-7, led to increased E-cadherin (Fig. 1C) and decreasedvimentin and fibronectin (Fig. 1D) expression. These observa-tions suggested that Hsp27 could regulate EMT in differentcancers and in prostate cells. As elevated expression of Hsp27is associated with poor prognosis of metastatic prostate can-cer (22), Hsp27 was stably overexpressed in the human andro-gen receptor (AR)-positive prostate cancer cell line, and LNCaPand EMT was examined. Hsp27 overexpression in LNCaPcells induced changes in cell morphology, protein, and mRNAexpression characteristic of EMT. Examination of cells byphase contrast and immunofluorescent microscopy showeda shift from an epithelial, cobblestone-like morphology inmock-treated (mock) cells to an elongated and spindled fibro-blasticmorphology inHsp27-overexpressing (Hsp27) cells (Fig.2A). This effect was accompanied by decreases in cell–cellcontact and a loss of E-cadherin and the tight junction protein,ZO-1 (Fig. 2A and B). Hsp27 cells also showed increased

Role of Hsp27 in EMT and Prostate Cancer Metastasis

www.aacrjournals.org Cancer Res; 73(10) May 15, 2013 3111




expression of the mesenchymal markers phospho-GSK-3b,vimentin, fibronectin, Twist, and N-cadherin at both proteinand mRNA levels (Fig. 2B). Moreover, consistent with previousstudies (27), we found that the expression of Hsp27 increasedcell migration (Fig. 2C) and matrix metalloproteinase (MMP)activity (Fig. 2D), 2 important factors in tumor migration andmetastasis (28).

Silencing Hsp27 leads to mesenchymal-to-epithelial inprostate cancer cells

To effectively relate Hsp27 expression levels to specificcellular behaviors, we developed a LNCaP cell line with stablereduction (up to 95%) of Hsp27 expression using Hsp27targeting shRNA (sh-Hsp27). sh-Hsp27 cells displayed a clearmorphologic transition to an enhanced epithelial or cobble-stone-like pattern, with well-organized cell contact and polar-ity (Fig. 3A). These changes were accompanied with increasedE-cadherin and decreased phospho-GSK-3B, vimentin, fibro-nectin, and Twist at protein and mRNA levels (Fig. 3A and B).Hsp27 knockdown cells also exhibited reduced cell invasion(Supplementary Fig. S1), migration, and MMP activity com-pared with controls (Fig. 3C and D). To control for nonspecificchanges in EMT status potentially induced by stable Hsp27

knockdown, similar experiments were conducted in LNCaPcells treated with siRNA targeting Hsp27. LNCaP cells treatedwith Hsp27 siRNA also showed induction of markers of epi-thelial, as opposed to mesenchymal, differentiation; includinghigher levels of E-cadherin and lower levels of vimentin,fibronectin, and Twist, compared with control siRNA(siCtr; Supplementary Fig. S2A and SB). Hsp27 siRNA alsoreduced cell invasion and MMP activity (Supplementary Fig.S3A and SB) in LNCaP cells. Taken together, these data suggestthat Hsp27 is a positive regulator of EMT.

IL-6 induces EMT in an Hsp27-dependent mannerIL-6 is elevated in biopsies and sera of patients with met-

astatic prostate cancer (6, 29) and its overexpression in thebreast cancer cells induces EMT via STAT3-Twist (5). There-fore, we sought to determine whether IL-6 induces EMT inprostate cancer cells and the involvement of Hsp27 in thispathway. As in MCF-7 cells (5), we found that LNCaP cellsexposed to IL-6 showed decreased levels of E-cadherin andincreased levels of fibronectin and Twist as early as 6 hoursafter treatment (Fig. 4A). Concomitantwith these changes, IL-6treatment of LNCaP cells increased expression of Hsp27(Fig. 4A). In addition, STAT3 and Twist transcriptional

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Figure 1. Hsp27 modulates EMT in cancer cell lines. A and B, Hsp27 was overexpressed in RWPE-2 and ARCaPE and Mock was used as a control. A,total proteins were examined by Western blot and vinculin was used as a loading control. B, target genes were evaluated by quantitiative PCRof total RNA and normalized to GAPDH. C and D, DU145, A549, and MCF-7 cells were treated with Hsp27 or control siRNA. C, total proteins wereextracted and examined by Western blot and vinculin was used as a loading control. D, target genes were evaluated by qRT-PCR of total RNAand normalized to GAPDH.

