Polycomb Repressor Complex-2 Is a Novel Target for ... · Inhibition of PRC-2 expres-sion by...

Human Cancer Biology

Polycomb Repressor Complex-2 Is a Novel Target forMesothelioma Therapy

Clinton D. Kemp1, Mahadev Rao1, Sichuan Xi1, Suzanne Inchauste1, Haresh Mani2, Patricia Fetsch2,Armando Filie2,Mary Zhang1, Julie A. Hong1, Robert L.Walker3, Yuelin J. Zhu3, R. Taylor Ripley1, Aarti Mathur1,Fang Liu1, Maocheng Yang1, Paul A. Meltzer3, Victor E. Marquez4, Assunta De Rienzo5, Raphael Bueno5, andDavid S. Schrump1

AbstractPurpose: Polycomb group (PcG) proteins are critical epigenetic mediators of stem cell pluripotency,

which have been implicated in the pathogenesis of human cancers. This study was undertaken to examine

the frequency and clinical relevance of PcG protein expression inmalignant pleuralmesotheliomas (MPM).

Experimental Design:Microarray, quantitative reverse transcriptase PCR (qRT-PCR), immunoblot, and

immunohistochemistry techniques were used to examine PcG protein expression in cultured MPM,

mesothelioma specimens, and normal mesothelial cells. Lentiviral short hairpin RNA techniques were

used to inhibit EZH2 and EED expression in MPM cells. Proliferation, migration, clonogenicity, and

tumorigenicity of MPM cells either exhibiting knockdown of EZH2 or EED, or exposed to 3-deazanepla-

nocin A (DZNep), and respective controls were assessed by cell count, scratch and soft agar assays, and

murine xenograft experiments. Microarray and qRT-PCR techniques were used to examine gene expression

profiles mediated by knockdown of EZH2 or EED, or DZNep.

Results: EZH2 and EED, which encode components of polycomb repressor complex-2 (PRC-2), were

overexpressed inMPM lines relative to normal mesothelial cells. EZH2was overexpressed in approximately

85% of MPMs compared with normal pleura, correlating with diminished patient survival. Overexpression

of EZH2 coincided with decreased levels of miR-101 andmiR-26a. Knockdown of EZH2 or EED, or DZNep

treatment, decreased global H3K27Me3 levels, and significantly inhibited proliferation, migration, clono-

genicity, and tumorigenicity of MPM cells. Common as well as differential gene expression profiles were

observed following knockdown of PRC-2 members or DZNep treatment.

Conclusions: Pharmacologic inhibition of PRC-2 expression/activity is a novel strategy for mesothe-

lioma therapy. Clin Cancer Res; 18(1); 77–90. �2011 AACR.

Introduction

Malignant pleural mesotheliomas (MPM) are highlylethal neoplasms attributable to asbestos exposure, as wellas ill-defined environmental and genetic factors (1).Because of industrialization and long latency associatedwith these malignancies, the global incidence of MPM

continues to increase (2). Currently, results of conventionaltreatments for MPM are far from optimal; median survivalsof patients undergoing aggressive multimodality therapyfor MPM range from 14 to 28months, depending on tumorhistology and stage, extent of surgical resection, andresponse to chemotherapy (3).

During recent years, considerable insight has beenachieved regarding the molecular pathogenesis of MPM.These neoplasms exhibit significant, recurrent karyotypicabnormalities (4, 5), perturbedmicroRNA (miRNA) expres-sion (6, 7), loss of tumor suppressor genes (4), and upre-gulation of oncogene signaling (5), resulting in cell-cycledysregulation, resistance to apoptosis (8), and enhancedinvasion potential of mesothelioma cells (9). Epigenomicalterations have also been implicated in the pathogenesis ofMPM. For example, changes in chromosomal copy number,as well as miRNA expression, correlate with global and site-specific alterations in DNAmethylation in these neoplasms(7, 10, 11). In several studies, DNA methylation signaturesin MPM seem to coincide with exposure to specific carcino-gens (12, 13). In addition, MPM exhibit a global increase in

Authors' Affiliations: 1Thoracic Oncology Section, Surgery Branch, Cen-ter for Cancer Research; 2Laboratory of Pathology; 3Genetics Branch;4Laboratory of Medicinal Chemistry; National Cancer Institute, NIH,Bethesda, Maryland; and 5Division of Thoracic Surgery, Brigham andWomen's Hospital, Boston, Massachusetts

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

Corresponding Author: David S. Schrump, Thoracic Oncology Section,Surgery Branch, Center for Cancer Research, National Cancer Institute, 10Center Drive, Room 4-3942MSC 1201, Bethesda, MD 20892. Phone: 301-496-2128; Fax: 301-451-6934; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-0962

�2011 American Association for Cancer Research.

