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

Published OnlineFirst October 10, 2011; DOI: 10.1158/1541-7786.MCR-11-0379

Molecular Cancer Signaling and Regulation Research

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

Abstract Clusterin is a stress-activated, cytoprotective chaperone that confers broad-spectrum treatment resistance in cancer. However, the molecular mechanisms mediating CLU transcription following anticancer treatment stress remain incompletely deﬁned. We report that Y-box binding protein-1 (YB-1) directly binds to CLU promoter regions to transcriptionally regulate clusterin expression. In response to endoplasmic reticulum stress inducers, 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, prostate cancer cells. Knockdown of either YB-1 or clusterin sensitized prostate cancer cells to paclitaxel, whereas their overexpression 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 cells from increased paclitaxel-induced apoptosis following clusterin knockdown. Collectively, these data indicate that YB-1 transactivation of clusterin in response to stress is a critical mediator of paclitaxel resistance in prostate cancer. Mol Cancer Res; 9(12); 1755–66. 2011 AACR.

Introduction for most cancer deaths. Improved understanding of the molecular basis underlying metastasis and resistance to ADT Prostate cancer is the most common noncutaneous cancer or chemotherapy will facilitate to design new therapeutic and the second leading cause of cancer-related death in men strategies to inhibit the emergence of this CRPC phenotype. of Western countries. The incidence of prostate cancer is CRPC progression is a complex process by which cells increasing as a result of aging populations and high-fat diet in acquire the ability to both survive and proliferate in the the Western world (1). While advanced prostate cancer is absence of gonadal androgens and involves mechanisms initially sensitive to androgen deprivation therapy (ADT), attributed to reactivation of the androgen receptor (AR) most eventually recur in a castration-resistant prostate cancer axis (5), alternative growth factor pathways (6, 7), stress- (CRPC). At present, only second-line ADT with abiraterone induced prosurvival genes (8–10), and cytoprotective chap- (2), and the taxanes docetaxel (3), and cabazitaxel (4) have erone networks (11, 12). Clusterin is a stress-induced been shown to delay progression and prolong survival in cytoprotective chaperone expressed in many human cancers. CRPC. However, therapeutic benefits are brief with under In prostate cancer, clusterin levels increase following castra- 4-month gains in survival before treatment resistance, pro- tion and in CRPC models (11). Clusterin levels are low in gression, and death occurs. Many strategies used to kill untreated, favorable grade tissues but increase with higher cancer cells, including ADT or taxane chemotherapy, induce Gleason grade (13) and within weeks after treatment stress a treatment-resistant phenotype that is the underlying basis with ADT (14) or taxane chemotherapy (15). Overexpres- sion of clusterin in human prostate cancer LNCaP cells Authors' Affiliations: 1The Vancouver Prostate Centre and Department of accelerates progression after ADT (11) or chemotherapy Urologic Sciences, University of British Columbia, 2Department of Molec- (16), identifying CLU as an antiapoptotic gene upregulated ular Oncology, BC Cancer Research Center, Vancouver, British Columbia, Canada; 3Department of Urology, Graduate School of Medical Sciences, by treatment-related stress that confers therapeutic resistance Kyushu University, Fukuoka, Japan; and 4Australian Prostate Cancer when overexpressed. Miyake and colleagues first reported Research Centre-Queensland, Institute of Health and Biomedical Innova- that antisense treatment against clusterin significantly tion, Queensland University of Technology, Brisbane, Queensland, Australia enhanced paclitaxel sensitivity in human prostate cancer PC-3 cells (10). Moreover, clusterin knockdown reduced Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). tumor growth and sensitized to chemotherapeutic agents in various cancer cells including renal cell cancer (17), bladder Corresponding Author: Martin E. Gleave, The Vancouver Prostate Centre and Department of Urologic Sciences, University of British Columbia, 2775 cancer (18), lung cancer (19), and breast cancer (20). Laurel Street, Level 6, Vancouver, BC V6H 3Z6, Canada. Phone: 1-604- Y-box binding protein-1 (YB-1) is a stress-activated tran- 875-4818; Fax: 1-604-875-5654; E-mail: [email protected] scription factor implicated in and linked to unfavorable doi: 10.1158/1541-7786.MCR-11-0379 clinical outcome in various cancers (21). In prostate cancer, 2011 American Association for Cancer Research. YB-1 is induced by ADT (22) and its overexpression confers