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activities were increased by IL-6 treatment (Fig. 4B). Takentogether, these data suggest that this pathway is active inLNCaP cells and that IL-6 induces EMT in LNCaP cells mostlikely via STAT3 and Twist transcription, with potentialinvolvement of Hsp27.Next, we set out to define the role of Hsp27 in IL-6-induced

EMT by analyzing epithelial andmesenchymal markers as wellas Twist activity in IL-6 stimulated Hsp27 stable knockdowncells. IL-6 treatment of LNCaP cells induced a morphologicchange to a fibroblastic phenotype in control (sh-Ctr), but notin Hsp27 knockdown (sh-Hsp27), cells (Fig. 4C). This effect oncell morphology correlated with changes in EMT markers. Incontrol cells, IL-6 treatment increased Twist and vimentinwithin 6 hours; by 24 hours, E-cadherin levels decreased andfibronectin levels increased (Fig. 4D). In contrast, in Hsp27knockdown cells, E-cadherin levels increased over time withIL-6 treatment, and levels of Twist, vimentin, and fibronectinprotein and mRNA expression were markedly reduced almostto undetectable levels, compared with control cells in both thepresence and absence of IL-6 (Fig. 4D). In addition, knockdownof Hsp27 in LNCaP cells prevented IL-6 inducedwound healing(Fig. 4E). These data suggest that Hsp27 is required for bothIL-6–dependent and IL-6–independent EMT in prostate can-cer cells.

Hsp27 expression is required for STAT3 binding to theTwist promoterTo further dissect mechanisms by which Hsp27 regulates

IL-6–mediated EMT responses, we examined effects of Hsp27

knockdown on IL-6 pathway activity. We observed that incontrol cells (sh-Ctr), IL-6 induced phosphorylation of STAT3on both tyrosine and serine residues, as well as Hsp27, in atime-dependent manner (Fig. 5A). In contrast, stable Hsp27knockdown (sh-Hsp27) drastically decreased IL-6–stimulatedSTAT3 phosphorylation (Fig. 5A). Decreased STAT3 phosphor-ylation correlated with reduced IL-6 induced STAT3 nucleartranslocation in Hsp27 knockdown cells (Fig. 5B). ChIP assayswere conducted to investigate the effect of Hsp27 knock-down on STAT3 binding to the Twist promoter (19). Usingprimers that covered the proximal 3 STAT3-binding sites in theTwist promoter (19), we found that IL-6 induced STAT3binding to the Twist promoter DNA in control, but not Hsp27knockdown, LNCaP cells (Fig. 5C). Finally, we monitored theeffect of Hsp27 knockdown on IL-6–induced Twist transcrip-tional activity using a luciferase transactivation assay. Twisttranscriptional activity was analyzed using serial truncationsof the human Twist promoter as described by Cheng andcolleagues (19) in the presence or absence of IL-6. We foundthat for each Twist truncation assessed, IL-6 induced morethan a 2-fold increase of promoter activation in control-trans-fected cells, whereas Hsp27 knockdown almost completelyabrogated IL-6–induced Twist transcriptional activity (Fig.5D left panel). Conversely, Hsp27 overexpression (Hsp27 WT)enhanced IL-6–induced Twist transcriptional activity com-pared with control (Mock) cells (Fig. 5D right panel). Takentogether, our data provide evidence that Hsp27 is required forSTAT3 binding to the Twist promoter and activating Twisttranscription in the presence of IL-6.

