Nuclear Receptor Corepressor 1 Expression and Output...

Biology of Human Tumors

Nuclear Receptor Corepressor 1 Expression andOutput Declines with Prostate CancerProgressionSandraM. Lopez1,2, Alexander I. Agoulnik3,4, Manqi Zhang1, Leif E. Peterson5, Egla Suarez1,Gregory A. Gandarillas1, Anna Frolov6, Rile Li7, Kimal Rajapakshe8, Christian Coarfa8,Michael M. Ittmann7,9, Nancy L.Weigel8, and Irina U. Agoulnik1,8,10

Abstract

Purpose: Castration therapy in advanced prostate cancereventually fails and leads to the development of castration-resistant prostate cancer (CRPC), which has no cure. Character-istic features of CRPC can be increased androgen receptor (AR)expression and altered transcriptional output. We investigatedthe expression of nuclear receptor corepressor 1 (NCOR1) inhuman prostate and prostate cancer and the role of NCOR1 inresponse to antiandrogens.

Experimental Design: NCOR1 protein levels were comparedbetween matched normal prostate and prostate cancer in 409patient samples. NCOR1 knockdown was used to investigate itseffect on bicalutamide response in androgen-dependent prostatecancer cell lines and transcriptional changes associated with theloss of NCOR1. NCOR1 transcriptional signature was also exam-ined in prostate cancer gene expression datasets.

Results:NCOR1 protein was detected in cytoplasm and nucleiof secretory epithelial cells in normal prostate. Both cytoplasmicand nuclear NCOR1 protein levels were lower in prostatecancer than in normal prostate. Prostate cancer metastases showsignificant decrease in NCOR1 transcriptional output. Inhibitionof LNCaP cellular proliferation by bicalutamide requires NCOR1.NCOR1-regulated genes suppress cellular proliferation andmedi-ate bicalutamide resistance. In the mouse, NCOR1 is required forbicalutamide-dependent regulation of a subset of the AR targetgenes.

Conclusions: In summary, we demonstrated that NCOR1function declines with prostate cancer progression. Reduction inNCOR1 levels causes bicalutamide resistance in LNCaP cells andcompromises response to bicalutamide in mouse prostate in vivo.Clin Cancer Res; 22(15); 3937–49. �2016 AACR.

IntroductionMetastatic prostate cancer is treated with androgen ablation.

Although it is initially successful, the resistance to the treatmentalmost invariably develops. Recurrent castration-resistant pros-

tate cancer (CRPC) is themajor cause ofmortality of patients withprostate cancer, andbetter understanding of its biology is requiredfor the development of novel therapies. Multiple studies showthat the common mechanisms in prostate cancer progression arean increase in the androgen receptor (AR) expression and areprograming of AR transcriptional output (1–3); both are accel-eratedby androgen ablation. AR is a transcription factor that bindsa broad range of androgen response elements (ARE) in regulatoryregions of genes and intergenic loci. It assembles promoter-specific complexes composed of coactivators, corepressors, andgeneral transcription factors and regulates gene expression (4).Inhibition of AR transcriptional activity eventually leads to anactivation of prosurvival Akt signaling both in patients withprostate cancer and AR-dependent cell lines, caused largely bydownregulationof PHLPP (5) and INPP4B (6). Castration triggersincreased expression of AR coactivators, such as TIF2 and CBP,that stimulate AR activity at low androgen levels (7). Anotheradaptation of prostate cancer to castration can be an increasedsynthesis and/or retention of androgens in tumor cells (8, 9).

NCOR1and its close homolog,NCOR2,werefirst discovered ascorepressors of unliganded thyroid receptor. NCOR1 has bothnuclear and cytoplasmic fractions that have distinct cellularfunctions. Nuclear–cytoplasmic shuttling is regulated in part byNCOR1 phosphorylation. In HeLa cells, NCOR1 is distributedbetween cytoplasm and the nucleus; PKA activation causesNCOR1 phosphorylation and its complete nuclear translocation(10). In thyroid tumor cells, cytoplasmic NCOR1 interacts with

1DepartmentofCellularBiologyandPharmacology,HerbertWertheimCollege of Medicine, Florida International University, Miami, Florida.2Johns Hopkins University School of Medicine, Baltimore, Maryland.3Department of Human and Molecular Genetics, Herbert WertheimCollege of Medicine, Florida International University, Miami, Florida.4Department of Obstetrics and Gynecology, Baylor College of Med-icine, Houston, Texas. 5Center for Biostatistics, Houston MethodistResearch Institute, Houston, Texas. 6Dan L. Duncan Cancer Center-Biostatistics,BaylorCollegeofMedicine,Houston,Texas. 7Departmentof Pathology and Immunology, Baylor College of Medicine, Houston,Texas. 8Department of Molecular and Cellular Biology, Baylor Collegeof Medicine, Houston, Texas. 9Michael E. DeBakey Veterans AffairsMedical Center, Houston, Texas. 10Biomolecular Sciences Institute,School of Integrated Science and Humanity, Florida internationalUniversity, Miami, Florida.

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

Corresponding Author: Irina U. Agoulnik, Department of Cellular Biology andPharmacology, Herbert Wertheim College of Medicine, 11200 S.W. 8th Street,HLS I 419C, Miami, FL 33199. Phone: 305-348-1475; Fax: 305-348-0688; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-15-1983

�2016 American Association for Cancer Research.

ClinicalCancerResearch

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PI3K regulatory subunit p85a and downregulates PI3K signaling(11). In the nucleus, NCOR1 binds various transcription factorsand modulates their activity. Both NCOR1 and NCOR2 arerecruited to PSA promoter in an AR-dependent manner (12) andcan inhibit both agonist and partial antagonist–dependent ARactivity (13). In addition to common activities, these paralogshave unique functions: NCOR2 is required for DNA repair (14),whereas NCOR1 regulates mitochondrial activity in mouse(15, 16). A recent report suggests that prostate cancer responseto castration therapies is dependent on functional AR–NCOR1complexes. The ubiquitin ligase SIAH2 that clears promoter-associated AR–NCOR1 complexes for degradation modulatescastration response (1).