ClinicalCancer

Research

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Published OnlineFirst October 25, 2011; DOI: 10.1158/1078-0432.CCR-11-0962

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histone H3 lysine 27 trimethylation (H3K27Me3; ref. 14),suggestive of aberrant expression/activity of polycombgroup (PcG) proteins (15). This study was undertaken toexamine the frequency and potential clinical relevance ofPcG expression in MPM.

Materials and Methods

Cell linesH28, H2052, and H2452 MPM cell lines were obtained

from American Type Culture Collection and cultured inRPMI plus 10% fetal calf serum with Pen/Strep (completemedia). LP3 and LP9 normal mesothelial cells wereobtained from the Coriell Institute for Medical Research(Camden, NJ) and cultured according to vendor instruc-tions. NCI-SB-NMES1-and 2 were isolated from histolog-ically normal pleura and cultured as described (16). NCI-SB-MES1-4 were established from primary MPM andcultured in complete RPMI media. These cell lines havebeen validated by HLA typing and molecular phenotyp-ing relative to the respective primary tumors. All tissuesfor cell lines or gene expression analysis were procured inaccordance with Institutional Review Board (IRB)-approved protocols.

RNA isolation and microarray analysisTotal RNA was isolated from cell lines or snap-frozen

surgical specimens using the RNeasy minikit (Qiagen).Normal pleural RNA from individual patients withoutMPM was obtained from ProteoGenex. Gene expressionprofiles were analyzed using Gene-Chip Human GenomeU133 2.0 plus Arrays (Affymetrix) or Illumina Arraysaccording to vendor instructions; higher order analysis wascarried out using Gene Sprint and Ingenuity Pathwaysoftware.

Real-time quantitative RT-PCRRNA was subjected to reverse transcription using a

BioRad RT kit, and gene expression was analyzed using theTaqMan real-time quantitative reverse transcriptase PCR(qRT-PCR) kit and primers/probes listed in SupplementaryTable S1.

MicroRNA isolation and quantitationqRT-PCR analysis was used to determine the relative

expression levels of miR-101, miR-26a, miR-26b, andRNU44 in MPM specimens using previously describedtechniques (17). Briefly, miRNA was isolated using TRIzolreagent (Invitrogen) and the RT2 qPCR-Grade miRNAIsolation Kit (Qiagen). Equal amounts of miRNA wereconverted into cDNA using miR-101, miR-26a, miR-26b,and RNU44 RT primers (Applied Biosystems), according tothe manufacturer’s protocol. Quantitative PCR was doneusing primers and materials from Applied Biosystems. TheCt valueswere used to calculate the relative fold difference inmiRNA levels. All experiments were done using biologicaltriplicates. The data were normalized to RNU44 expressionlevels.

Generation of stable cells expressing shRNA constructsMPM cell lines were transfected with commercially val-

idated short hairpinRNAs (shRNA) targeting EZH2, or EED,or sham sequences (Sigma) according to themanufacturer’sinstructions. Cell lines were selected with puromycin andknockdown confirmed by immunoblot analysis.

Immunohistochemistry of primary tumor samplesFormalin-fixed, paraffin-embedded tissue blocks from

primary MPM specimens and unmatched normal pleurafrom autopsy specimens resected on IRB-approved proto-cols at the National Cancer Institute (NCI), as well as tissuemicroarrays (TMA) containing normal mesothelia, MPM,and peritoneal mesotheliomas (PM; US Biomax) werestained with mouse anti-EZH2 (BD Biosciences) at a 1:50dilution following heat-induced epitope retrieval in a citratebuffer solution. Detection was done using the VentanaNexus automated immunostainer (Ventana Medical Sys-tems) with a diaminobenzidine chromogen and hematox-ylin counterstain. Appropriate tissue controlswere includedin each run. Hematoxylin and eosin stains were done formorphologic assessment. The immunostains were scoredby a board-certified pathologist (HM) in a blindedmanner,with extent of staining being graded from0 to 4 on the basisof percentage of positive cells: 0–no positive cells, 1–lessthan 25% positive, 2–26% to 50% positive, 3–51% to 75%positive, and 4–76% to 100% positive.

ImmunoblottingSubmitted as Supplementary Materials.

Proliferation assaysA total of 1,500 cells were plated in triplicate in tissue

culture treated 96-well plates and allowed to proliferate for5 days. Cell proliferationwasmeasured on days 1 to 5 using

Translational Relevance

Polycomb group (PcG) proteins have been implicatedin mediating stem cell pluripotency and aggressive phe-notype of a variety of human cancers. In this study, wesought to evaluate the frequency and clinical relevance ofPcG protein expression in pleural mesotheliomas. Ouranalysis revealed that EZH2, a core component of poly-comb repressor complex-2 (PRC-2), is overexpressed inthe majority of pleural mesotheliomas, correlating withdecreased patient survival. Inhibition of PRC-2 expres-sion by knockdown or pharmacologic techniques sig-nificantly inhibited proliferation, migration, clonogeni-city, and tumorigenicity of pleural mesothelioma cells.Collectively, these data show that aberrant PcG proteinexpression contributes to the pathogenesis of mesothe-liomas and suggest that targeting PRC-2 expression/activity may be a novel strategy for the treatment andpossible prevention of these neoplasms.