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Figure 2. Hsp27 induces EMT in LNCaP cells. A, morphology of LNCaP cells stably transfected with control vector (mock) or Hsp27 (Hsp27) was examinedbyphase contrastmicroscopy. Immunofluorescenceshows expression of E-cadherin, ZO-1, andvimentin [green, target; blue, 4', 6-diamidino-2-phenylindole(DAPI)]. B, total protein or RNA was analyzed by Western blotting or quantitative PCR. Equal protein loading was confirmed by actin expression and EMTmarker mRNA expression was normalized to GAPDH. C, cells were plated in transwell chambers for 48 hours and cells located in bottom chamber werecounted. D, MMP activity was assessed by gelatin zymography.






Hsp27 and Twist are associated with aggressive humanprostate cancer

Increased expression of Hsp27 is found in prostate cancerand is associated with poor prognosis and metastasis (23,24). However, correlation between increased Hsp27 andTwist expression in human tumors has not been reported.Using IHC, we observed a direct correlation between Glea-son score and Hsp27 and Twist-staining intensity; Gleason 4or 5 cancers had significantly higher Hsp27 and Twiststaining than Gleason 3 cancers (Fig. 6A and B) and Hsp27and Twist staining positively correlated across all Gleasongrades (Pearson coefficient ¼ 0.218). In addition, weobserved increased pSTAT3 staining in Gleason 4 versus 3tumors (Fig. 6A). Quantification of positive versus negativepSTAT3 signal in our TMAs showed that 83.33% of Gleason 3tumors were positive for pSTAT3 staining, whereas 98.31% ofGleason 4 tumors were positive for pSTAT3. In addition,there was a positive correlation between pSTAT3 and Hsp27(Pearson coefficient ¼ 0.114) or Twist expression (Pearsoncoefficient ¼ 0.112) across all Gleason grades. These resultssuggest a direct relationship between Hsp27 in Twist sig-naling in aggressive human prostate cancer.

Hsp27 is required for metastasis in a mouse model ofprostate cancer

Our data suggested that Hsp27 expression is a feature ofaggressive human prostate cancer tumors and EMT inprostate cancer cell lines. Therefore, we wanted to deter-

mine whether Hsp27 knockdown affects metastasis in amouse model of highly metastatic prostate cancer usingPC-3M cells. In vitro, Hsp27 knockdown reduced migrationof PC-3M cells (Supplementary Fig. S4). In vivo, after sys-temic delivery of luciferase expressing PC-3M cells, athymicnude mice were treated with either control scrambledantisense oligonucleotide (Scr ASO) or ASO targeting Hsp27(OGX-427, OncoGeneX Pharmaceuticals), and tumor spreadwas monitored over 10 weeks using a bioluminescent imag-ing system. We found a drastic reduction in luminescentsignal, indicating the presence of tumor cells, throughout theentire body of mice treated with OGX-427 compared with ScrASO at 10 weeks after tumor injection (Fig. 7A). Quantifi-cation of luminescence at 6, 8, and 10 weeks after injectionshowed a significant reduction in signal at each time point inmice treated with OGX-427 compared with Scr ASO (Fig. 7B).These results indicate that Hsp27 enhances dissemination ofprostate cancer tumor cells in vivo and further link increasedHsp27 expression with cancer aggressiveness and metastaticcapacity.

OGX-427 treatment decreases circulating tumor cells inpatients with mCRPC

Decreases in CTC counts in patients with prostate cancerundergoing treatment is associated with improved survival(30). To provide clinical relevance to our work, we evaluatedthe effects of OGX-427 on CTC counts in patients with prostatecancer. In a phase I study of OGX-427, 19 patients with

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Figure 3. Stable knockdown of Hsp27 in LNCaP cells prevents EMT. A, the morphology of LNCaP cells transfected with either Hsp27 shRNA (sh-Hsp27)or control shRNA (sh-Ctr) was examined by phase contrast microscopy. Immunofluorescence shows expression of E-cadherin, ZO-1, and vimentin[green, target; blue, 4', 6-diamidino-2-phenylindole (DAPI)]. B, total protein or RNA was analyzed by Western blotting or qPCR. Equal protein loading wasconfirmedby actin expression andEMTmarkermRNAexpressionwasnormalized toGAPDH.C, cells were plated in transwell chambers for 48 hours and cellslocated in lower chamber were counted. D, MMP activity was assessed by gelatin zymography.