In this article, we show that response of prostate cancer celllines LNCaP and LAPC4 to the AR antagonist bicalutamide(Casodex) requires expression of NCOR1. We demonstrate thatNCOR1 modulates bicalutamide-dependent transcriptome. Weshow that the loss of NCOR1 changes the expression of anumber of genes strongly implicated in prostate cancer pro-gression. In agreement with previous reports, we discovered theAR and SIAH2 gene expression signatures among NCOR1regulated genes (1, 13). We show that the NCOR1 proteinlevels are significantly lower in prostate cancer than in normalprostate with the corresponding increase in activated Akt. Inadvanced prostate cancer, NCOR1 signatures are significantlydiminished in multiple cohorts. Our data indicate that NCOR1is required for optimal bicalutamide response in mouse pros-tate, and the loss of its function is associated with prostatecancer progression.

Materials and MethodsCell culture and reagents

LNCaP, LAPC4, PC3, VCaP, DU145, RWPE, PNT1A, and HeLacell lines were purchased from ATCC and maintained in therecommendedmedia. LNCaPAR-V7/pLenti was described previously(17). Media was purchased from Life Technologies. FBS andsteroid-depleted charcoal-stripped serum (CSS) were purchasedfrom Sigma. Bicalutamide (Casodex) and MDV3100 (enzaluta-mide) were purchased from Selleckchem.

siRNA transfectionssiRNAs were transfected using Lipofectamine 2000 (Life Tech-

nologies) or electroporated in R buffer (Lonza) with the Nucleo-fector Device (Lonza). TARP1 was downregulated using siRNAss226672 and s54578 (Life Technologies). NCOR1 was targetedwith s201and s203 (Life Technologies) andon-target SMARTpoolL-003518-00-0050 (Thermo Fisher Scientific). Checkpoint kinase1 (CHEK1)–specific siRNAs were s504 and s503 (Life Technolo-gies). T-cell receptor g chain alternative reading frame protein(TARP) was downregulated using s226672 and s54578 (LifeTechnologies). NCOR2, UGT2B15, and UGT2B17 were down-regulated using SMARTpools (Thermo Fisher Scientific) and pre-validated siRNA from Life Technologies (s18467). Control non-coding siRNAs were purchased from Life Technologies andThermo Fisher Scientific.

Western blottingFor AR, total Akt, pAkt (S473), actin, and tubulin, 30 mg of

protein was resolved on SDS-PAGE and transferred to nitrocel-lulose membrane. For NCOR1 and NCOR2, 50 mg of protein wassimilarly processed. Membranes were blocked with 2% milk inTBST and incubated with 1:1,000 dilution of NCOR1 (BethylLaboratories), 1:1,000 dilution of NCOR2, 1:1,000 of total Akt(Cell Signaling Technology), 1:1,000 pAkt (S473), 1:2,000 dilu-tion of N20 AR (Santa Cruz Biotechnology), 1:2,000 tubulin(Millipore), and 1:1,000 actin (Sigma) primary antibodies over-night at 4�C. Membranes were washed and incubated with HRP-conjugated secondary antibodies (Promega) for 1 hour, andsignals were captured on a Gel Logic 2000 imaging system withCarestream Molecular Imaging Software (Carestream).

Chromatin immunoprecipitation assayChromatin immunoprecipitation assay was performed as

described previously (6) using AR antibody N-20 (Santa CruzBiotechnology) and NCOR1 antibody (Bethyl Laboratories).

DNA synthesis and proliferation assaysThe [3H]thymidine incorporation was performed exactly as

described previously (7). Cellular proliferation and motility wascompared using Roche xCelligence RTCA (Roche Diagnostics) asdescribed previously (6, 18). Cellular impedance in these assays isproportionate to the number of cells covering the E-plate andCIM-plate membrane.

Cell motility assays were performed using xCelligence RTCA(Roche Diagnostics) as described before (18) and Cellomics CellMotility Kit as recommendedby themanufacturer (ThermoFisherScientific).

Gene expression array analysisTo determine NCOR1-regulated genes, 2 � 106 LNCaP cells

were transfected with 800 pmol of either noncoding control orNCOR1 SMARTpool siRNA (Thermo Fisher Scientific) using RBuffer (Lonza) andNucleofector Device (Lonza) exactly as recom-mended by the manufacturer. After 24 hours, cells were treatedwith either ethanol or 1 mmol/L bicalutamide for 48 hours, andRNA was purified using TRIzol reagent (Life Technologies). ForDHT-regulated gene expression, LNCaP cells were plated in RPMImedium supplemented with 10% CSS. Twenty-four hours later,cells were treated with 10 nmol/L DHT or ethanol vehicle. Cellswere harvested 24 or 48 hours after treatment. RNAswere used for

Translational Relevance

Castration therapies are the standard-of-care treatment formen with advanced prostate cancer. The short- and long-termmorbidities of this treatment are substantial. In addition, timelost for ineffective treatment can lead to the progression of thedisease. With the advances of personalizedmedicine, it will bepossible to more precisely characterize the tumor type forindividual patients. Thus, markers that would predict thera-peutic response are important. We examined the role ofandrogen receptor (AR) coregulator NCOR1 in response toantiandrogen treatments. In multiple datasets, NCOR1 muta-tions and loss of expression has been reported and, in thisarticle, we showdecline inNCOR1 protein and transcriptionaloutput in prostate cancer tissues. We present data that suggestthat patients with the loss ofNCOR1 function due tomutationor loss of protein expression may be predictive of resistance tocastration therapy in AR-expressing tumors.

Lopez et al.

Clin Cancer Res; 22(15) August 1, 2016 Clinical Cancer Research3938




expression analysis with Affymetrix 133A2.0Arrays atGenomic&RNA Profiling Core, Baylor College of Medicine (Houston, TX).Gene expression was marked as changed if the difference wasstatistically significant (P < 0.01) and the change was twofold ormore. Gene expression data were deposited into GEO repository,series numbers GSE60721 and GSE60722.

Gene set enrichment analysisGene set enrichment analysis (GSEA) was performed using

JAVA program (http://www.broadinstitute.org/gsea) as describedpreviously (19). The AR gene signature was generated from genesupregulated in LNCaP after 48-hour DHT treatment (P < 0.01;Supplementary Table S3). The SIAH2 gene signature was gener-ated by extracting genes changed more than twofold (P < 0.01) inLNCaP cell following SIAH2 knockdown (ref. 1; SupplementaryTable S4).