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castration resistance (23). Moreover, YB-1 is associated with constructed as described previously (23). The pRC/ drug resistance to both cisplatin (24, 25) and paclitaxel (26, CMV-clusterin plasmid expressing the clusterin protein was 27). YB-1 confers paclitaxel resistance in PC-3 cells (27), kindly provided from Dr. Martin P. Tenniswood (State whereas its knockdown sensitized breast cancer SUM149 University of New York at Albany, Rensselaer, NY; ref. 30). cells to paclitaxel (28). Various lengths of the promoter and partial first exon While the above findings functionally link YB-1 and of the wild-type CLU gene were amplified by PCR clusterin to treatment resistance in prostate cancer, the using genomic DNA and the following primer pairs: precise mechanisms mediating these effects remain unde- 50-CTCGAGCATGGCAGGTAGTGAGCTCCCTG-30 fined. Here, we define functional links between stress- and 50-AGATCTGTGTCCAGAGGGGTTTGCT-30 for induced increases in YB-1 and clusterin and analyze their clusterin–Luc 1,998/702; 50-AGATCTGATTTCCT- biological relevance, as related to taxane resistance in prostate AACTGGGAAGG-30 and 50-AAGCTTGAGCTGTGT- cancer. CATCCCTCTCTGCCT-30 for clusterin–Luc 707/ þ254. The resultant PCR products were cloned and ligated Materials and Methods into the pGL3-basic vector (Promega). Clusterin–Luc 1,998/þ254 was constructed by inserting clusterin–Luc Cell culture and transfection 707/þ254 fragment digested by BglII and HindIII into The human prostate cancer cell lines, PC-3 and DU145, BglII and HindIII site of clusterin–Luc 1,998/702. were purchased from the American Type Culture Collection Clusterin–Luc 1,116/702 was constructed from clus- (2008 and 1989, ATCC authentication by isoenzymes terin–Luc 1,198/702 by deletion of the SmaI and PshAI analysis) and maintained in Dulbecco's Modified Eagle's fragments. Medium (DMEM; Thermo Scientific) supplemented with 5% FBS. LNCaP cells were kindly provided by Dr. Leland siRNAs W.K. Chung (1992, MD Anderson Cancer Center, Hous- The following double-stranded 25-bp siRNA oligonu- ton, TX), tested and authenticated by whole-genome and cleotides were commercially generated (Invitrogen): 50- whole-transcriptome sequencing on Illumina Genome Ana- UUUGCUGGUAAUUGCGUGGAGGACC-30 for YB- lyzer IIx platform in 2009. LNCaP cells were maintained in 1siRNA#1;50-UGGAUAGCGUCUAUAAUGGUUA- RPMI-1640 (Thermo Scientific) supplemented with 5% CGG-30 forYB-1siRNA#2.ThesequenceofsiRNA FBS. Docetaxel-resistant derivatives of LNCaP and DU145 corresponding to the human clusterin initiation site cells (LNCaP/DTX and DU145/DTX cells) were estab- in exon II was 50-GCAGCAGAGUCUUCAUCAU-30 lished by long-term culture under the appropriate medium (Dharmacon Research Inc.). Stealth RNAi Negative Con- containing gradually increasing concentrations of docetaxel trol Medium GC Duplex #2 (Invitrogen) was used as a and maintained under media containing 3 ng/mL and 2 ng/ control siRNA. mL of docetaxel, respectively. The LNCaP/DTX and DU145/DTX cells were found to be about 2.5-fold more Quantitative reverse transcriptase PCR resistant to docetaxel than their parental cells. Cells were RNA extraction and reverse transcriptase PCR (RT-PCR) transfected with the indicated siRNA or the indicated were carried out as described previously (31). Real-time plasmid as previously described (29). monitoring of PCR amplification of cDNA was carried out using the following primer pairs and probes: YB-1 Antibodies and reagents (Hs00898625_g1), CLU (Hs00156548_m1), and GAPDH Antibodies against ATF4 (sc-200), Myc (9E10, sc-815), (Hs03929097_g1; Applied Biosystems) on the ABI PRISM GRP78 (sc-1051), and clusterin (sc-6419) were purchased 7900 HT Sequence Detection System (Applied Biosystems) from Santa Cruz Biotechnology. Anti-Lamin B1 antibody with TaqMan Gene Expression Master Mix (Applied Bio- was purchased from Abcam. Anti-phosphorylated YB-1 (p- systems). Target gene expression was normalized to glycer- YB-1, #2900), anti-cleaved PARP (#9541), anti-PARP aldehyde-3-phosphate dehydrogenase (GAPDH) levels in (#9542), and anti-cleaved caspase-3 (#9661) antibodies respective samples as an internal control. The results are were purchased from Cell Signaling Technology. Anti- representative of at least 3 independent experiments. YB-1, anti-caspase-3, and anti-b-actin antibodies were purchased from Epitomics, BD Biosciences, and Sigma, respec- Western blot analysis tively. Paclitaxel and docetaxel were obtained from Sigma. Whole-cell extracts were obtained by lysis of cells in an MG132 and 17-N-allylamino-17-demethoxy-geldanamy- appropriate volume of ice-cold RIPA buffer composed of 50 cin (17-AAG) were purchased from Calbiochem. mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS con- Plasmids taining 1 mmol/L Na3VO4, 1 mmol/L NaF, 1 mmol/L The pCMV-myc-YB-1 plasmid expressing the N-termi- phenylmethylsulfonylfluoride, and protease inhibitor cock- nal, myc-tagged YB-1 protein, as well as the pCMV-YB-1- tail tablets (Complete; Roche Applied Science). Nuclear and myc-nuc and pCMV-YB-1-myc-cyto plasmids expressing cytoplasmic extracts were obtained using CelLytic NuCLE- the C-terminal, myc-tagged YB-1 protein [with or without AR Extraction Kit (Sigma) according to manufacturer's nuclear-localizing signaling (NLS), respectively], were protocol. Cellular extracts were clarified by centrifugation

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at13,000x g for 10 minutes and protein concentrations of the Flow cytometric analysis extracts determined by a BCA protein assay kit (Thermo Prostate cancer cells transfected with the indicated Scientiﬁc). Thirty micrograms of the extracts were boiled for amount of siRNA and expression plasmid were plated 5 minutes in SDS sample buffer and separated by SDS-PAGE in 6-well plates, and cells were treated with 10 nmol/L of and transferred onto a polyvinylidene diﬂuoride membrane following standard methods. Membranes were probed with dilutions of primary antibodies followed by incubation with A horseradish peroxidase–conjugated secondary antibodies. 1.2 YB-1 LNCaP After extensive washing, proteins were visualized by a chemi- LNCaP 1 CLU luminescent detection system (GE Healthcare). 0.8

0.6 Luciferase reporter assay 0.4 CLU Prostate cancer cells were transfected with the indicated * * 0.2 * reporter plasmid, expression plasmid, or siRNA, and 0.05 mg * YB-1