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metastatic CRPC were treated with doses of OGX-427 rangingfrom400 to 1,000mgandbloodwas collected from17 evaluablepatients for CTC analyses (CTC at baseline and at least 1posttreatment time point). The median baseline CTC countwas 40 CTC/7.5 mL blood (range 4–938), which decreased to amedian of 15 CTC/7.5 mL (range 0–446) as a best CTC declinewhile on therapy. Mean CTC count was 152/7.5 mL (SD¼ 239)at baseline, which decreased to 66/7.5 mL (SD¼ 118) as a bestCTC decline on therapy (t test ¼ 0.088, 2-tailed). The medianbest percentage decline in CTC from baseline by patient was58% (range �43%–100%). One patient had a decline in CTCfrom 39 CTC/7.5 mL at baseline to 0 CTC/7.5 mL by the fifthtreatment cycle (OGX-427 dose ¼ 400 mg; summary, Supple-mentary Table S1). These CTC data provide the first clinicalevidence of anticancer activity after Hsp27 inhibition, and arecompatible with our preclinical data supporting a role forHsp27 in EMT and dissemination of tumor cells to metastaticsites.

DiscussionEpithelial-to-mesenchymal transition is a critical compo-

nent of prostate cancer progression, facilitating developmentof lethal metastatic CRPC. Using Hsp27 overexpressing, as wellknockdown prostate cancer cell lines, we showed that Hsp27 isa critical mediator of IL-6–dependent and -independent EMT.Investigation into the mechanism of Hsp27 function in IL-6induced EMT showed that Hsp27 mediates STAT3 phosphor-ylation, nuclear translocation and STAT3 binding to the Twistpromoter. These data were supported by immunohistochem-istry showing increased Hsp27 and Twist staining in highGleason grade tumors from patients with prostate cancer. Wehave also shown a role for Hsp27 in promoting prostate cancermetastasis in vivo, as tumor cell dissemination was signifi-cantly suppressed in mice treated with the Hsp27 inhibitor,OGX-427. More importantly, targeting Hsp27 in men withmetastatic CRPC using OGX-427 led to reduced CTC counts,

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Figure 4. Hsp27 is required for IL-6 induced EMT. A, LNCaP cells were serum-starved overnight and treated with �50 ng/mL of IL-6 for 0 to 24 hours.Total proteins were analyzed by Western blotting. b-Actin was used as a loading control. B, LNCaP cells were transfected with Twist or STAT3-luciferase promoter plasmids for 24 hours, then stimulated with IL-6 for 6 hours. Luciferase activity was measured using the Dual-Luciferase ReporterAssay System. C, serum-starved LNCaP sh-Ctr or sh-Hsp27 cells were stimulated with �50 ng/mL of IL-6 for 24 hours and cell morphology wasexamined by phase contrast microscopy. D, serum-starved cells were treated with �50 ng/mL of IL-6 for 0 to 24 hours. Total protein or RNA wasanalyzed by Western blotting or qPCR. Equal protein loading was confirmed by actin expression and EMT marker mRNA expression was normalized toGAPDH. E, serum-starved cells were treated with �50 ng/mL of IL-6 and percentage of migrating cells in a scratch assay was quantified at 0 and 24hours after stimulation.






providing the first clinical evidence of anticancer metastaticactivity for this drug in humans. Collectively, our resultshighlight Hsp27 as a key modulator of prostate cancer cellplasticity, driving EMT and endowing prostate cancer cellswith metastatic potential.

