Clusterin Is a Critical Downstream Mediator of Stress-Induced YB …mcr.aacrjournals.org/content/molcanres/early/2011/11/22/1541-7786... · Signaling and Regulation Clusterin Is a

Signaling and Regulation

Clusterin Is a Critical Downstream Mediator ofStress-Induced YB-1 Transactivation in Prostate Cancer

Masaki Shiota1, Amina Zoubeidi1, Masafumi Kumano1, Eliana Beraldi1, Seiji Naito3, Colleen C. Nelson1,4,Poul H.B. Sorensen2, and Martin E. Gleave1

AbstractClusterin is a stress-activated, cytoprotective chaperone that confers broad-spectrum treatment resistance in

cancer. However, the molecular mechanisms mediating CLU transcription following anticancer treatmentstress remain incompletely defined. We report that Y-box binding protein-1 (YB-1) directly binds to CLUpromoter regions to transcriptionally regulate clusterin expression. In response to endoplasmic reticulum stressinducers, including paclitaxel, YB-1 is translocated to the nucleus to transactivate clusterin. Furthermore,higher levels of activated YB-1 and clusterin are seen in taxane-resistant, compared with parental, prostatecancer cells. Knockdown of either YB-1 or clusterin sensitized prostate cancer cells to paclitaxel, whereas theiroverexpression increased resistance to taxane. Clusterin overexpression rescued cells from increased paclitaxel-induced apoptosis following YB-1 knockdown; in contrast, however, YB-1 overexpression did not rescue cellsfrom increased paclitaxel-induced apoptosis following clusterin knockdown. Collectively, these data indicatethat YB-1 transactivation of clusterin in response to stress is a critical mediator of paclitaxel resistance inprostate cancer. Mol Cancer Res; 1–12. �2011 AACR.

Introduction

Prostate cancer is the most common noncutaneous cancerand the second leading cause of cancer-related death in menof Western countries. The incidence of prostate cancer isincreasing as a result of aging populations and high-fat diet inthe Western world (1). While advanced prostate cancer isinitially sensitive to androgen deprivation therapy (ADT),most eventually recur in a castration-resistant prostate cancer(CRPC). At present, only second-line ADTwith abiraterone(2), and the taxanes docetaxel (3), and cabazitaxel (4) havebeen shown to delay progression and prolong survival inCRPC. However, therapeutic benefits are brief with under4-month gains in survival before treatment resistance, pro-gression, and death occurs. Many strategies used to killcancer cells, including ADT or taxane chemotherapy, inducea treatment-resistant phenotype that is the underlying basis

for most cancer deaths. Improved understanding of themolecular basis underlyingmetastasis and resistance to ADTor chemotherapy will facilitate to design new therapeuticstrategies to inhibit the emergence of this CRPC phenotype.CRPC progression is a complex process by which cells

acquire the ability to both survive and proliferate in theabsence of gonadal androgens and involves mechanismsattributed to reactivation of the androgen receptor (AR)axis (5), alternative growth factor pathways (6, 7), stress-induced prosurvival genes (8–10), and cytoprotective chap-erone networks (11, 12). Clusterin is a stress-inducedcytoprotective chaperone expressed inmany human cancers.In prostate cancer, clusterin levels increase following castra-tion and in CRPC models (11). Clusterin levels are low inuntreated, favorable grade tissues but increase with higherGleason grade (13) and within weeks after treatment stresswith ADT (14) or taxane chemotherapy (15). Overexpres-sion of clusterin in human prostate cancer LNCaP cellsaccelerates progression after ADT (11) or chemotherapy(16), identifying CLU as an antiapoptotic gene upregulatedby treatment-related stress that confers therapeutic resistancewhen overexpressed. Miyake and colleagues first reportedthat antisense treatment against clusterin significantlyenhanced paclitaxel sensitivity in human prostate cancerPC-3 cells (10). Moreover, clusterin knockdown reducedtumor growth and sensitized to chemotherapeutic agents invarious cancer cells including renal cell cancer (17), bladdercancer (18), lung cancer (19), and breast cancer (20).Y-box binding protein-1 (YB-1) is a stress-activated tran-

scription factor implicated in and linked to unfavorableclinical outcome in various cancers (21). In prostate cancer,YB-1 is induced by ADT (22) and its overexpression confers

Authors' Affiliations: 1The Vancouver Prostate Centre and Department ofUrologic Sciences, University of British Columbia, 2Department of Molec-ular Oncology, BC Cancer Research Center, Vancouver, British Columbia,Canada; 3Department of Urology, Graduate School of Medical Sciences,Kyushu University, Fukuoka, Japan; and 4Australian Prostate CancerResearch Centre-Queensland, Institute of Health and Biomedical Innova-tion, Queensland University of Technology, Brisbane, Queensland,Australia

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author:Martin E. Gleave, The Vancouver Prostate CentreandDepartment of Urologic Sciences, University of British Columbia, 2775Laurel Street, Level 6, Vancouver, BC V6H 3Z6, Canada. Phone: 1-604-875-4818; Fax: 1-604-875-5654; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-11-0379

�2011 American Association for Cancer Research.