Relative mRNA expression mRNA Relative 0 of pRL-TK as an internal control. After 48 hours, the luci- Control YB-1 YB-1 β-Actin ferase activities were measured using a Dual-Luciferase siRNA siRNA #1 siRNA #2 Reporter Assay System (Promega) and a microplate lumin- B ometer (EG&G Berthold). The firefly luciferase activities 1.2 PC-3 YB-1 PC-3 were corrected by the corresponding Renilla luciferase activ- 1 CLU ities. The results are representative of at least 3 independent 0.8 experiments. 0.6 * * 0.4 CLU Chromatin immunoprecipitation assay 0.2 * * YB-1 Prostate cancer cells were treated with paraformaldehyde expression mRNA Relative 0 Control YB-1 YB-1 β and digested with micrococcal nuclease to achieve a DNA siRNA siRNA #1 siRNA #2 -Actin smear of 200 to 1,000 bp. Chromatin immunoprecipitation (ChIP) assay on the CLU and RPL30 genes was conducted C 2.5 LNCaP CLU LNCaP using SimpleChIP Enzymatic Chromatin IP Kit (Agarose Beads) according to the manufacturer's protocol (Cell Sig- 2 * naling Technology). The quantitative RT-PCR (qRT-PCR) 1.5 CLU assay was conducted using ABI PRISM 7900 HT Sequence 1 Detection System with 2 mLof20mL DNA extraction, the IB; Myc 0.5 Myc–YB-1 primer pairs described later, and RT2 Real-Time SYBR β Relative mRNA expression mRNA Relative 0 -Actin Green/Rox PCR master mix (Qiagen). The results are myc myc–YB-1 representative of at least 3 independent experiments. The D primer pairs for clusterin 02 (GPH025704( )02A) tar- 6 PC-3 CLU PC-3 geting around 1,403 bp, clusterin 01 (GPH025704() 5 * þ 01A) targeting around 417 bp, and clusterin 07 4 þ þ (GPH025704( )07A) targeting around 6,603 bp were 3 CLU obtained from Qiagen. The primer pairs for RPL30 gene 2 (exon 3) were included in the SimpleChIP Enzymatic IB; Myc 1 Myc–YB-1 Chromatin IP Kit. Relative mRNA expression mRNA Relative 0 β-Actin Myc–YB-1 Myc–YB-1 Immunofluorescence Prostate cancer cells were grown on coverslips and treated with 10 nmol/L of paclitaxel for 1 hour. After media were Figure 1. YB-1 regulates clusterin expression in prostate cancer cells. fi LNCaP (A) and PC-3 (B) cells were transfected with 40 nmol/L of the refreshed, cells were recovered for 1 hour, and xed in indicated siRNA. At 48 hours after transfection, qRT-PCR was carried out paraformaldehyde for 10 minutes at room temperature. using the primer pairs and probes for YB-1, CLU, and GAPDH. Each Immunofluorescence was carried out as described previously transcript level from cells transfected with control siRNA was set as 1. (32) using YB-1 (1:100) antibody. Boxes, mean; bars, SD. , P < 0.05 (compared with cells transfected with control siRNA). Whole-cell extracts were analyzed by SDS-PAGE and Western blot analysis with specific antibodies. LNCaP (C) and PC-3 Cell growth assay (D) cells were transfected with 1.0 mg/mL of the indicated expression Prostate cancer cells transfected with the indicated amount plasmid. At 72 hours after transfection, qRT-PCR was carried out using of siRNA and expression plasmid were plated in 96-well the primer pairs and probes for CLU and GAPDH. Each transcript level plates and treated with paclitaxel at indicated concentration. from cells transfected with Myc expression plasmid was set as 1. Boxes, mean; bars, SD. , P < 0.05 (compared with cells transfected with Myc Afterincubationfor48hours,cellgrowthwasmeasuredusing expression plasmid). Whole-cell extracts were analyzed by SDS-PAGE thecrystalvioletassayasdescribedpreviously(31).Theresults and Western blot analysis with specific antibodies. IB, immunoblot; CLU, are representative of at least 3 independent experiments. clusterin.

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paclitaxel. After incubation for 48 hours, cell-cycle Results fraction was analyzed as described previously (33). The results are representative of at least 3 independent YB-1 regulates clusterin expression in prostate cancer experiments. cells YB-1 is known to regulate the expression of various genes Statistical analysis through transcriptional and translational mechanisms. To All data were assessed using the Student t test. Levels of investigate the functional link between YB-1 and clusterin, statistical signiﬁcance were set at P < 0.05. we examined clusterin expression levels after YB-1

A -2,000 +1 +8,000 CLU Figure 2. YB-1 binds to CLU CLU -02 CLU -01 CLU +07 promoter region and regulates CLU CLE AP-1–binding site Y-box transcription. A, schematic representation of the promoter –1,998 +254 0 CLU–Luc –1,998/+254 Luciferase region and 5 end of the CLU gene. Double bars, Y-boxes (50-ATTG-30); –1,998 –702 – CLU–Luc –1,998/-702 Luciferase gray box, CLE; white box, AP-1 binding site; rail, primer pairs used –1,116 –702 in (D). Clusterin–Luc plasmids CLU–Luc –1,116/-702 Luciferase (1,998/þ254, 1,998/702, –707 +254 1,116/702, and 707/þ254) CLU–Luc –707/+254 Luciferase used in (B) and (C) are shown. B, LNCaP and PC-3 cells were m B 2.5 LNCaP myc 3 PC-3 myc cotransfected with 0.5 g/mL of the various clusterin–Luc plasmids, 0.5 myc–YB-1 * * myc–YB-1 2 * 2.5 mg/mL of myc, or myc–YB-1 expression plasmid and 0.05 mg/mL 2 1.5 * * of pRL-TK. The luciferase activity of 1.5 * clusterin–Luc 1,998/þ254 alone 1 was set as 1. Boxes, mean; bars, 1 SD. , P < 0.05 (compared with cells transfected with myc 0.5 0.5 expression plasmid). C, LNCaP and Relative luciferase activity luciferase Relative