RNA extraction and quantitative PCR analysisRNA was extracted using TRI Reagent (Thermo Fisher Scien-

tific) as recommended by the manufacturer. RNA was used toprepare cDNA using Verso cDNA Expression Kit (Thermo FisherScientific). Primers and probes are listed in SupplementaryTable S5.

Animal studiesThe mice were maintained under standard conditions at

Florida International University (FIU; Miami, FL) animal facil-ities. All procedures were reviewed and approved by the Insti-tutional Animal Care and Use Committee at FIU and conductedin accordance with the National Academy of Science Guide forCare and Use of Laboratory Animals. Mice with Ncor1-floxedallele (Ncor1fl; ref. 16) were kindly provided by Dr. JohanAuwerx (Switzerland); B6.Cg-Tg(Pbsn-cre)4Prb/Nci transgenicmice with Cre recombinase gene driven by a derivative of the ratprostate–specific probasin (Pbsn) promoter (20) were obtainedfrom the NCI Mouse Repository (Bethesda, MD). The mice wereintercrossed to obtain wild-type Ncor1fl/fl males and Ncor1fl/fl,Pbsn-cre males with conditional inactivation of Ncor1 gene inprostate epithelium. Genotyping of the animals was conductedas described in the original publication (20). Ten-month-oldwild-type and mutant males were treated by a single oral gavagewith either oil alone or bicalutamide in sesame oil at 50 mg/kg.Each of the four groups contained seven to eight males. Forty-eight hours later, mice were euthanized, and ventral, lateral,and dorsal lobes of the prostate were dissected. Total RNA wasextracted with TRIzol Reagent (Life Technologies). cDNA wassynthesized using a Verso cDNA Kit (Thermo Fisher Scientific).qRT-PCR was performed using primers and probes listed inSupplementary Table S5.

IHC and tissue microarray analysisTissue microarray (TMA) analysis was described previously

(21). Briefly, sections were deparaffinized and antigen retrievalperformed in 10 mmol/L TRIS-HCl buffer (pH 8). Microarrayswere stained using NCOR1 antibody (Millipore) and counter-stained with hematoxylin. An automated slide scanner was usedto digitize the staining, and slides were scored as describedpreviously (22). Immunostaining and analysis for pAkt (S473)was described previously (23).

Association of an NCOR1 silencing signature with metastaticprogression

A gene signature of NCOR1 silencing was inferred (t test, P <0.05, fold change exceeding 1.5�) using the R statistical system.The association of the NCOR1 silencing transcriptome footprintwith metastatic progression was evaluated using three prostatecancer patient cohorts containing both primary and metastaticprostate cancer: Taylor and colleagues (ref. 2; GSE21034), Var-ambally and colleagues (ref. 24; GSE3325), and Cai and collea-gues (ref. 25; GSE32269). For each gene in NCOR1 silencingtranscriptomic response and for each prostate cancer specimen,we first computed the z-score for its expression within the cohort,as described previously (2), and next computed the sum z-scorefor each specimen. Specifically, the z-scores of genes repressed inthe NCOR1 silencing signature were subtracted from the z-scoresof genes induced in the NCOR1 silencing signature, resulting in acorresponding NCOR1 silencing activity score for each specimen.We next evaluated the difference between the distribution ofactivity scores in patients with primary prostate cancer and met-astatic cancer patients using the t test (P <0.05) as implemented inthe R statistical system.

Statistical AnalysisComparison of NCOR1 levels between tissue types was done

on paired observations, that is, patients without matching tumorand normal tissue measurements were excluded from analysis.Wilcoxon signed-rank testwasused to evaluate thedifferences dueto skewedness of the data. The association of cytoplasmic NCOR1with pAkt (S473) levels was evaluated using the Spearman cor-relation. All analyseswere performedusing the SPSS15.0 softwarepackage (SPSS for Windows, version 15.0., SPSS Inc).

Comparisons of mean levels of expression of specific mRNAsand combination indices (CI) were done using independentsamples Student t test. P < 0.05 was considered statisticallysignificant.

ResultsNCOR1 expression modulates response to bicalutamide inandrogen-dependent prostate cancer cell lines

Clearance of AR/NCOR1 complexes froma subset of promotersby SIAH2 interferes with androgen ablation (1). We asked wheth-er direct NCOR1 depletion would similarly alter cellularresponses to androgen ablation. First, we compared NCOR1expression in several AR-positive and AR-negative cell lines. Asshown in Fig. 1A and Supplementary Fig. S1A, all cell linesexpressed NCOR1 protein, with somewhat higher expression inVCaP and LAPC4 prostate cancer cell lines. Next, we evaluatedwhether the loss of NCOR1 altered AR protein levels in androgen-responsive cell lines, LNCaP and LAPC4. No change of ARexpression was observed following NCOR1 loss in both LNCaP(Fig. 1B) and LAPC4 cells (Supplementary Fig. S1B and S1C). InAR-negative cell lines, HeLa and DU145, knockdown of NCOR1did not affect proliferation of cell lines grown in complete media(Fig. 1C and D). Depletion of NCOR1 in LNCaP cells increasedproliferation in medium supplemented with CSS (Fig. 1E) but,similar to HeLa and DU145, had no effect on cells' growthin complete medium. A striking increase in proliferation follow-ing NCOR1 loss was observed in LNCaP cells treated with bica-lutamide (Fig. 1E). We next tested whether NCOR1 mediates