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castration resistance (23). Moreover, YB-1 is associated withdrug resistance to both cisplatin (24, 25) and paclitaxel (26,27). YB-1 confers paclitaxel resistance in PC-3 cells (27),whereas its knockdown sensitized breast cancer SUM149cells to paclitaxel (28).While the above findings functionally link YB-1 and

clusterin to treatment resistance in prostate cancer, theprecise mechanisms mediating these effects remain unde-fined. Here, we define functional links between stress-induced increases in YB-1 and clusterin and analyze theirbiological relevance, as related to taxane resistance in prostatecancer.

Materials and Methods

Cell culture and transfectionThe human prostate cancer cell lines, PC-3 and DU145,

were purchased from the American Type Culture Collection(2008 and 1989, ATCC authentication by isoenzymesanalysis) and maintained in Dulbecco's Modified Eagle'sMedium (DMEM; Thermo Scientific) supplemented with5% FBS. LNCaP cells were kindly provided by Dr. LelandW.K. Chung (1992, MD Anderson Cancer Center, Hous-ton, TX), tested and authenticated by whole-genome andwhole-transcriptome sequencing on Illumina Genome Ana-lyzer IIx platform in 2009. LNCaP cells were maintained inRPMI-1640 (Thermo Scientific) supplemented with 5%FBS. Docetaxel-resistant derivatives of LNCaP and DU145cells (LNCaP/DTX and DU145/DTX cells) were estab-lished by long-term culture under the appropriate mediumcontaining gradually increasing concentrations of docetaxeland maintained under media containing 3 ng/mL and 2 ng/mL of docetaxel, respectively. The LNCaP/DTX andDU145/DTX cells were found to be about 2.5-fold moreresistant to docetaxel than their parental cells. Cells weretransfected with the indicated siRNA or the indicatedplasmid as previously described (29).

Antibodies and reagentsAntibodies against ATF4 (sc-200), Myc (9E10, sc-815),

GRP78 (sc-1051), and clusterin (sc-6419) were purchasedfrom Santa Cruz Biotechnology. Anti-Lamin B1 antibodywas purchased from Abcam. Anti-phosphorylated YB-1 (p-YB-1, #2900), anti-cleaved PARP (#9541), anti-PARP(#9542), and anti-cleaved caspase-3 (#9661) antibodieswere purchased from Cell Signaling Technology. Anti-YB-1, anti-caspase-3, and anti-b-actin antibodies were pur-chased from Epitomics, BD Biosciences, and Sigma, respec-tively. Paclitaxel and docetaxel were obtained from Sigma.MG132 and 17-N-allylamino-17-demethoxy-geldanamy-cin (17-AAG) were purchased from Calbiochem.

PlasmidsThe pCMV-myc-YB-1 plasmid expressing the N-termi-

nal, myc-tagged YB-1 protein, as well as the pCMV-YB-1-myc-nuc and pCMV-YB-1-myc-cyto plasmids expressingthe C-terminal, myc-tagged YB-1 protein [with or withoutnuclear-localizing signaling (NLS), respectively], were

constructed as described previously (23). The pRC/CMV-clusterin plasmid expressing the clusterin protein waskindly provided from Dr. Martin P. Tenniswood (StateUniversity of New York at Albany, Rensselaer, NY; ref. 30).Various lengths of the promoter and partial first exon

of the wild-type CLU gene were amplified by PCRusing genomic DNA and the following primer pairs:50-CTCGAGCATGGCAGGTAGTGAGCTCCCTG-30and 50-AGATCTGTGTCCAGAGGGGTTTGCT-30 forclusterin–Luc �1,998/�702; 50-AGATCTGATTTCCT-AACTGGGAAGG-30 and 50-AAGCTTGAGCTGTGT-CATCCCTCTCTGCCT-30 for clusterin–Luc �707/þ254. The resultant PCR products were cloned and ligatedinto the pGL3-basic vector (Promega). Clusterin–Luc�1,998/þ254 was constructed by inserting clusterin–Luc�707/þ254 fragment digested by BglII and HindIII intoBglII and HindIII site of clusterin–Luc �1,998/�702.Clusterin–Luc �1,116/�702 was constructed from clus-terin–Luc�1,198/�702 by deletion of the SmaI and PshAIfragments.

siRNAsThe following double-stranded 25-bp siRNA oligonu-

cleotides were commercially generated (Invitrogen): 50-UUUGCUGGUAAUUGCGUGGAGGACC-30 for YB-1 siRNA #1; 50-UGGAUAGCGUCUAUAAUGGUUA-CGG-30 for YB-1 siRNA #2. The sequence of siRNAcorresponding to the human clusterin initiation sitein exon II was 50-GCAGCAGAGUCUUCAUCAU-30(Dharmacon Research Inc.). Stealth RNAi Negative Con-trol Medium GC Duplex #2 (Invitrogen) was used as acontrol siRNA.