0 activity lucifearase Relative 0 PC-3 cells were cotransfected with CLU–Luc CLU–Luc CLU–Luc CLU–Luc CLU–Luc CLU–Luc CLU–Luc CLU–Luc 0.5 mg/mL of the various clusterin– –1998/+254 –1998/-702 –1116/-702 –707/+254 –1998/+254 –1998/-702 –1116/-702 –707/+254 Luc plasmids, 20 nmol/L of the indicated siRNA, and 0.05 mg/mL of C PC-3 1.2 LNCaP CLU–Luc –1,998/+254 1.2 CLU–Luc –1,998/+254 pRL-TK. The luciferase activity of – þ 1 1 clusterin Luc 1,998/ 254 alone was set as 1. Boxes, mean; bars, 0.8 0.8 SD. , P < 0.05 (compared with 0.6 0.6 * * cells transfected with control * siRNA). D, ChIP assays were 0.4 * 0.4 conducted on nuclear extracts from 0.2 0.2 LNCaP and PC-3 cells using 2.0 mg of the indicated antibodies and 20 Relative luciferase activity luciferase Relative Relative luciferase activity luciferase Relative 0 0 mL of Protein G agarose. The qRT- Control YB-1 YB-1 Control YB-1 YB-1 PCR was carried out using siRNA siRNA #1 siRNA #2 siRNA siRNA #1 siRNA #2 immunoprecipitated DNAs, soluble chromatin, and speciﬁc primer pairs D 120 LNCaP IgG 120 PC-3 IgG for the CLU and RPL30 genes. The Histone H3 Histone H3 100 100 results of immunoprecipitated YB-1 YB-1 samples were corrected for the 80 80 * results of the corresponding soluble * chromatin samples. Boxes, mean; 60 60 bars, SD. , P < 0.05 (compared with immunoprecipitants by IgG). 40 40 CLU, clusterin. * 20 * 20 Immunoprecipitants/input (%) Immunoprecipitants/input (%) 0 0 CLU –02 CLU –01 CLU +07 RPL30 CLU –02 CLU –01 CLU +07 RPL30

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knockdown. Clusterin expression, both at transcript and YB-1 knockdown reduced CLU transcriptional activity in protein levels, decreased after YB-1 silencing in prostate the clusterin–Luc 1,998/þ254 constructs in both LNCaP cancer LNCaP and PC-3 cells (Fig. 1A and B). Conversely, and PC-3 cells (Fig. 2C). These data indicate that YB-1 YB-1 overexpression increased clusterin mRNA and protein transcriptionally activates clusterin expression. levels in both LNCaP and PC-3 cells (Fig. 1C and D). Next, we set out to determine whether YB-1 binds to CLU promoter by ChIP assay and the primer pairs against CLU YB-1 binds to clusterin promoter region and regulates gene. As shown in Fig. 2D, YB-1 bound to CLU gene regions clusterin transcription around 1,400 and 400 bp from transcription start site Because YB-1 has been reported to act both transcrip- represented by clusterin 02 and clusterin 01, respective- tionally (21) and translationally (34), to begin defining ly, in both LNCaP and PC-3 cells. In contrast, YB-1 did not mechanisms by which YB-1 regulates clusterin expression, bind to the CLU gene around þ6,600 bp region from we searched for putative YB-1 binding sites (Y-boxes) in the transcription start site or PRL30 gene in either cell lines CLU promoter region between 2,000 bp and þ500 bp (Fig. 2D). from transcription start site. Ten Y-boxes were identified in CLU promoter regions (Fig. 2A). Subsequently, we cloned Nuclear YB-1 regulates clusterin transcription the CLU promoter region and constructed various lengths of To further confirm transcriptional regulation of clusterin clusterin reporter plasmids (clusterin–Luc 1,998/þ254, by YB-1, we used different YB-1 expression plasmid that 1,998/702, 1,116/702, and 707/þ254) as shown expresses YB-1 proteins with or without a NLS (23). As in Fig 2A and then conducted reporter assays with YB-1 shown in Fig 3A and in corroboration with data in Fig 2, þ overexpression. The results indicate increased luciferase reporter gene assays indicate that YB-1–NLS plasmids activity with clusterin–Luc 1,998/702 and clusterin– increased clusterin–Luc luciferase activity more effectively Luc 702/þ254, but not clusterin–Luc 1,116/702, in than YB-1–NLS in both LNCaP and PC-3 cells (Fig. 3A). response to YB-1 overexpression in both LNCaP and PC-3 Similarly, CLU transcription was induced more effectively þ cells (Fig. 2B). These findings suggest that the cis-element of by YB-1–NLS plasmids compared with YB-1–NLS in CLU promoter region containing 702/þ254 bp and both LNCaP and PC-3 cells (Fig. 3B), finding also con- 1,998/1,116 bp was activated by YB-1. Conversely, firmed at the protein level (Fig. 3C).