NCOR1 in Castration Resistance

www.aacrjournals.org Clin Cancer Res; 22(15) August 1, 2016 3939




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Figure 1.NCOR1 suppresses proliferation of LNCaP cells in CSS-supplemented medium and in the presence of bicalutamide (Bic). A, fifty micrograms of proteinextracted from LAPC4, LNCaP, VCaP, DU145, PC-3, and HeLa cells was resolved on SDS-PAGE and analyzed for NCOR1 and tubulin expression. B, LNCaPcells were transfected with control (C) or two independent NCOR1-specific siRNAs (s201 and s203). Cells were harvested 48 hours later and analyzed forNCOR1, AR, and tubulin expression by Western blot analysis. C, HeLa cells were transfected with either control or NCOR1-specific siRNA, and cellswere grown for 48 hours in complete medium. DNA synthesis was compared by measuring rates of [3H] thymidine incorporation. Cells transfected inparallel were analyzed for levels of NCOR1 by Western blotting. D, DU145 cells were transfected and analyzed exactly as in C. E, LNCaP cells weretransfected with control or NCOR1-specific siRNA and grown for 24 hours in medium supplemented with CSS or FBS as indicated. Cells were then treated for24 hours with either vehicle (ethanol) or 1 mmol/L bicalutamide and [3H] thymidine incorporation measured. Cells transfected in parallel and treatedwith vehicle were analyzed for NCOR1 and tubulin expression by Western blot analysis. F, LNCaP cells were transfected with either control or NCOR1-specificsiRNA and 24 hours later treated with either vehicle (ethanol), 1 mmol/L bicalutamide, 10 mmol/L bicalutamide, or 10 mmol/L MDV3100 for an additional24 hours. B–F, experiments were repeated at least three times with two different NCOR1-specific siRNAs. Each point in DNA synthesis assay wasdone in triplicates. Average and SDs are shown. Unless the exact value is shown, asterisk (�) denotes differences between control and NCOR1 siRNAtransfected cell with P < 0.05.

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response to pure AR antagonist MDV3100 (enzalutamide). Bothbicalutamide and MDV3100 at 10 mmol/L similarly suppressedDNA synthesis in LNCaP cells transfected with control siRNA(no statistically significant difference). However, MDV3100inhibited much better then bicalutamide after NCOR1 knock-down (Fig. 1F). NCOR1 knockdown stimulated the proliferationof LAPC4 cells under all treatment conditions (SupplementaryFig. S1D). Opposite to NCOR1, NCOR2 knockdown did notstimulate LNCaP cell growth under any conditions (Supplemen-tary Fig. S1E).

Bicalutamide also reduced LNCaP motility, and NCOR1knockdown reversed this effect (Supplementary Fig S2). LNCaPremained noninvasive after NCOR1 knockdown (not shown). InPC-3 and DU145 cell lines, which express NCOR1 but not AR,NCOR1 knockdown did not alter cell motility and invasion (notshown).

Loss of NCOR1 alters bicalutamide-regulated gene expressionprofile

To understand how NCOR1 mediates bicalutamide resistance,we explored changes in gene expression caused by bicalutamidetreatment and knockdown of NCOR1 using Affymetrix micro-array. LNCaP cells were transfected with either control- orNCOR1-specific siRNAs and treated with either ethanol or 1mmol/L bicalutamide generating four groups: Control siRNA_Ve-hicle (control siRNA transfected, treated with ethanol), ControlsiRNA_Bic (control siRNA transfected, treated with bicaluta-mide), NCOR1 siRNA_Vehicle (NCOR1 siRNA transfected, trea-ted with ethanol), and NCOR1 siRNA_Bic (NCOR1 siRNA trans-fected, treated with bicalutamide; Fig. 2A). Gene expressionanalysis revealed pathways significantly affected by NCOR1 losswith or without bicalutamide treatment (Supplementary TableS1);we observed significant alteration inmetabolic, cell signaling,and prostate cancer–associated pathways (Supplementary TableS1). We next examined whether NCOR1 regulated AR transcrip-tional activity using GSEA analysis. To determine DHT-regulatedgenes, we treated the LNCaP cell line with DHT or vehicle andperformedmicroarray analysis. DHT treatment caused significantchanges in genes associated with cell cycle, prostate cancer, andbiosynthesis of steroids (Supplementary Table S2). A list of genesthat were significantly increased by DHT treatment (P < 0.01;Supplementary Table S3) was used to analyze changes in geneexpression in Control siRNA_Bic and NCOR1 siRNA_Bic pair(Fig. 2B). As expected, AR signature was significantly representedamong bicalutamide-regulated genes with or without NCOR1expression (Supplementary Fig. S3A and S3B). As seen from Fig.2B, the AR signature was represented among NCOR1-regulatedgenes [normalized enrichment score (NES)¼ 1.4378, FDR q value¼ 0.1126, P < 0.0001]. Using a published list of SIAH2-regulatedgenes in LNCaP cells (1), we created the SIAH2 signature com-prised of top SIAH2 up- and downregulated genes (Supplemen-tary Table S4). As shown in Supplementary Fig. S3C, SIAH2signature is present among NCOR1-regulated genes in bicaluta-mide-treated LNCaP cells (NES¼ 1.2258, FDR q value ¼ 0.2101,P ¼ 0.046).

NCOR1-regulated genes modulate cellular proliferation andresponse to castration and bicalutamide treatment

UDP glucuronosyltransferases 2B17 and 2B15 are expressed inluminal epithelium of human prostate and in LNCaP cells (26).They stimulate androgen removal and are implicated in prostate

cancer (27).UGT2B15 andUGT2B17 are directly repressed by ARin an agonist-dependent manner (28). Loss of NCOR1 causeddecrease in UGT2B15 expression under all treatment conditions(Fig. 3A and B). Expression ofUGT2B17 declined in parallel withNCOR1 loss only in FBS or in CSS and R1881-supplementedmedia, suggesting that agonist-bound AR is needed for thisregulation (Fig. 3C and D). Interestingly, the UGT2B17 andUGT2B15 expression was significantly lower in C4-2 cells, anLNCaP-derived cell line that proliferates and produces PSA insteroid-depleted medium (Supplementary Fig. S4). Consistentwith previous reports (28), concomitant knockdown of bothUGT2B15 and UGT2B17 resulted in increased AR transcriptionalactivity (Supplementary Fig. S5).

The most upregulated gene upon NCOR1 depletion was pro-tocadherin 11 Y-linked (PCDH11Y). Previous reports showedthat PCDH11Y overexpression allowed LNCaP cells proliferate inmedium supplemented with CSS andmade them resistant to TPAand serum starvation–induced apoptosis (29–31). We showedthat PCDH11Y was suppressed by androgens in LNCaP cells atandrogen concentrations as low as 0.1 nmol/L (Fig. 3E and F).Androgendepletion, bicalutamide treatment, andNCOR1knock-down stimulated PCDH11Y expression (Fig. 3G, H, and I).