Quantitative reverse transcriptase PCRRNA extraction and reverse transcriptase PCR (RT-PCR)

were carried out as described previously (31). Real-timemonitoring of PCR amplification of cDNA was carried outusing the following primer pairs and probes: YB-1(Hs00898625_g1),CLU (Hs00156548_m1), andGAPDH(Hs03929097_g1; Applied Biosystems) on the ABI PRISM7900HT Sequence Detection System (Applied Biosystems)with TaqMan Gene Expression Master Mix (Applied Bio-systems). Target gene expression was normalized to glycer-aldehyde-3-phosphate dehydrogenase (GAPDH) levels inrespective samples as an internal control. The results arerepresentative of at least 3 independent experiments.

Western blot analysisWhole-cell extracts were obtained by lysis of cells in an

appropriate volume of ice-cold RIPA buffer composed of 50mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 0.5%sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS con-taining 1 mmol/L Na3VO4, 1 mmol/L NaF, 1 mmol/Lphenylmethylsulfonylfluoride, and protease inhibitor cock-tail tablets (Complete; Roche Applied Science). Nuclear andcytoplasmic extracts were obtained using CelLytic NuCLE-AR Extraction Kit (Sigma) according to manufacturer'sprotocol. Cellular extracts were clarified by centrifugation

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at 13,000x g for 10minutes andprotein concentrations of theextracts determined by a BCA protein assay kit (ThermoScientific). Thirty micrograms of the extracts were boiled for5minutes in SDS sample buffer and separatedbySDS-PAGEand transferred onto a polyvinylidene difluoride membranefollowing standard methods. Membranes were probed withdilutions of primary antibodies followed by incubation withhorseradish peroxidase–conjugated secondary antibodies.After extensive washing, proteins were visualized by a chemi-luminescent detection system (GE Healthcare).

Luciferase reporter assayProstate cancer cells were transfected with the indicated

reporter plasmid, expression plasmid, or siRNA, and 0.05mgof pRL-TK as an internal control. After 48 hours, the luci-ferase activities were measured using a Dual-LuciferaseReporter Assay System (Promega) and a microplate lumin-ometer (EG&G Berthold). The firefly luciferase activitieswere corrected by the corresponding Renilla luciferase activ-ities. The results are representative of at least 3 independentexperiments.

Chromatin immunoprecipitation assayProstate cancer cells were treated with paraformaldehyde

and digested with micrococcal nuclease to achieve a DNAsmear of 200 to 1,000 bp. Chromatin immunoprecipitation(ChIP) assay on the CLU and RPL30 genes was conductedusing SimpleChIP Enzymatic Chromatin IP Kit (AgaroseBeads) according to the manufacturer's protocol (Cell Sig-naling Technology). The quantitative RT-PCR (qRT-PCR)assay was conducted using ABI PRISM 7900 HT SequenceDetection System with 2 mL of 20 mL DNA extraction, theprimer pairs described later, and RT2 Real-Time SYBRGreen/Rox PCR master mix (Qiagen). The results arerepresentative of at least 3 independent experiments. Theprimer pairs for clusterin �02 (GPH025704(�)02A) tar-geting around �1,403 bp, clusterin �01 (GPH025704(�)01A) targeting around �417 bp, and clusterinþ07(GPH025704(þ)07A) targeting around þ6,603 bp wereobtained from Qiagen. The primer pairs for RPL30 gene(exon 3) were included in the SimpleChIP EnzymaticChromatin IP Kit.

ImmunofluorescenceProstate cancer cells were grown on coverslips and treated

with 10 nmol/L of paclitaxel for 1 hour. After media wererefreshed, cells were recovered for 1 hour, and fixed inparaformaldehyde for 10 minutes at room temperature.Immunofluorescence was carried out as described previously(32) using YB-1 (1:100) antibody.

Cell growth assayProstate cancer cells transfectedwith the indicated amount

of siRNA and expression plasmid were plated in 96-wellplates and treated with paclitaxel at indicated concentration.After incubation for48hours, cell growthwasmeasuredusingthecrystalviolet assayasdescribedpreviously (31).The resultsare representative of at least 3 independent experiments.

Flow cytometric analysisProstate cancer cells transfected with the indicated

amount of siRNA and expression plasmid were platedin 6-well plates, and cells were treated with 10 nmol/L of

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Figure 1. YB-1 regulates clusterin expression in prostate cancer cells.LNCaP (A) and PC-3 (B) cells were transfected with 40 nmol/L of theindicated siRNA. At 48 hours after transfection, qRT-PCRwas carried outusing the primer pairs and probes for YB-1, CLU, and GAPDH. Eachtranscript level from cells transfected with control siRNA was set as 1.Boxes, mean; bars, �SD. �, P < 0.05 (compared with cells transfectedwith control siRNA). Whole-cell extracts were analyzed by SDS-PAGEand Western blot analysis with specific antibodies. LNCaP (C) and PC-3(D) cells were transfected with 1.0 mg/mL of the indicated expressionplasmid. At 72 hours after transfection, qRT-PCR was carried out usingthe primer pairs and probes for CLU and GAPDH. Each transcript levelfrom cells transfected with Myc expression plasmid was set as 1. Boxes,mean; bars, �SD. �, P < 0.05 (compared with cells transfected with Mycexpression plasmid). Whole-cell extracts were analyzed by SDS-PAGEandWestern blot analysis with specific antibodies. IB, immunoblot; CLU,clusterin.