Figure 3. Nuclear YB-1 regulates A 3 LNCaP 4 PC-3 CLU nuc * nuc transcription. A, LNCaP and * 3.5 2.5 cyto cyto PC-3 cells were cotransfected with 3 0.5 mg/mL of clusterin–Luc 1,998/ 2 * 2.5 þ254 plasmid, 0.5 mg/mL of the * 1.5 2 indicated expression plasmid, and activity activity 1.5 0.05 mg/mL of pRL-TK. The 1 1 luciferase activity of myc-nuc 0.5 0.5 Relative CLU luciferase CLU Relative expression plasmid was set as 1. luciferase CLU Relative P < 0 0 Boxes, mean; bars, SD. , 0.05 myc YB-1 myc YB-1 (compared with cells transfected with myc expression plasmid). B, B 3.5 LNCaP nuc 2 PC-3 nuc LNCaP and PC-3 cells were * * 3 cyto cyto transfected with 1.0 mg/mL of the 2.5 1.5 * indicated expression plasmid. At 48 hours after transfection, qRT-PCR 2 * 1 was carried out using the primer 1.5 expression pairs and probes for CLU and 1 expression GAPDH 0.5 Relative CLU mRNA mRNA CLU Relative . Each transcript level from 0.5 mRNA CLU Relative cells transfected with myc-nuc was 0 0 set as 1. Boxes, mean; bars, SD. myc YB-1 myc YB-1 , P < 0.05 (compared with cells transfected with myc expression C LNCaP PC-3 plasmid). C, LNCaP and PC-3 cells were transfected with 1.0 mg/mL of the indicated expression plasmid. At 48 hours after transfection, whole-cell extracts were analyzed CLU CLU by SDS-PAGE and Western blot analysis with speciﬁc antibodies. IB;Myc IB; Myc nuc, nucleus; cyto, cytoplasm; IB, YB-1–myc YB-1–myc immunoblot; CLU, clusterin. β-Actin β-Actin

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Endoplasmic reticulum stress inducers increase clusterin MG132 treatment induced time-dependent expression of expression through YB-1 endoplasmic reticulum (ER) stress markers ATF4 and Clusterin expression is induced by various stresses includ- GRP78 in both LNCaP and PC-3 cells (Supplementary ing proteasome inhibitors like MG132 (35), Hsp90 inhi- Fig. S1A and S1B); similar time-dependent increases in bitors (31), and chemotherapeutics like paclitaxel (15). We clusterin mRNA and protein expression were also observed, next investigated whether these stressors induced clusterin whereas YB-1 was not altered at either mRNA or protein expression via YB-1 in prostate cancer cells. levels. On the basis of the functional links between YB-1 and

A LNCaP PC-3 – 6 12 24 Paclitaxel (h) – 6 12 24 Paclitaxel (h) CLU CLU

YB-1 YB-1

ATF4 ATF4 Figure 4. Paclitaxel treatment increases clusterin expression GRP78 GRP78 through YB-1. A, LNCaP and PC-3 cells were cultured with 10 nmol/L β-Actin β-Actin of paclitaxel for 1 hour, then changed into fresh media, and further incubated for the indicated B 2 LNCaP YB-1 2.5 PC-3 YB-1 duration. After the cells were Paclitaxel * Paclitaxel * CLU harvested, whole-cell extracts were CLU 2 1.5 * * analyzed by SDS-PAGE and 1.5 * Western blot analysis with speciﬁc 1 antibodies. B, LNCaP and PC-3 1 cells cultured as described in (A). 0.5 0.5 After the cells were harvested, qRT-PCR was carried out using the Relative mRNA mRNA expression Relative 0 mRNA expression Relative 0 primer pairs and probes for YB-1, Vehicle 6 h 12 h 24 h Vehicle 6 h 12 h 24 h CLU, and GAPDH. Each transcript Control siRNA Control siRNA level from cells treated with vehicle C 1.4 LNCaP 3 LNCaP YB-1 YB-1 siRNA #1 CLU YB-1 siRNA #1 was set as 1. Boxes, mean; bars, 1.2 2.5 * SD. , P < 0.05 (compared with 1 2 cells treated with vehicle). C, 0.8 LNCaP and PC-3 cells were 1.5 0.6 * transfected with 40 nmol/L of the 1 0.4 indicated siRNA and incubated for 0.2 0.5 48 hours. Then, cells were cultured Relative mRNA mRNA expression Relative 0 mRNA expression Relative 0 with 10 nmol/L of paclitaxel for 1 Vehicle 6 h 12 h 24 h Vehicle 6 h 12 h 24 h hour, then changed into fresh media, and further incubated for the Control siRNA Control siRNA 1.4 PC-3 2.5 PC-3 indicated duration. After the cells YB-1 siRNA #1 YB-1 siRNA #1 1.2 YB-1 CLU * were harvested, qRT-PCR was 2 1 carried out using the primer pairs YB-1 CLU 0.8 1.5 * and probes for , ,and * GAPDH. Each transcript level from 0.6 1 cells transfected with control siRNA 0.4 0.5 and treated with vehicle was set 0.2 as 1. Boxes, mean; bars, SD. Relative mRNA mRNA expression Relative Relative mRNA mRNA expression Relative 0 0 , P < 0.05 (compared with cells Vehicle 6 h 12 h 24 h Vehicle 6 h 12 h 24 h treated with vehicle). D, LNCaP and D PC-3 cells were transfected and LNCaP PC-3 cultured as described in (C). After Control YB-1 Control YB-1 the cells were harvested, whole-cell siRNA siRNA #1 siRNA siRNA #1 extracts were analyzed by SDS- – 6 12 24 – 6 12 24 Paclitaxel (h) – 6 12 24 – 6 12 24 Paclitaxel (h) PAGE and Western blot analysis CLU CLU with speciﬁc antibodies. CLU, clusterin. YB-1 YB-1