High levels of Hsp27 have been reported in many cancersincluding breast (21), ovarian (31), glial (32), and prostate (33,34), and it has been implicated in EMT in breast cancer (35),lung cancer (14), and kidney fibrosis (15). Our data show thatHsp27 is also required for EMT in prostate cancer cell linesand can drive EMT independently of exogenous factors likeIL-6. This may be related to its function in actin rearrange-ment, cytoskeleton organization, and cell migration (27). Ourresults showing that Hsp27 silencing increased expression ofthe tight junction protein ZO-1 and prevented cell migration,further support these reports. Such effects on Hsp27-deficientcells may be dependent not on IL-6, but the ability of Hsp27 to

interact with STAT3. Multiple reports have shown directinteractions between Hsp27 and STAT3 in the absence ofIL-6 (7, 16). Furthermore, Hsp27 overexpression enhances cellmigration via the modulation of Fak-dependent actin organi-zation and STAT3-dependent MMP-2 expression (25). Factorsthat facilitate Hsp27 and STAT3 interactions to promote IL-6independent EMT are under further investigation.

We also identified a role for Hsp27 downstream of IL-6, a keyregulator of EMT with clinical relevance to aggressive carci-noma. In prostate cancer, IL-6 mediates the development andprogression of CRPC (6, 36) and elevated IL-6 levels in sera ofpatients with prostate cancer correlate with tumor burden andmetastases (6, 29). In normal prostate cell lines, ectopicexpression of IL-6 induces cell transformation, EMT, tumorformation, and tumor progression to metastasis (37). In breastcancer cell lines, IL-6 overexpression leads to a reduction inE-cadherin, increases of vimentin, fibronectin, and Twist, as

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Shiota et al.





well as aberrant activation of STAT3 (5). Here, we show thatexogenous IL-6 treatment increased Hsp27, fibronectin, andTwist expression and decreased E-cadherin expression inLNCaP cells, indicating that IL-6 also induces EMT in prostatecancer.A key regulator of Twist transcription is STAT3, which is

constitutively active in many tumors to control the expressionof cell survival as well as EMT genes (19, 38). Recent evidencehighlighted the importance of the IL-6/STAT3 pathway inprostate cancer stem cells, identifying epigenetic modifica-tions of key genes that interact with STAT3 to promoteinvasiveness (39). Interestingly, Hsp27 binds STAT3 in breast(16) and prostate cancer cells (7), and in vivo, Hsp27 over-expression in the LNCaP xenograft mouse model confershormone resistance postcastration via the activation of STAT3(7). In this study, we showed that Hsp27 is required for IL-6/STAT3/Twist-induced EMT in prostate cancer cells. In partic-ular, we found that Hsp27 knockdown decreased IL-6 inducedSTAT3 phosphorylation and nuclear translocation, and pre-vented STAT3 binding to the Twist promoter. Our data fromhigh Gleason grade prostate cancer tumors suggest thatHsp27 may modulate the Twist pathway in human cancer aswell, mediating effects of EMT inducers like IL-6 duringmetastatic disease. Accordingly, Hsp27 was also required forin vitro wound healing, further indicating its importance in IL-6–dependent EMT. These results are similar to previousreports showing knockdown of Hsp27 inhibits VEGF andTGF-b��induced cell migration and STAT3-mediated cellinvasion in human prostate cancer cell lines (40, 41). Our

data therefore suggest that Hsp27 promotes IL-6 signaling intumor microenvironments to induce EMT, highlighting animportant mechanism in prostate cancer progression andmetastasis. The dramatic effect on reduction of CTC numberin patients with CRPC treated with the Hsp27 inhibitor,OGX-427, indicate that Hsp27 may indeed drive metastasis incancers associated with high levels of IL-6.