Clusterin Mediates Taxane Resistance by YB-1

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paclitaxel. After incubation for 48 hours, cell-cyclefraction was analyzed as described previously (33). Theresults are representative of at least 3 independentexperiments.

Statistical analysisAll data were assessed using the Student t test. Levels of

statistical significance were set at P < 0.05.

Results

YB-1 regulates clusterin expression in prostate cancercellsYB-1 is known to regulate the expression of various genes

through transcriptional and translational mechanisms. Toinvestigate the functional link between YB-1 and clusterin,we examined clusterin expression levels after YB-1

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Figure 2. YB-1 binds to CLUpromoter region and regulates CLUtranscription. A, schematicrepresentation of the promoterregion and 50 end of the CLU gene.Double bars, Y-boxes (50-ATTG-30);gray box, CLE; white box, AP-1–binding site; rail, primer pairs usedin (D). Clusterin–Luc plasmids(�1,998/þ254, �1,998/�702,�1,116/�702, and �707/þ254)used in (B) and (C) are shown. B,LNCaP and PC-3 cells werecotransfected with 0.5 mg/mL of thevarious clusterin–Luc plasmids, 0.5mg/mL of myc, or myc–YB-1expression plasmid and 0.05mg/mLof pRL-TK. The luciferase activity ofclusterin–Luc �1,998/þ254 alonewas set as 1. Boxes, mean; bars,�SD. �, P < 0.05 (compared withcells transfected with mycexpression plasmid). C, LNCaP andPC-3 cells were cotransfected with0.5 mg/mL of the various clusterin–Luc plasmids, 20 nmol/L of theindicated siRNA, and 0.05 mg/mL ofpRL-TK. The luciferase activity ofclusterin–Luc �1,998/þ254 alonewas set as 1. Boxes, mean; bars,�SD. �, P < 0.05 (compared withcells transfected with controlsiRNA). D, ChIP assays wereconducted on nuclear extracts fromLNCaP and PC-3 cells using 2.0 mgof the indicated antibodies and 20mL of Protein G agarose. The qRT-PCR was carried out usingimmunoprecipitated DNAs, solublechromatin, and specific primer pairsfor the CLU and RPL30 genes. Theresults of immunoprecipitatedsamples were corrected for theresults of the corresponding solublechromatin samples. Boxes, mean;bars, �SD. �, P < 0.05 (comparedwith immunoprecipitants by IgG).CLU, clusterin.

Shiota et al.





knockdown. Clusterin expression, both at transcript andprotein levels, decreased after YB-1 silencing in prostatecancer LNCaP and PC-3 cells (Fig. 1A and B). Conversely,YB-1 overexpression increased clusterin mRNA and proteinlevels in both LNCaP and PC-3 cells (Fig. 1C and D).

YB-1 binds to clusterin promoter region and regulatesclusterin transcriptionBecause YB-1 has been reported to act both transcrip-

tionally (21) and translationally (34), to begin definingmechanisms by which YB-1 regulates clusterin expression,we searched for putative YB-1 binding sites (Y-boxes) in theCLU promoter region between �2,000 bp and þ500 bpfrom transcription start site. Ten Y-boxes were identified inCLU promoter regions (Fig. 2A). Subsequently, we clonedtheCLU promoter region and constructed various lengths ofclusterin reporter plasmids (clusterin–Luc �1,998/þ254,�1,998/�702,�1,116/�702, and�707/þ254) as shownin Fig 2A and then conducted reporter assays with YB-1overexpression. The results indicate increased luciferaseactivity with clusterin–Luc �1,998/�702 and clusterin–Luc �702/þ254, but not clusterin–Luc �1,116/�702, inresponse to YB-1 overexpression in both LNCaP and PC-3cells (Fig. 2B). These findings suggest that the cis-element ofCLU promoter region containing �702/þ254 bp and�1,998/�1,116 bp was activated by YB-1. Conversely,

YB-1 knockdown reduced CLU transcriptional activity inthe clusterin–Luc�1,998/þ254 constructs in both LNCaPand PC-3 cells (Fig. 2C). These data indicate that YB-1transcriptionally activates clusterin expression.Next, we set out to determine whether YB-1 binds toCLU

promoter by ChIP assay and the primer pairs against CLUgene. As shown in Fig. 2D, YB-1 bound toCLU gene regionsaround �1,400 and �400 bp from transcription start siterepresented by clusterin�02 and clusterin�01, respective-ly, in both LNCaP and PC-3 cells. In contrast, YB-1 did notbind to the CLU gene around þ6,600 bp region fromtranscription start site or PRL30 gene in either cell lines(Fig. 2D).