ATF4 ATF4

GRP78 GRP78

β-Actin β-Actin

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clusterin expression (Figs. 2 and 3), we next measured was not a general upstream activator of there markers of ER changes in clusterin expression following ER stress with stress but rather speciﬁcally upregulated clusterin. MG132 when YB-1 was silenced with siRNA. YB-1 knock- Similarly, ER stress induction using the Hsp90 inhibitor, down attenuated clusterin transcript (Supplementary Fig. 17-AAG, also increased clusterin protein and mRNA expres- S1C) and protein (Supplementary Fig. S1D) induction after sion concurrently with ER stress markers ATF4 and GRP78 MG132 treatment compared with control siRNA. Interest- in both LNCaP and PC-3 cells; again YB-1 expression was ingly, induction of the ER stress markers ATF4 and GRP78 not affected (Supplementary Fig. S2A and S2B). When YB-1 were not affected by YB-1 silencing, suggesting that YB-1 was silenced, however, clusterin transcript and protein

A LNCaP PC-3 0 5 15 30 60 Paclitaxel (min) 0 5 15 30 60 Paclitaxel (min) p-YB-1 p-YB-1

YB-1 YB-1

β-Actin β-Actin

B LNCaP PC-3 LNCaP PC-3

YB-1 Vehicle Paclitaxel Vehicle Paclitaxel

DAPI YB-1

Lamin B1

Merge β-Actin

Vehicle Paclitaxel Vehicle Paclitaxel

Vehicle Vehicle C 120 LNCaP 100 LNCaP CLU-02 Paclitaxel CLU-01 Paclitaxel 100 80 80 60 60 * 40 40 * 20 20 Immunoprecipitants/input (%) 0 Immunoprecipitants/input (%) 0 IgG Histone YB-1 IgG Histone YB-1 H3 H3

Vehicle PC-3 Vehicle 100 PC-3 100 CLU-02 Paclitaxel CLU-01 Paclitaxel 80 * 80 60 60 * 40 40

20 20 Immunoprecipitants/input (%) 0 Immunoprecipitants/input (%) 0 IgG Histone YB-1 IgG Histone YB-1 H3 H3

Figure 5. Paclitaxel treatment induces YB-1 nuclear translocation and transactivation. A, LNCaP and PC-3 cells were cultured with 10 nmol/L of paclitaxel for 1 hour, then changed into fresh media, and further incubated for the indicated duration. After the cells were harvested, whole-cell extracts were analyzed by SDS-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 changed into fresh media, and further incubated for 1 hour. After the cells were fixed, immunofluorescence was conducted for YB-1 (green). Then, cells were stained with 40,6-diamidino-2-phenylindole (DAPI; blue) and observed by fluorescence microscopy. Representative images of high magnification fields (400)are shown. Bars, 10 mm. For Western blotting, after cells were harvested and fractioned into nuclear and cytoplasmic extracts, extracts were analyzed by SDS-PAGE and Western blot analysis with specific antibodies. C, ChIP assays were conducted on nuclear extracts from LNCaP and PC-3 cells cultured as described 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 corresponding soluble chromatin samples. Boxes, mean; bars, SD. , P < 0.05 (compared with immunoprecipitants from cell treated with vehicle). CLU, clusterin.

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induction by 17-AAG was attenuated compared with con- confirm that paclitaxel-induced stress leads to nuclear trans- trol siRNA; again, induction of the ER stress markers ATF4 location of YB-1 with binding to CLU promoter. and GRP78 were not affected by YB-1 silencing (Supple- mentary Fig. S2C and S2D). YB-1 and clusterin are augmented in docetaxel-resistant Similar patterns of ER stress and clusterin induction, cells and involved in paclitaxel resistance without changes in YB-1 levels, were seen following treat- We next examined the expression levels of YB-1 and ment with paclitaxel in both LNCaP and PC-3 cells (Fig. 4A clusterin in prostate cancer cells resistant to docetaxel and B); however, YB-1 silencing again attenuated clusterin (LNCaP/DTX and DU145/DTX). As shown in Fig. 6, transcript and protein induction by paclitaxel, without higher expression levels of both YB-1 and clusterin mRNA affecting induction of ER stress markers ATF4 and GRP78 (Fig. 6A) and protein (Fig. 6B) were seen in both docetaxel- (Fig. 4C and D). Collectively, these data indicate that resistant cell lines. clusterin induction following ER stress is mediated, in part, To determine the biological significance of the functional by YB-1. link between YB-1 and clusterin, we evaluated the effects of YB-1/clusterin knockdown and overexpression on stress Paclitaxel treatment induces YB-1 nuclear translocation induced prostate cancer cell death. As shown in Supplemen- and transactivation tary Fig. S3, YB-1 and clusterin siRNAs potentially and Because treatment stress did not increase expression levels specifically silenced their target gene in intact and over- of YB-1, we next examined the effects of treatment stress expressed cell lines. As expected, YB-1 overexpression led to with paclitaxel on YB-1 intracellular localization and trans- higher clusterin levels, which could be silenced by clusterin activation. We examined phosphorylation status of YB-1 siRNA; in contrast, clusterin overexpression did not affect after paclitaxel treatment. As shown in Fig. 5A, phosphor- YB-1 levels. As shown in Fig. 7A, both YB-1 and clusterin ylated YB-1 was increased by paclitaxel treatment. Next, we knockdown sensitized LNCaP and PC-3 cells to paclitaxel, investigated YB-1 localization with or without paclitaxel whereas both YB-1 and clusterin overexpression conferred treatment. Then, the translocation into nucleus was revealed paclitaxel resistance to both cell lines. Importantly, when by immunofluorescence against endogenous YB-1 protein clusterin was silenced, YB-1 overexpression did not signif- and using Western blot analysis after LNCaP or PC-3 cell icantly rescue cell survival, suggesting that clusterin is a fractionation (Fig. 5B). To confirm that paclitaxel treatment critical downstream mediator of stress-induced YB-1 trans- leads to transactivation of YB-1 and clusterin expression, we activation. Consistent with this view, clusterin overexpres- conducted ChIP assay after paclitaxel stress. As shown sion almost completely rescued cell survival following YB-1 in Fig.5C, YB-1 binding to clusterin promoters (clusterin knockdown. These findings were confirmed by other meth- 02 and clusterin 01) was enhanced after paclitaxel ods shown in Fig. 7B where knockdown of either YB-1 or treatment in both LNCaP and PC-3 cells. These data clusterin plus paclitaxel stress led to activation of caspase-3 and increased cleavage of PARP. Clusterin overexpression with YB-1 knockdown attenuated caspase-3 activation and A PARP cleavage whereas YB-1 overexpression with clusterin YB-1 YB-1 3 1.6 * knockdown did not. Similarly, paclitaxel induction of apo- CLU CLU * fl 2.5 * 1.4 ptosis, as measured by sub-G0 to -G1 fraction using ow 1.2 2 1 cytometry, increased after either YB-1 or clusterin knock- 1.5 * 0.8 down, an effect that was rescued only by overexpression of 1 0.6 clusterin but not by YB-1 (Fig. 7C). Collectively, these data 0.4 fi 0.5 0.2 con rm that clusterin is a critical downstream mediator of