While it is required for STAT3 binding to Twist down-stream of IL-6, Hsp27 also induced EMT and metastasisin vivo, as it did in vitro, independently of this factor. Asaforementioned, this could be explained by Hsp27/STAT3interactions that mediate aspects of cell migration andinvasion. In addition, since Hsp27 is a molecular chaperonethat stabilizes protein complexes (12) it is possible thatHsp27 stabilizes STAT3 binding with its kinases, explainingthe dramatic effect of Hsp27 knockdown on STAT3 phos-phorylation in the presence or absence of IL-6. Alternatively,Hsp27 may play a role in other STAT3 independent signalpathways that can promote EMT. For example, Hsp27 main-tains a population of breast cancer stem cells that displayEMT characteristics by increasing degradation of IkBa,enhancing nuclear translocation of NF-kB, and stabilizingSnail, thereby repressing E-cadherin expression (35). While itremains unknown how NF-kB signaling mediates EMT inprostate cancer, reports have shown that NF-kB is activateddownstream of Egr-1 in LNCaP and PC-3 prostate cancercells (42) and Egr-1 induces EMT in breast cancer (43).

In summary, Hsp27 is a component of several pathwaysknown to induce EMT in prostate cancer. This has widespread

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Figure 6. Hsp27 expression is associated with aggressive human prostate cancer. A, representative sections of prostate cancer tumors from radicalprostectomy specimens with Gleason scores of 3 or 4 were stained for IHC specific for Hsp27 Twist and pSTAT3. B, Hsp27 and Twist staining intensitywas quantified in 2 cores each of 30 Gleason grade 3, 27 grade 4, and 10 grade 5; radical prostectomy prostate cancer tumors and ANOVAstatistical analysis was used to show significance. Error bars represent SD. C, pSTAT3 positivity was quantified in 2 cores each of 30 Gleason grade 3and 27 grade 4 tumors. Fisher's test was used to analyze statistical significance of positive versus negative staining and risk associated withpositive staining and increased Gleason grade. D, Pearson's coefficient was calculated to analyze correlation between Hsp27, Twist, and pSTAT3or Twist and pSTAT3 across Gleason grades.






implications, as EMT is not simply a feature of metastatic cells;it is also a characteristic of cancer stem cells and associatedwith cancer dormancy and treatment resistance (2). Thesedata provide support for the role of Hsp27 in IL-6 signaling andEMT and as a therapeutic target for CRPC.

Disclosure of Potential Conflicts of InterestK.N. Chi has a commercial research grant inOncoGeneX Pharmaceuticals Inc.

and is a consultant/advisory board member of OncoGenex Technologies Inc. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: A. ZoubeidiDevelopment of methodology: K. Nip, A. Zardan, J. Bazov, A. ZoubeidiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Shiota, A. Takeushi, K.N. Chi, L. FazliAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Shiota, A. Zardan, A. Takeushi, T. Cordonnier, J.Bazov, K.N. Chi, M. Gleave, A. Zoubeidi

Writing, review, and/or revision of themanuscript:M. Shiota, J.L. Bishop, A.Zardan, K.N. Chi, M. Gleave, A. ZoubeidiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): T. Cordonnier, E. BeraldiStudy supervision: A. Zoubeidi

AcknowledgmentsThe authors thank Dr. Lu-Hai Wang (Mount Sinai School of Medicine, New

York, NY) for the Twist–Luc reporter plasmid.

Grant SupportThis study was supported by Prostate Cancer Foundation USA. and Michael

Smith Foundation for Health Research (A. Zoubeidi); The Japanese PostdoctoralFellowship for Research Abroad (M. Shiota); and Prostate Cancer Foundation BCPost-doctoral fellowship (T. Cordonnier).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 18, 2012; revised February 15, 2013; accepted March 1, 2013;published OnlineFirst March 14, 2013.

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2013;73:3109-3119. Published OnlineFirst March 14, 2013.Cancer Res Masaki Shiota, Jennifer L. Bishop, Ka Mun Nip, et al. and Circulating Tumor Cells in Prostate CancerHsp27 Regulates Epithelial Mesenchymal Transition, Metastasis,

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