Following NCOR1 knockdown, the expression of TARP wassignificantly reduced. As previously reported, we show that TARPexpression is induced by R1881 (32) almost 60-fold and sup-pressed by bicalutamide in medium with FBS (Fig. 4A). Intrigu-ingly, TARP expression was consistently lower in medium sup-plemented with FBS than CSS plus R1881, suggesting additionalmodes of regulation by signaling pathways (Fig. 4A). NCOR1knockdown significantly reduced TARP expression both in medi-um supplemented with CSS plus R1881 and with FBS. TARPis regulated similarly by NCOR1 and AR in LAPC4 cells (Supple-mentary Fig. S6A). As seen from Fig. 4B, TARP depletion increasedLNCaP cell proliferation. Moreover, loss of TARP expressionreduced LNCaP cells' sensitivity to bicalutamide treatment(Fig. 4B).

We next tested whether the AR splice variant V7 (AR-V7) caninduceTARP expression andwhetherNCOR1 is required forTARPoptimal induction using previously described LNCaPAR-V7/pLenti

cell line inducibly expressing AR-V7 (17). As seen from Fig. 4C,doxycycline induction of AR-V7 expression increased TARPexpression levels, and NCOR1 knockdown reduced AR and AR-V7–dependent induction of TARP. To analyze whether NCOR1regulates AR-V7–specific transcription, we measured EDN2 geneexpression. As previously reported (17), EDN2 was inducedspecifically by AR-V7, and optimal induction required NCOR1expression (Fig. 4D).

NCOR1 knockdown resulted in significant loss of expression ofCHEK1. CHEK1 kinase was not regulated by androgens in ourLNCaP and LAPC4 cell lines (Fig. 4E and Supplementary Fig.S6B), and no recruitment sites were reported within 30 kbupstream or downstream of its gene locus (3). Depletion ofNCOR1 (Fig. 4F) significantly reduced CHEK1 expression inLNCaP cells (Fig. 4G). In LAPC4 cells, NCOR1 depletion reducedCHEK1 expression in CSS (Supplementary Fig. S6B). As seenfrom Fig. 4H, loss of CHEK1 in LNCaP cells stimulated prolifer-ation but did not abolish responsiveness to bicalutamide.

We next tested whether NCOR1 modulates AR activity bychanging its recruitment to PSA and INPP4B promoters, geneswhose expression is regulated by both AR andNCOR1 (6, 12).Weobserved NCOR1 recruitment to PSA and INPP4B promoters






(6, 12). However, NCOR1 knockdown did not significantly alterAR recruitment to these loci (Supplementary Fig S7).

NCOR1 mediates bicalutamide response in mouse prostateTo test whether NCOR1 is required for response to bicaluta-

mide inmouse prostate, we obtainedNcor1fl/fl, Prbn-cremalemicewith conditional deletion of Ncor1 gene in prostate epitheliumand controlNcor1fl/fl littermate males with functionalNcor1 gene.Mutant males did not display any overt abnormality: total bodyweight and the weights of testes or seminal vesicles did not differ

between two genotypes. Males with both genotypes were fertileandhadnormal prostates. Levels ofNcor1 expression evaluated byqRT-PCR were significantly lower in males with conditionaldeletion ofNcor1 (Fig. 5A), whereas Ar expression did not changewhen compared with the control group (Fig. 5B). After 48-hourtreatment with bicalutamide at 50mg/kg, no change was detectedin Tmprss2 gene expression (Fig. 5C), confirming hypothesis thatSIAH2/NCOR1/AR complexes do not regulate its expression inmouse prostate (1). Another three SIAH2/AR target genes inmouse prostate are ApoF, Nkx3.1, and Spink1 (1). In control

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Figure 2.Modulation of gene expression by NCOR1 and bicalutamide (Bic). A, LNCaP cells were transfected with either noncoding control or NCOR1 siRNA and treatedwith either ethanol or 1 mmol/L bicalutamide for 48 hours. Gene expression was evaluated using Affymetrix 133A 2.0 Arrays, and differentially expressedgenes were clustered. B, GSEA of the AR target gene signature among NCOR1-regulated genes in bicalutamide-treated LNCaP cells (Control siRNA_Bic andNCOR1 siRNA_Bic pair).

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animals, the expression of AR target gene, ApoF, was reducedafter bicalutamide treatment; downregulation of Ncor1 inmutant prostates abolished bicalutamide-dependent repres-sion of ApoF. In control animals, we observed statisticallysignificant downregulation of Nkx3.1 and Spink1 expressionafter treatment with bicalutamide. In mutant prostates, changesin expression of these genes were not statistically significant.Total reduction in Ncor1 was modest in prostate, which con-tains multiple cell types, but there is a substantial change in theregulation of epithelial-specific genes. Thus, we observed that

the loss of Ncor1 compromised response to bicalutamide for asubset of AR target genes (Fig. 5D–F).

NCOR1 protein levels decline with prostate cancer progressionin patients with prostate cancer

Wenext analyzedNCOR1protein levels in normal prostate andprostate cancer samples from radical prostatectomy specimensusing TMAs. Consistent with previous observations, strong stain-ingwas detected both in cytoplasm and nuclei of prostate luminalepithelium (Supplementary Fig. S8). In primary prostate tumors,

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Figure 3.NCOR1 regulates AR-repressed genes. A, LNCaP cells were transfected with either control (C) or NCOR1-specific siRNAs. Cells were plated in mediumsupplemented with FBS and treated with ethanol (vehicle) or 1 mmol/L bicalutamide (Bic) for 48 hours. RNA was analyzed for UGT2B15 expression. B, cells weretransfected as in A, plated in CSS-supplemented medium and treated with ethanol vehicle or 1 nmol/L R1881. RNA was analyzed for UGT2B15 expression.C, RNAs from A were analyzed for UGT2B17 expression. D, RNAs from B were analyzed for expression of UGT2B17. E, LNCaP cells were grown in mediumsupplemented with CSS, treated with vehicle or 1nmol/L R1881, and harvested at indicated time points. RNAwas extracted and analyzed for expression of PCDH11Y.F, LNCaP cells were grown in CSS-supplemented medium and treated with vehicle (V) or indicated concentrations of R1881 for 48 hours, RNA extracted, andanalyzed for PCDH11Y expression. G, LNCaP cells were grown in FBS-supplemented medium and treated with 1 mmol/L bicalutamide and RNA extracted atindicated time points. PCDH11Y expression was analyzed. H, LNCaP cells transfected and treated as in A were analyzed for PCDH11Y expression. I, LNCaP cellstransfected and treated as in B were analyzed for PCDH11Y expression. Every point is an average of three biologic replicates � SD; all experiments were done atleast three times and a representative experiment shown. In A–D, H, and I, at least three independent NCOR1 siRNAs were used. � , difference with correspondingcontrol with P < 0.05.