Nuclear YB-1 regulates clusterin transcriptionTo further confirm transcriptional regulation of clusterin

by YB-1, we used different YB-1 expression plasmid thatexpresses YB-1 proteins with or without a NLS (23). Asshown in Fig 3A and in corroboration with data in Fig 2,reporter gene assays indicate that YB-1–NLSþ plasmidsincreased clusterin–Luc luciferase activity more effectivelythan YB-1–NLS� in both LNCaP and PC-3 cells (Fig. 3A).Similarly, CLU transcription was induced more effectivelyby YB-1–NLSþ plasmids compared with YB-1–NLS� inboth LNCaP and PC-3 cells (Fig. 3B), finding also con-firmed at the protein level (Fig. 3C).

Figure 3. Nuclear YB-1 regulatesCLU transcription. A, LNCaP andPC-3 cells were cotransfected with0.5 mg/mL of clusterin–Luc�1,998/þ254 plasmid, 0.5 mg/mL of theindicated expression plasmid, and0.05 mg/mL of pRL-TK. Theluciferase activity of myc-nucexpression plasmid was set as 1.Boxes,mean; bars,�SD. �,P < 0.05(compared with cells transfectedwith myc expression plasmid). B,LNCaP and PC-3 cells weretransfected with 1.0 mg/mL of theindicated expression plasmid. At 48hours after transfection, qRT-PCRwas carried out using the primerpairs and probes for CLU andGAPDH. Each transcript level fromcells transfected with myc-nuc wasset as 1. Boxes, mean; bars, �SD.�, P < 0.05 (compared with cellstransfected with myc expressionplasmid). C, LNCaP and PC-3 cellswere transfected with 1.0 mg/mL ofthe indicated expression plasmid.At 48 hours after transfection,whole-cell extracts were analyzedby SDS-PAGE and Western blotanalysis with specific antibodies.nuc, nucleus; cyto, cytoplasm; IB,immunoblot;CLU, clusterin.

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Endoplasmic reticulum stress inducers increase clusterinexpression through YB-1Clusterin expression is induced by various stresses includ-

ing proteasome inhibitors like MG132 (35), Hsp90 inhi-bitors (31), and chemotherapeutics like paclitaxel (15). Wenext investigated whether these stressors induced clusterinexpression via YB-1 in prostate cancer cells.

MG132 treatment induced time-dependent expression ofendoplasmic reticulum (ER) stress markers ATF4 andGRP78 in both LNCaP and PC-3 cells (SupplementaryFig. S1A and S1B); similar time-dependent increases inclusterin mRNA and protein expression were also observed,whereas YB-1 was not altered at either mRNA or proteinlevels. On the basis of the functional links between YB-1 and

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Figure 4. Paclitaxel treatmentincreases clusterin expressionthrough YB-1. A, LNCaP and PC-3cells were cultured with 10 nmol/Lof paclitaxel for 1 hour, thenchanged into fresh media, andfurther incubated for the indicatedduration. After the cells wereharvested, whole-cell extracts wereanalyzed by SDS-PAGE andWestern blot analysis with specificantibodies. B, LNCaP and PC-3cells cultured as described in (A).After the cells were harvested,qRT-PCR was carried out using theprimer pairs and probes for YB-1,CLU, and GAPDH. Each transcriptlevel from cells treated with vehiclewas set as 1. Boxes, mean; bars,�SD. �, P < 0.05 (compared withcells treated with vehicle). C,LNCaP and PC-3 cells weretransfected with 40 nmol/L of theindicated siRNA and incubated for48 hours. Then, cells were culturedwith 10 nmol/L of paclitaxel for 1hour, then changed into freshmedia, and further incubated for theindicated duration. After the cellswere harvested, qRT-PCR wascarried out using the primer pairsand probes for YB-1, CLU, andGAPDH. Each transcript level fromcells transfected with control siRNAand treated with vehicle was setas 1. Boxes, mean; bars, �SD.�, P < 0.05 (compared with cellstreated with vehicle). D, LNCaP andPC-3 cells were transfected andcultured as described in (C). Afterthe cells were harvested, whole-cellextracts were analyzed by SDS-PAGE and Western blot analysiswith specific antibodies. CLU,clusterin.