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Clin Cancer Res; 18(1) January 1, 2012 Clinical Cancer Research78




the CellTiter 96 Aqueous Non-Radioactive Cell Prolifera-tion Assay (Promega). Each experiment was conducted 3times.

Cell migration assaysCell lines were plated in triplicate to near confluent

monolayers in 6-well plates and migration assessed asdescribed (18). For 3-deazaneplanocin A (DZNep) experi-ments, DZNep at indicated concentrations was added at thetimeof the scratch and then daily until the conclusion of theexperiment. Each experiment was conducted 3 times.

In vitro DZNep treatmentDZNepwas dissolved inwater to a stock concentration of

10 mmol/L and subsequently diluted to experimental con-centrations in complete RPMI media. Cells were plated inappropriate tissue culture dishes; the following day, mediawere replacedwith fresh completemediaor completemediacontaining DZNep at the appropriate concentrations.Media were changed daily with fresh DZNep for the 3-daytreatment.

Soft agar clonogenicitySubmitted as Supplementary Materials.

Murine xenograft experimentsSubmitted as Supplementary Materials.

Superarray analysesSubmitted as Supplementary Materials.

Gene set enrichment analysisSubmitted as Supplementary Materials.

Statistical analysisSEM is indicated by bars on all figures and was calculated

using Microsoft Office Excel 2007. All experiments weredone with at a minimum of triplicate samples, and all Pvalues were calculated with 2-tailed t tests, unless otherwiseindicated.

Results

Isolation and characterization of NCI cell linesNCI-SB-NMES1 and NCI-SB-NMES2 (NMES1 and 2)

were isolated from histologically normal pleura from a78-year-old female smoker and a 36-year-old female non-smoker, respectively, who underwent thoracotomy for earlystage non-small cell lung cancer at the NCI. These lines arecapable of growth in tissue culture for approximately 20 to30 passages before senescence. NCI-SB-MES1-4 (MES1-4)cell lines were established from histologically confirmedMPM resected at the NCI. MES1, 2 and 4 were derived fromepithelial tumors, whereas MES3 was established frombiphasic MPM. No patient underwent neoadjuvant chemo-therapy or radiation therapy prior to resection and isolationof tumor cell lines. Demographic data pertaining to thepatients from whom these lines were established, as well as

molecular phenotypes of the lines are summarized in Sup-plementary Table S2. The photomicrographic appearancesof NMES1 and 2 and MES1-4 are similar to those corre-sponding to commercially available normal mesothelialcell cultures, LP3 and LP9, and MPM cell lines H28,H2052, and H2452, respectively (Supplementary Fig. S1Aand B).

Overexpression of EZH2 and EED in MPM cellsAffymetrix U133 2.0 plus microarrays were used to

examine global gene expression profiles, and specificallyidentify genes encoding PcG proteins in MES1-4, H28,H2052, and H2452 MPM cells relative to LP9 andNMES1. Consistent with previous studies pertaining togene expression analysis of MPM (11), unsupervisedhierarchical cluster analysis of triplicate samples for eachcell line showed that gene expression profiles in MPMlines were distinctly different than those observed inNMES1 and LP9 cells (Fig. 1A). Interestingly, MPM linesexhibited overexpression of EZH2 (also known as KMT6)and, to a lesser extent, EED and SUZ12, which encodecore components of polycomb repressor complex-2(PRC-2), recently implicated in the pathogenesis of avariety of human cancers (ref. 15; Fig. 1B). qRT-PCRexperiments were undertaken to further examine EZH2and EED expression in MPM cells; primers for EZH2recognized both splice variants. This analysis confirmedthat EZH2 and EED were overexpressed in MPM linesrelative to cultured normal mesothelial cells (Fig. 1C).Subsequent qRT-PCR experiments revealed that bothEZH2 splice variants were coordinately expressed at com-parable levels in MPM cells (data not shown). Immuno-blot analysis (Fig. 1D) showed that EZH2 protein levels incultured MPM cells were higher than those observed innormal mesothelial cells. However, EED levels did notseem to be increased in MPM cells. Additional immuno-blot experiments revealed a global increase in the PRC-2–mediated repressive chromatin mark, H3K27Me3 in MPMcells (Fig. 1D).