Relative mRNA mRNA expression Relative 0 mRNA expression Relative 0 stress-induced YB-1 transactivation. LNCaP LNCaP/DTX DU145 DU145/DTX Discussion B YB-1 and clusterin are both stress-activated survival fac- YB-1 YB-1 tors functionally associated with anticancer treatment resistance (21, 36). In prostate cancer, both are induced by CLU CLU castration and confer castration resistance when overexpressed (10, 22, 23). In addition, both YB-1 and clusterin β-Actin β-Actin are linked to cytotoxic resistance including cisplatin (18, 24, 25) and taxanes (16, 19, 20, 26–28). YB-1 is a Figure 6. YB-1 and clusterin are increased in docetaxel-resistant cells. transcription factor that binds to inverted CCAAT "Y" boxes A, qRT-PCR using cDNAs from LNCaP, LNCaP/DTX, DU145, and present in the promoter region of a variety of genes, includ- DU145/DTX cells was carried out using the primer pairs and probes for ing MHC class II gene, epidermal growth factor receptor YB-1, CLU, and GAPDH. Each transcript level from parental cells was set as 1. Boxes, mean; bars, SD. , P < 0.05 (compared with parental cells). (EGFR), proliferating cell nuclear antigen (PCNA), B, whole-cell extracts were analyzed by SDS-PAGE and Western blot DNA polymerase A, topoisomerase IIA, MDR-1, and analysis with speciﬁc antibodies. CLU, clusterin. matrix metalloproteinase (MMP)-2. The cascades of gene

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Clusterin Mediates Taxane Resistance by YB-1

A Myc pRC 0.6 LNCaP Myc pRC 0.6 PC-3 0.6 PC-3 0.6 LNCaP myc–YB-1 pRC-CLU myc–YB-1 * * * pRC-CLU * 0.55 * 0.55 * 0.55 0.55 0.5 0.5 0.5 0.5

0.45 0.45 0.45 0.45 Relative cell viability cell Relative Relative cell viability cell Relative Relative cell viability cell Relative Relative cell viability cell Relative 0.4 0.4 0.4 0.4 Control CLU Control YB-1 Control CLU Control YB-1 siRNA siRNA siRNA siRNA #1 siRNA siRNA siRNA siRNA #1

LNCaP PC-3 B Paclitaxel Paclitaxel Control CLU Control YB-1 Control CLU Control YB-1 siRNA siRNA siRNA siRNA #1 siRNA siRNA siRNA siRNA #1

Myc–YB-1 Myc–YB-1 Myc Myc–YB-1 Myc–YB-1 Cleaved PARP Cleaved PARP

PARP PARP

Cleaved caspase-3 Cleaved caspase-3

Caspase-3 Caspase-3

β-Actin β-Actin

C Myc LNCaP LNCaP Myc PC-3 pRC PC-3 40 40 pRC 35 40 Myc–YB-1 pRC-CLU 35 Myc–YB-1 35 pRC-CLU 30 35 30 30 * 25 30 25 * 25 25 20 fraction (%)

fraction (%) * fraction (%) 1 20 fraction (%)

20 1 20 1 1 * 15 * /G 15 /G * /G

15 15 0 /G 0 0 0 10 10 10 10 5 5 5 5 Sub G Sub G Sub G Sub G 0 0 0 0 Control CLU Control YB-1 Control CLU Control YB-1 siRNA siRNA siRNA siRNA #1 siRNA siRNA siRNA siRNA #1

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, and 0.5 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 for 48 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 (compared with cells transfected with mock expression plasmid). B, whole-cell extracts from LNCaP and PC-3 cells were transfected as described in (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 and PC-3 cells transfected as described in (A) were seeded into 6-well plates. The following day, 10 nmol/L of paclitaxel were 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. expression regulated by YB-1 have been linked to genotoxic (CRE), and a "clusterin-specific element" (CLE) recognized stress, drug resistance, and metastatic invasion (21). by HSF-1/HSF-2 heterocomplexes (37) as well as steroid While YB-1 and clusterin are functionally linked to response elements including glucocorticoid and androgen treatment resistance in cancer, the role of YB-1 in stress- response element sites (38). Our initial analysis of the CLU induced clusterin transactivation and cytoprotection is promoter region identified 10 putative YB-1 binding sites undefined. Promoter regions of CLU gene are conserved (Y-boxes), which led us to investigate the role of YB-1 in during evolution and include stress-associated sites like stress-induced clusterin transactivation. activator protein-1 (AP-1), AP-2, stimulatory element YB-1 is known to be activated by many varied stressors via (SP-1), heat shock element (HSE), cAMP response element phosphorylation by AKT (39) and p90RSK (40),