Shiota et al.





clusterin expression (Figs. 2 and 3), we next measuredchanges in clusterin expression following ER stress withMG132 when YB-1 was silenced with siRNA. YB-1 knock-down attenuated clusterin transcript (Supplementary Fig.S1C) and protein (Supplementary Fig. S1D) induction afterMG132 treatment compared with control siRNA. Interest-ingly, induction of the ER stress markers ATF4 and GRP78were not affected by YB-1 silencing, suggesting that YB-1

was not a general upstream activator of there markers of ERstress but rather specifically upregulated clusterin.Similarly, ER stress induction using the Hsp90 inhibitor,

17-AAG, also increased clusterin protein andmRNA expres-sion concurrently with ER stress markers ATF4 and GRP78in both LNCaP and PC-3 cells; again YB-1 expression wasnot affected (Supplementary Fig. S2A and S2B).WhenYB-1was silenced, however, clusterin transcript and protein

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Figure 5. Paclitaxel treatment induces YB-1 nuclear translocation and transactivation. A, LNCaP andPC-3 cells were culturedwith 10 nmol/L of paclitaxel for 1hour, then changed into fresh media, and further incubated for the indicated duration. After the cells were harvested, whole-cell extracts were analyzed bySDS-PAGE and Western blot analysis with specific antibodies. B, LNCaP and PC-3 cells cultured with 10 nmol/L of paclitaxel for 1 hour, then changedinto fresh media, and further incubated for 1 hour. After the cells were fixed, immunofluorescence was conducted for YB-1 (green). Then, cells were stainedwith 40,6-diamidino-2-phenylindole (DAPI; blue) and observed by fluorescence microscopy. Representative images of high magnification fields (400�) areshown. Bars, 10 mm. For Western blotting, after cells were harvested and fractioned into nuclear and cytoplasmic extracts, extracts were analyzed bySDS-PAGE and Western blot analysis with specific antibodies. C, ChIP assays were conducted on nuclear extracts from LNCaP and PC-3 cells cultured asdescribed in (B) using 2.0 mg of the indicated antibodies and 20 mL of Protein G agarose. The qRT-PCR was carried out using immunoprecipitated DNAs,soluble chromatin, and specific primer pairs for the CLU gene. Results of immunoprecipitated samples were corrected for the results of the correspondingsoluble chromatin samples. Boxes, mean; bars, �SD. �, P < 0.05 (compared with immunoprecipitants from cell treated with vehicle). CLU, clusterin.






induction by 17-AAG was attenuated compared with con-trol siRNA; again, induction of the ER stress markers ATF4and GRP78 were not affected by YB-1 silencing (Supple-mentary Fig. S2C and S2D).Similar patterns of ER stress and clusterin induction,

without changes in YB-1 levels, were seen following treat-ment with paclitaxel in both LNCaP and PC-3 cells (Fig. 4Aand B); however, YB-1 silencing again attenuated clusterintranscript and protein induction by paclitaxel, withoutaffecting induction of ER stress markers ATF4 and GRP78(Fig. 4C and D). Collectively, these data indicate thatclusterin induction following ER stress is mediated, in part,by YB-1.

Paclitaxel treatment induces YB-1 nuclear translocationand transactivationBecause treatment stress did not increase expression levels

of YB-1, we next examined the effects of treatment stresswith paclitaxel on YB-1 intracellular localization and trans-activation. We examined phosphorylation status of YB-1after paclitaxel treatment. As shown in Fig. 5A, phosphor-ylated YB-1 was increased by paclitaxel treatment. Next, weinvestigated YB-1 localization with or without paclitaxeltreatment. Then, the translocation into nucleus was revealedby immunofluorescence against endogenous YB-1 proteinand using Western blot analysis after LNCaP or PC-3 cellfractionation (Fig. 5B). To confirm that paclitaxel treatmentleads to transactivation of YB-1 and clusterin expression, weconducted ChIP assay after paclitaxel stress. As shownin Fig.5C, YB-1 binding to clusterin promoters (clusterin�02 and clusterin �01) was enhanced after paclitaxeltreatment in both LNCaP and PC-3 cells. These data

confirm that paclitaxel-induced stress leads to nuclear trans-location of YB-1 with binding to CLU promoter.

YB-1 and clusterin are augmented in docetaxel-resistantcells and involved in paclitaxel resistanceWe next examined the expression levels of YB-1 and

clusterin in prostate cancer cells resistant to docetaxel(LNCaP/DTX and DU145/DTX). As shown in Fig. 6,higher expression levels of both YB-1 and clusterin mRNA(Fig. 6A) and protein (Fig. 6B) were seen in both docetaxel-resistant cell lines.To determine the biological significance of the functional