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Figure 4.NCOR1 is required for optimal expression of TARP and CHEK1. A, LNCaP cells transfected with either control or NCOR1-specific siRNAs were placed in mediumsupplemented with either CSS or FBS. LNCaP cells grown in CSS-supplemented medium were treated with ethanol vehicle (Veh) or 1 nmol/L R1881. Cells grown inFBS-supplemented medium were treated with ethanol vehicle or 1 mmol/L bicalutamide (bic). RNA was extracted and levels of TARP mRNA compared byqRT-PCR. Level of TARP expression in cells grown in CSS-supplementedmedium transfectedwith control siRNA and treatedwith vehicle was assigned a value of 1 andall other values adjusted accordingly. B, cells transfected as in A were plated into E-plate of xCelligence analyzer at 10,000 cells per well 12 hours after transfectionand treated with vehicle or 1 mmol/L bicalutamide. Cellular impedance was measured every 30 minutes and average and SE calculated for each time point.Statistical significancewas calculated for the last time point.C–D, parental LNCaP and LNCaPAR-V7/pLenti cells were transfectedwith control or NCOR1 siRNA and grownin CSS-supplemented medium. Cells were treated with either vehicle (V), 1 nmol/L R1881, or doxycycline (DOX) and RNA evaluated for TARP (C) and EDN2 (D)expression. E, LNCaP cells were treated with 1 nmol/L R1881 for 24 hours and RNA extracted and examined for CHEK1 expression. F–G, LNCaP cells grown incomplete medium were transfected with either control, CHEK1, or NCOR1 siRNAs and expression of NCOR1 (F) and CHEK1 (G) compared 48 hours after transfection.H, LNCaP cells were transfected with CHEK1 or control siRNA. Twelve hours later, 10,000 cells were plated into each well of E-plate of xCelligence analyzer.Ethanol vehicle or 1 mmol/L bicalutamide were added and changes in cellular impedance measured every 30 minutes during 50 hours. An average and SE fromfour biologic replicates were calculated for each time point. The statistical significance was calculated for the last cellular impedance measurement between cellstransfected with control and NCOR1 siRNAs. �, P < 0.05.

Lopez et al.





both cytoplasmic and nuclear levels of NCOR1 were significantlyreduced (Fig. 6A). We correlated previously reported pAkt levelsdetermined using the same array (23) with NCOR1 levels. Inagreementwith theobservation thatNCOR1 inhibits Akt pathwayin thyroid cancer cells (11), we detected statistically significantnegative correlation between the levels of pAkt (S473) andNCOR1 proteins in prostate cancer tissues (Fig. 6B). Consistentwith this finding, NCOR1 knockdown in LNCaP and LAPC4 cellsincreased levels of pAkt (S473; Fig. 6C). On the other hand,NCOR1 knockdown did not change NCOR2 protein levels inLNCaP cells (Fig. 6C). In addition, no correlation betweenNCOR1 andNCOR2was detected in patients with prostate cancerusing Grasso (primary and CRPC; ref. 33), Stanborough (primaryand metastatic; ref. 34), and Taylor (2) cohorts (SupplementaryTable S7).

To determine whether NCOR1-regulated genes change withprostate cancer progression, we inferred transcriptome signatureof NCOR1 silencing. Using previously employed methodology

(2), we evaluated the NCOR1 silencing signature activity in threecohorts containing both primary and metastatic prostate cancerpatients: Taylor and colleagues (2), Varambally and colleagues(24), and Cai and colleagues (25). In each cohort, the metastaticpatients exhibited significantly higher activity scores for theNCOR1 silencing gene signature (P < 0.005 for each cohort), aspresented in Fig. 6D–F, indicating a diminished activity ofNCOR1 in metastatic prostate cancer.

DiscussionNCOR1 is a steroid receptor coregulatory protein, which was

first described as a repressor of steroid receptor activity (13, 35).Unique to AR, NCOR1 was shown to repress both agonist- andantagonist-dependent transcriptional activity on ARE-drivenreporters (13). We investigated whether NCOR1 modulatesendogenous AR transcriptional activity and plays a role inresponse to bicalutamide treatment. Using independently

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Figure 5.NCOR1 loss in mouse dampensresponse to bicalutamide (bic) of someAR-regulated genes. A, Ncor1fl/fl andNcor1fl/fl,Pbsn-cre mice were treatedwith single oral gavage of eithersesame oil or 50mg/kg bicalutamide insesame oil. Ventral, lateral, and dorsalprostate lobes were dissected 48 hourslater and RNA analyzed for Ncor1 byqRT-PCR. B, RNA from A was analyzedby qRT-PCR for Ar (B), Tmprss2 (C),ApoF (D), Nkx3.1 (E), and Spink1 (F).Values were normalized for 18S.Significance was evaluated using t test,and all statistically significantdifferences are marked on bar graphswith corresponding P values.






derived androgen-dependent cell lines LNCaP and LAPC4, wedetermined that NCOR1 suppresses cellular proliferation.Interestingly, in the LNCaP cells, which express moderate levelsof NCOR1, the loss of NCOR1 stimulated proliferation in bothCSS-supplemented medium and in full medium treated withbicalutamide. In the high NCOR1–expressing LAPC4 cells,depletion increased proliferation under all conditions (Sup-plementary Fig. S1D). Importantly, the loss of NCOR1 did notalter AR protein levels in either cell line, suggesting NCOR1-dependent changes in AR activity rather than AR level. Deple-tion of NCOR2 had the opposite effect on the proliferation ofLNCaP cells (Supplementary Fig. S1E), confirming previousreports of nonredundant roles for these corepressors. Bicaluta-mide and the pure AR inhibitor MDV3100 suppressed LNCaPproliferation to a similar degree. However, in the absence of