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stimulating its translocation to the nucleus. Stresses linked to and prostate cancer cell growth without affecting normal activation of YB-1 include genotoxic stress with radiation immortalized breast epithelial cells and primary breast epi- (41, 42) and cisplatin (43) as well as paclitaxel (26, 28). In thelial cells (49). Clusterin is being targeted using the this study, we show that ER stress associated with paclitaxel, antisense oligonucleotide (OGX-011), now in phase III proteasome inhibition, or Hsp90 inhibition led to YB-1 clinical trials, in CRPC after randomized phase II studies nuclear translocation. Because clusterin is a potent inhibitor reported a significant survival benefit when OGX-011 was of protein aggregation and functions like small Hsps to added to docetaxel (50). Previously, we reported that clus- chaperone and stabilize proteins under stress, we used these terin downregulation using OGX-011 resensitized doce- ER stress inducers to define the role of YB-1 in regulation of taxel-resistant prostate cancer cells to docetaxel and mitox- CLU transcription. We further focused on paclitaxel acti- antrone and sensitized breast cancer cells to paclitaxel (20). vation of YB-1 and clusterin as taxane-based chemotherapy Although inhibition of either YB-1 or clusterin sensitizes is the standard first-line therapy for men with metastatic prostate cancer cells to paclitaxel, clusterin overexpression CRPC (3). can reverse increases in paclitaxel-induced apoptosis follow- Although it was previously reported that clusterin is ing YB-1 knockdown; in contrast, however, YB-1 over- overexpressed in taxane-resistant cells (44, 45), the status expression did not rescue cells from increased paclitaxel- of YB-1 in taxane-resistant cells was unknown. This study induced apoptosis following clusterin knockdown, suggest- revealed that YB-1 is overexpressed in taxane-resistant pros- ing that clusterin may be a more relevant therapeutic target. tate cancer cells. Furthermore, we show that both YB-1 and In summary, YB-1 transcriptionally enhances clusterin clusterin are functionally involved in paclitaxel resistance, expression following ER stress in prostate cancer cells, findings compatible with previous reports (16, 26, 27). For resulting in inhibition of treatment-induced apoptosis and example, because YB-1 regulates P-glycoprotein (P-gp) taxane resistance. Although inhibition of either YB-1 or expression, and paclitaxel is a substrate of P-gp, it is reason- clusterin sensitized prostate cancer cells to paclitaxel, clus- able to speculate that YB-1 may affect paclitaxel sensitivity terin seems to play a more dominant role. Collectively, these via P-gp. However, prostate cancer cell lines like LNCaP and data indicate that YB-1 transactivation of clusterin in PC-3 cells express P-gp at undetectable levels (46), and response to stress is a critical mediator of paclitaxel resistance hence, it is likely that YB-1 affects paclitaxel sensitivity in prostate cancer. through factors other than P-gp. Clusterin is an extracellular chaperone which directly interacts with paclitaxel and con- Disclosure of Potential Conflicts of Interest fers paclitaxel resistance (47). Our studies indicate that clusterin acts as a critical mediator of stress-induced YB-1 The University of British Columbia has submitted patent applications, listing activity and paclitaxel resistance. Clusterin overexpression Dr. Gleave as inventor, on the antisense sequence targeting CLU described in this article. This IP has been licensed to OncoGenex Technologies, a Vancouver-based reversed increases in paclitaxel-induced apoptosis following biotechnology company in which Dr. Gleave has founding shares. YB-1 knockdown; in contrast, however, YB-1 overexpression did not rescue cells from increased paclitaxel-induced Acknowledgments apoptosis following clusterin knockdown. In addition, these data indicate that both YB-1 and clusterin confer taxane The authors thank Dr. Martin P. Tenniswood (State University of New York at Albany, Rensselaer, NY) for providing the pRC/CMV-clusterin expression plasmid resistance irrespective of p53 and AR expression status and Dr. Bob Shukin for sequencing cloned CLU promoter region. because LNCaP cells express wild-type p53 as well as AR, whereas PC-3 cells express neither p53 nor AR. Grant Support Many strategies used to kill cancer cells induce stress responses that promote the emergence of a treatment resis- This study was supported by the Terry Fox New Frontiers Program, the Canadian Institutes of Health Research, the Pacific Northwest Prostate Cancer SPORE, and the tant phenotype, and novel therapeutic approaches targeting Japanese Postdoctoral Fellowship for Research Abroad. these stress responses are being developed. With respect to The costs of publication of this article were defrayed in part by the payment of page YB-1, a small-molecular inhibitor of integrin-linked kinase charges. This article must therefore be hereby marked advertisement in accordance with (ILK), QLT0267, was found to inhibit YB-1 with suppres- 18 U.S.C. Section 1734 solely to indicate this fact. sion of breast cancer cell growth (48). Alternatively, a Received August 10, 2011; revised September 27, 2011; accepted October 3, 2011; molecular decoy to YB-1 also inhibited both breast cancer published OnlineFirst October 10, 2011.

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

Masaki Shiota, Amina Zoubeidi, Masafumi Kumano, et al.

Mol Cancer Res 2011;9:1755-1766. Published OnlineFirst October 10, 2011.

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