link between YB-1 and clusterin, we evaluated the effects ofYB-1/clusterin knockdown and overexpression on stressinduced prostate cancer cell death. As shown in Supplemen-tary Fig. S3, YB-1 and clusterin siRNAs potentially andspecifically silenced their target gene in intact and over-expressed cell lines. As expected, YB-1 overexpression led tohigher clusterin levels, which could be silenced by clusterinsiRNA; in contrast, clusterin overexpression did not affectYB-1 levels. As shown in Fig. 7A, both YB-1 and clusterinknockdown sensitized LNCaP and PC-3 cells to paclitaxel,whereas both YB-1 and clusterin overexpression conferredpaclitaxel resistance to both cell lines. Importantly, whenclusterin was silenced, YB-1 overexpression did not signif-icantly rescue cell survival, suggesting that clusterin is acritical downstream mediator of stress-induced YB-1 trans-activation. Consistent with this view, clusterin overexpres-sion almost completely rescued cell survival following YB-1knockdown. These findings were confirmed by other meth-ods shown in Fig. 7B where knockdown of either YB-1 orclusterin plus paclitaxel stress led to activation of caspase-3and increased cleavage of PARP. Clusterin overexpressionwith YB-1 knockdown attenuated caspase-3 activation andPARP cleavage whereas YB-1 overexpression with clusterinknockdown did not. Similarly, paclitaxel induction of apo-ptosis, as measured by sub-G0 to -G1 fraction using flowcytometry, increased after either YB-1 or clusterin knock-down, an effect that was rescued only by overexpression ofclusterin but not by YB-1 (Fig. 7C). Collectively, these dataconfirm that clusterin is a critical downstream mediator ofstress-induced YB-1 transactivation.

Discussion

YB-1 and clusterin are both stress-activated survival fac-tors functionally associated with anticancer treatment resis-tance (21, 36). In prostate cancer, both are induced bycastration and confer castration resistance when overex-pressed (10, 22, 23). In addition, both YB-1 and clusterinare linked to cytotoxic resistance including cisplatin(18, 24, 25) and taxanes (16, 19, 20, 26–28). YB-1 is atranscription factor that binds to invertedCCAAT "Y" boxespresent in the promoter region of a variety of genes, includ-ing MHC class II gene, epidermal growth factor receptor(EGFR), proliferating cell nuclear antigen (PCNA),DNA polymerase A, topoisomerase IIA, MDR-1, andmatrix metalloproteinase (MMP)-2. The cascades of gene

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Figure 6. YB-1 and clusterin are increased in docetaxel-resistant cells.A, qRT-PCR using cDNAs from LNCaP, LNCaP/DTX, DU145, andDU145/DTX cells was carried out using the primer pairs and probes forYB-1,CLU, andGAPDH. Each transcript level from parental cells was setas 1. Boxes, mean; bars,�SD. �, P < 0.05 (compared with parental cells).B, whole-cell extracts were analyzed by SDS-PAGE and Western blotanalysis with specific antibodies. CLU, clusterin.

Shiota et al.





expression regulated by YB-1 have been linked to genotoxicstress, drug resistance, and metastatic invasion (21).While YB-1 and clusterin are functionally linked to

treatment resistance in cancer, the role of YB-1 in stress-induced clusterin transactivation and cytoprotection isundefined. Promoter regions of CLU gene are conservedduring evolution and include stress-associated sites likeactivator protein-1 (AP-1), AP-2, stimulatory element(SP-1), heat shock element (HSE), cAMP response element

(CRE), and a "clusterin-specific element" (CLE) recognizedby HSF-1/HSF-2 heterocomplexes (37) as well as steroidresponse elements including glucocorticoid and androgenresponse element sites (38). Our initial analysis of the CLUpromoter region identified 10 putative YB-1 binding sites(Y-boxes), which led us to investigate the role of YB-1 instress-induced clusterin transactivation.YB-1 is known to be activated by many varied stressors via

phosphorylation by AKT (39) and p90RSK (40),

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Figure 7. YB-1 and clusterin are involved in paclitaxel resistance. A, LNCaP and PC-3 cells were transfected with 20 nmol/L of the indicated siRNA, and0.5 mg/mL of the indicated expression plasmid were seeded into 96-well plates. The following day, 10 nmol/L of paclitaxel were applied. After incubation for48 hours, cell survival was analyzed by a cell growth assay. Cell growth in the absence of paclitaxel corresponds to 1. Boxes, mean; bars, � SD.�,P < 0.05 (comparedwith cells transfectedwithmock expression plasmid). B, whole-cell extracts from LNCaP and PC-3 cells were transfected as describedin (A) and treated with 10 nmol/L of paclitaxel for 48 hours and analyzed by SDS-PAGE and Western blot analysis with specific antibodies. C, LNCaP andPC-3 cells transfected asdescribed in (A)were seeded into 6-well plates. The following day, 10 nmol/L of paclitaxelwere applied. After incubation for 48 hours,the cells were stained with propidium iodide and analyzed by flow cytometry. The percentage of sub-G0 to -G1 fraction is shown. Boxes, mean; bars, � SD.�, P < 0.05 (compared with cells transfected with mock expression plasmid). CLU, clusterin.