NCOR1, bicalutamide was much less effective than MDV3100in inhibiting proliferation (Fig. 1E and F). This difference maybe due to the different mechanisms of action of the twocompounds. Bicalutamide allows some AR DNA binding andrecruitment of protein complexes that include NCOR1. Deple-tion of NCOR1 reduces the inhibitory complexes. In contrast,MDV3100-bound AR does not bind to the DNA, and thus, theelimination of NCOR1 should have little or no effect on AR-dependent activity under these conditions, whereas AR-inde-pendent actions would be retained. Recently, MDV3100-regu-lated changes in gene expression have been reported for LNCaP(36). A comparison of MDV3100 transcriptional regulation(GSE44905 and GSE44924) in LNCaP and bicalutamide-dependent changes in our experiments showed that approxi-mately 20% of the genes were regulated by both ligands.

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Figure 6.NCOR1 expression and transcriptionalsignature in prostate cancer (PCa).A, TMAs constructed from tissuesobtained from radical prostatectomywere analyzed for NCOR1 expressionin nuclear and cytoplasmic (Cyto)compartments of luminal epitheliumof benign prostate and in tumor tissue.Wilcoxon signed rank test was used tocompare levels of NCOR1 in thesecompartments in normal and tumorcells and to calculate P values for eachcomparison. B, cytoplasmic stainingwith phospho-S473 was correlatedwith NCOR1 staining using a scatterplot with jitter and Spearmancorrelation coefficient calculated.C, LNCaP and LAPC4 cells weretransfected with either control (Ctrl)or NCOR1-specific siRNA. Cells weremaintained in FBS-supplementedmedium for 48 hours, harvested, andanalyzed for NCOR1, NCOR2, AR, totalAkt, pAkt (Ser473), and tubulinexpression. D–F, NCOR1 silencingsignatures primary and metastaticprostate cancer tissues in Taylor andcolleagues (GSE21034), Varamballyand colleagues (GSE3325), and Caiand colleagues (GSE32269) cohorts.

Lopez et al.





To identify NCOR1-regulated genes and cellular pathways thatmediate resistance to bicalutamide, we performedwhole-genomegene expression analysis in LNCaP cells. As seen from Fig. 2A,NCOR1 regulates both bicalutamide-dependent and -indepen-dent transcription. In a previous report, loss of the ubiquitinligase, SIAH2, reduced LNCaP proliferation, growth of colonies insoft agar, and sphere formation both in the presence and absenceof androgens (1). This was presumablymediated by an increase inNCOR1–AR complexes on selected promoters. Indeed, the DHT-upregulated gene signature was highly significantly representedamong NCOR1 target genes, suggesting a functional interactionbetween AR and NCOR1. This conclusion is supported by previ-ous reports of physical and functional AR–NCOR1 interactions inthe presence of androgens (1, 13) and bicalutamide (12). Signif-icantly, the SIAH2 target gene signature was also significantlyrepresented amongNCOR1-regulated genes, confirming the find-ing that SIAH2 targets NCOR1–AR complexes for degradation inthe presence of both androgen and bicalutamide (1).

Depletion of NCOR1 in control- and bicalutamide-treatedLNCaP cells led to changes in the expression of genes that playsignificant roles in prostate cancer progression. The most down-regulated gene was NCOR1, confirming successful knockdown.Many, but not all, of the genes identified are alsoAR regulated. Thesecond most downregulated gene was an androgen-inducedtumor suppressor, INPP4B, encoding inositol polyphosphate 4-phosphatase, type II. We have previously reported concordantinduction of INPP4B by AR andNCOR1 (6). Consistent with this,we found that NCOR1 protein levels decline with prostate cancerprogression. We and others reported that INPP4B levels decreasewith prostate cancer progression both in primary and especially inmetastatic prostate cancer (2, 6), consistent with the positivecorrelation between NCOR1 and INPP4B expression observedin prostate cancer cell lines (6). NCOR1 loss also caused asignificant induction of the PCDH11Y transcript, an androgen-repressed target. Androgen deprivation increases PCDH11Yexpression in LNCaP cells. High levels of endogenously or exog-enously expressed PCDH11Y stimulate androgen-independentLNCaP cell growth (29), confer resistance to apoptosis (31), andactivate oncogenic Wnt signaling (30). PCDH11Y mRNA innormal prostates, primary prostate tumors, and untreated pros-tate cancer is low but increases significantly in CRPC (29),consistent with a negative correlation between NCOR1 andPCDH11Y observed in LNCaP cells. AR-repressed genes,UGT2B15 and UGT2B17, cause DHT conjugation and excretion(28, 37) and are associated with prostate cancer risk (38, 39). Weshow that NCOR1 is required for optimal UGT2B15 expressionunder all conditions, whereas for UGT2B17, expression ofNCOR1 was required only in the presence of androgens. Bothincreased and decreased expression of these enzymes have beenreported in prostate cancer (34, 40) potentially due to usingsamples from prostate tumors at different stages and treatments.It is also possible that levels of UGT2B15 and UGT2B17 enzymesdecline only in a subset of prostate cancers as a mechanism toadapt to castration therapy by raising intracellular levels ofandrogens.