stimulating its translocation to the nucleus. Stresses linked toactivation of YB-1 include genotoxic stress with radiation(41, 42) and cisplatin (43) as well as paclitaxel (26, 28). Inthis study, we show that ER stress associated with paclitaxel,proteasome inhibition, or Hsp90 inhibition led to YB-1nuclear translocation. Because clusterin is a potent inhibitorof protein aggregation and functions like small Hsps tochaperone and stabilize proteins under stress, we used theseER stress inducers to define the role of YB-1 in regulation ofCLU transcription. We further focused on paclitaxel acti-vation of YB-1 and clusterin as taxane-based chemotherapyis the standard first-line therapy for men with metastaticCRPC (3).Although it was previously reported that clusterin is

overexpressed in taxane-resistant cells (44, 45), the statusof YB-1 in taxane-resistant cells was unknown. This studyrevealed that YB-1 is overexpressed in taxane-resistant pros-tate cancer cells. Furthermore, we show that both YB-1 andclusterin are functionally involved in paclitaxel resistance,findings compatible with previous reports (16, 26, 27). Forexample, because YB-1 regulates P-glycoprotein (P-gp)expression, and paclitaxel is a substrate of P-gp, it is reason-able to speculate that YB-1 may affect paclitaxel sensitivityvia P-gp.However, prostate cancer cell lines like LNCaP andPC-3 cells express P-gp at undetectable levels (46), andhence, it is likely that YB-1 affects paclitaxel sensitivitythrough factors other than P-gp. Clusterin is an extracellularchaperone which directly interacts with paclitaxel and con-fers paclitaxel resistance (47). Our studies indicate thatclusterin acts as a critical mediator of stress-induced YB-1activity and paclitaxel resistance. Clusterin overexpressionreversed increases in paclitaxel-induced apoptosis followingYB-1 knockdown; in contrast, however, YB-1 overexpres-sion did not rescue cells from increased paclitaxel-inducedapoptosis following clusterin knockdown. In addition, thesedata indicate that both YB-1 and clusterin confer taxaneresistance irrespective of p53 and AR expression statusbecause LNCaP cells express wild-type p53 as well as AR,whereas PC-3 cells express neither p53 nor AR.Many strategies used to kill cancer cells induce stress

responses that promote the emergence of a treatment resis-tant phenotype, and novel therapeutic approaches targetingthese stress responses are being developed. With respect toYB-1, a small-molecular inhibitor of integrin-linked kinase(ILK), QLT0267, was found to inhibit YB-1 with suppres-sion of breast cancer cell growth (48). Alternatively, amolecular decoy to YB-1 also inhibited both breast cancer

and prostate cancer cell growth without affecting normalimmortalized breast epithelial cells and primary breast epi-thelial cells (49). Clusterin is being targeted using theantisense oligonucleotide (OGX-011), now in phase IIIclinical trials, in CRPC after randomized phase II studiesreported a significant survival benefit when OGX-011 wasadded to docetaxel (50). Previously, we reported that clus-terin downregulation using OGX-011 resensitized doce-taxel-resistant prostate cancer cells to docetaxel and mitox-antrone and sensitized breast cancer cells to paclitaxel (20).Although inhibition of either YB-1 or clusterin sensitizesprostate cancer cells to paclitaxel, clusterin overexpressioncan reverse increases in paclitaxel-induced apoptosis follow-ing YB-1 knockdown; in contrast, however, YB-1 over-expression did not rescue cells from increased paclitaxel-induced apoptosis following clusterin knockdown, suggest-ing that clusterin may be a more relevant therapeutic target.In summary, YB-1 transcriptionally enhances clusterin

expression following ER stress in prostate cancer cells,resulting in inhibition of treatment-induced apoptosis andtaxane resistance. Although inhibition of either YB-1 orclusterin sensitized prostate cancer cells to paclitaxel, clus-terin seems to play a more dominant role. Collectively, thesedata indicate that YB-1 transactivation of clusterin inresponse to stress is a critical mediator of paclitaxel resistancein prostate cancer.

Disclosure of Potential Conflicts of Interest

The University of British Columbia has submitted patent applications, listingDr. Gleave as inventor, on the antisense sequence targeting CLU described in thisarticle. This IP has been licensed to OncoGenex Technologies, a Vancouver-basedbiotechnology company in which Dr. Gleave has founding shares.

Acknowledgments

The authors thank Dr. Martin P. Tenniswood (State University of New York atAlbany, Rensselaer, NY) for providing the pRC/CMV-clusterin expression plasmidand Dr. Bob Shukin for sequencing cloned CLU promoter region.

Grant Support

This study was supported by the Terry Fox New Frontiers Program, the CanadianInstitutes of Health Research, the Pacific Northwest Prostate Cancer SPORE, and theJapanese Postdoctoral Fellowship for Research Abroad.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received August 10, 2011; revised September 27, 2011; accepted October 3, 2011;published OnlineFirst October 10, 2011.

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Shiota et al.





Published OnlineFirst October 10, 2011.Mol Cancer Res Masaki Shiota, Amina Zoubeidi, Masafumi Kumano, et al. Stress-Induced YB-1 Transactivation in Prostate CancerClusterin Is a Critical Downstream Mediator of

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Clusterin Is a Critical Downstream Mediator of Stress-Induced YB …mcr.aacrjournals.org/content/molcanres/early/2011/11/22/1541-7786... · Signaling and Regulation Clusterin Is a