One of the most strongly downregulated transcripts whenNCOR1 is depleted was TARP mRNA, a mitochondrial proteinexpressed specifically in normal prostate, prostate cancer, andbreast cancer. At least four distinct protein-coding TARP variantsare produced from this locus in response to androgens (32, 41).Splice variant, NM_001003799.1, changed expression with

NCOR1 knockdown. TARP was strongly induced by androgensand by NCOR1 expression in LNCaP and LAPC4 cells. We foundthat TARP depletion significantly increased LNCaP cellular pro-liferation (Fig. 4B) and contributed to bicalutamide resistance.Our finding that AR-V7 also upregulates TARP and that NCOR1 isrequired for optimal induction suggests that in prostate cancersdriven by various AR splice variants, NCOR1 is an importantcoregulator. This is confirmed by our observation that the induc-tion of the AR-V7–specific target gene EDN2 requires NCOR1expression (Fig. 5G and H). In an earlier report, exogenousexpression of TARP increased cellular proliferation in an AR-negative prostate cancer cell line by increasing CAV1, AGREG,andCXCL1 and suppressing IL1b expression (42). In LNCaP cells,levels of IL1b, AREG, and CAV1 are low to undetectable, whereasthe loss of CXCL1 causes the opposite effect, increasing cellproliferation andanchorage-independent growth (43). TARPmayalso affect AR signaling, contributing to differences in its effectbetween AR-negative and AR-positive cells.

As shown in Fig. 4, NCOR1 also regulates CHEK1, and thisregulation is androgen independent. CHEK1 is required for cell-cycle arrest in response toDNAdamage andmay also regulate cellcycle (44). A decline in CHEK1 expression was reported inprostate cancer (45), and compound loss of CHEK1 and PTENtriggers progression fromhigh-grade prostatic intraepithelial neo-plasia to invasive prostate carcinoma (46). CHEK1 knockdownincreased LNCaP cellular proliferation but bicalutamide was stillable to suppress proliferation (Fig. 4H). It is possible that in theNCOR1-negative prostate tumors, a decline in CHEK1 mightcontribute to more aggressive proliferative phenotype.

Additional confirmation of a role for NCOR1 in bicalutamideactionwas obtainedusingmutantmicewith conditional prostate-specific Ncor1 gene deletion. We chose 48-hour time point forbicalutamide treatment because it would inhibit AR action with-out significant cell death in prostate epithelial compartment.Importantly, Ncor1 expression is not exclusive to luminal epithe-lium of mouse prostate, the site of Pbsn-cre expression. Similar towhat we saw in LNCaP and LAPC4 cells, NCOR1 deletion inmouse prostate did not reduce AR mRNA levels. However, someof the AR target genes that are highly expressed in prostateepithelium (1, 47) lost their response to bicalutamide in mutantmice. In agreement with the previous report that Siah2 regulatesgene expression by removing NCOR1 complexes from a subset ofgenes (1), Siah2-regulated AR target genes, ApoF, Nkx3.1, andSpink1, lost their ability to respond to bicalutamide treatment inNcor1 knockout mouse prostates, while Tmprss2 expression didnot change.

NCOR1 has emerged as an important regulatory protein inprostate and other cancers. Analysis of changes in signaling path-ways with prostate cancer progression by Taylor and colleaguesshowed that NCOR1 is lost in 8% of primary and 16%metastaticprostate tumors due to deletions, loss of expression, and/ormutations (2, 33, 48). A comparison of high-grade untreatedlocalized prostate cancers and lethal metastatic CRPC showed ahigh level of NCOR1 somatic mutations and copy numberalterations in CRPC, while none were detected in untreatedtumors (33). Additional evidence for NCOR10s contribution tothe development of resistance to antihormonal treatments wasfound in breast cancer. In ERa-positive breast tumors, lowNCOR1 expression is an independent predictor of tamoxifenresistance (49). Loss of NCOR1 at the protein level also correlatedwith acquired tamoxifen resistance in mouse models of breast






cancer (50) and with breast cancer recurrence in human patients(51). Tamborero and colleagues identified NCOR1 as a high-confidence cancer-driver gene using three unbiased screeningtechniques: MuSIC-SMG, OncodriveFM, and Active Driver(48). We have compared NCOR1 protein expression in normalprostate and prostate cancer in more than 400 paired tissues (Fig.6). NCOR1 was detected mostly in the prostate secretory epithe-lium (Supplementary Fig. S8). Consistent with previous reports,we observed NCOR1 staining in both the cytoplasm and thenuclei of the cells (10, 11), and levels significantly declined inboth compartments inmalignant cells (Fig. 6A). Significantly, theNCOR1-regulated transcriptional signature declines in advancedprostate cancer inmultiple cohorts (Fig. 6D–F). Similar to thyroidcancer cells (11), we observe a highly significant negative corre-lation between cytoplasmic levels of NCOR1 and levels of pAktstaining in prostate cancer tissues (Fig. 6B). These data suggest thata decline in NCOR1 protein level occurs with prostate cancerprogression and may contribute to patients' response to antian-drogen treatments.

Our data illustrate the importance of NCOR1 in prostateresponse to bicalutamide in two cell-based and inNcor1-deficientmice in vivo. Together with the observation that NCOR1 levelsdecline during prostate cancer progression in men, we demon-strate that NCOR1 is involved in the development of resistance tobicalutamide treatment and provide an additional mechanism toexplain the better response to MDV3100.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A.I. Agoulnik, N.L. Weigel, I.U. AgoulnikDevelopment of methodology: I.U. AgoulnikAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.M. Lopez, A.I. Agoulnik, M. Zhang, E. Suarez,G.A. Gandarillas, R. Li, M.M. Ittmann, N.L. Weigel, I.U. AgoulnikAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.M. Lopez, A.I. Agoulnik, M. Zhang, L.E. Peterson,A. Frolov, K. Rajapakshe, C. Coarfa, I.U. AgoulnikWriting, review, and/or revision of themanuscript: S.M. Lopez, A.I. Agoulnik,A. Frolov, K. Rajapakshe, C. Coarfa, M.M. Ittmann, N.L. Weigel, I.U. AgoulnikAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.M. Lopez, E. Suarez, G.A. Gandarillas,I.U. AgoulnikStudy supervision: I.U. Agoulnik

Grant SupportThis work was supported by R15 CA179287-01A1 (to I.U. Agoulnik)The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 14, 2015; revised February 9, 2016; accepted February 19,2016; published OnlineFirst March 11, 2016.

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2016;22:3937-3949. Published OnlineFirst March 11, 2016.Clin Cancer Res Sandra M. Lopez, Alexander I. Agoulnik, Manqi Zhang, et al. with Prostate Cancer ProgressionNuclear Receptor Corepressor 1 Expression and Output Declines

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