Analysis of EZH2 and EED expression in primarymesotheliomas

qRT-PCR experiments were done to examine expressionofEZH2 andEED in apanel of primaryMPMs resected at theNCI. Interestingly, this analysis revealed upregulation ofEZH2, but not EED, in 5 of 6 primary epithelial MPMrelative to normal pleura (Fig. 2A). Immunohistochemistry(IHC) experiments were undertaken to further examineEZH2 expression in these 6 specimens as well as 14 addi-tional MPM resected at the NCI. This analysis revealed nodetectable EZH2 expression in normal pleura or stroma; aspectrum of EZH2 expression was detected in mesothelio-ma cells (Fig. 2B). In general, EZH2 levels tended to coincidewith mRNA copy numbers, although some variations werenoted, suggesting that posttranscriptional mechanisms alsocontribute to EZH2 overexpression in MPM. Furthermore,EZH2 expression coincided with mRNA copy numbers inMES1-4 cells and correlated with tumors from which the

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lines were derived; a gradient of EZH2 expression observedin these primaryMPM specimens wasmaintained when therespective cell lines were passaged in vitro (SupplementaryFig. S2).Overall, 90%of the 20primaryMPM(16 epithelial,3 biphasic, and one sarcomatoid) specimens expressedEZH2 (Fig. 2C). Additional IHC experiments using TMArevealed that approximately 85% of 45 mesotheliomas (28MPM and 17 PMs) expressed EZH2; no EZH2 expressionwas observed in 12normal pleura or peritoneum specimensor stromal elements (Fig. 2D). The patterns of EZH2 expres-sion in PM were comparable with those observed in MPM(Supplementary Fig. S3).

Additional analysiswas done to ascertainwhether levels ofEZH2 protein expression by IHC coincided with tumorburden (T stage) using 20 NCI samples plus 27 of 28 MPMsfrom the commercial TMA for which T stage was available.Themajority of theMPMs on the TMAwere epithelial. EZH2expression was significantly higher in MPM relative to nor-mal pleura (P < 0001). Although EZH2 expression tended toincrease with disease burden, this trend was not statisticallysignificant (Z ¼ 0.5; Jonckheere–Terpstra test), possiblybecause of the small number of samples evaluated (Fig. 3A).

Additional analysis was undertaken to ascertain whetherintratumoral EZH2 expression detected by Illumina array

Figure 1. Components of PRC-2 are overexpressed in mesothelioma cells. A, heat map showing that gene expression profiles of mesothelioma cells aredistinctly different from cultured normal mesothelia. B, microarray results depicted as fold upregulation of EZH2, EED, and SUZ12 in cultured MPM linesrelative to normal mesothelial cells. C, qRT-PCR analysis of EZH2 and EED expression inMPMcell lines relative to cultured normal pleura ormesothelial cells.D, immunoblot of EZH2, EED, and H3K27Me3 expression in MPM lines relative to cultured normal mesothelia. Upregulation of EZH2 coincided with a globalincrease in H3K27Me3 levels in MPM cells.

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Figure2. Analysis ofEZH2andEEDexpression in primarymesotheliomaspecimensandnormalmesothelia. A, qRT-PCRanalysis ofEZH2andEED expressionin 6 primary mesothelioma specimens including 4 from which MES1-4 cell lines were derived, compared with normal pleura samples. EZH2 levels weresignificantly higher in primary mesotheliomas relative to normal pleura (P < 0.05). This phenomenon was not evident following analysis of EED expression inthesespecimens.B, representative results of immunohistochemical analysis of EZH2expression in normal pleura andprimaryMPM.C, immunohistochemicalanalysis of 20 primaryMPMspecimens resected at theNCI, including tumors fromwhichMES1-4 cell lineswere derived relative to normal pleura. Themajorityofmesothelioma specimens expressedEZH2; noEZH2 expressionwas detectable in normal pleura. D, TMAanalysis of EZH2 expression inMPM, PM, normalpleura, or peritoneum.






techniques correlated with survival in 39 patients withlocally advanced MPM undergoing potentially curativeresections at Brigham and Women’s Hospital (De Rienzo

and colleagues, submitted); 24 patients had epithelialmesotheliomas, whereas 7 patients had biphasic and8 patients had sarcomatoid malignancies. Increased

Figure 3. EZH2 expression in primaryMPMs relative toT stage andsurvival ofmesotheliomapatients. A, EZH2expression by IHCanalysis relative to Tstage.B,correlation between EZH2 expression in primaryMPM assessed by Illuminamicroarray techniques and patient survival. Overexpression of either of the EZH2spice variants coincides with diminished survival. Variant 1: median ¼ 1.62; top quartile: 2.94 � 0.85; bottom quartile: 1.14 � 0.15; P ¼ 2.96e-5. Variant 2:median¼2.14; topquartile: 4.05�0.81; bottomquartile: 1.53�2.44;P¼5.46e-7.C, analysis ofmicro-RNAexpression inMPMandnormal pleura specimens.RNU-44 was used as endogenous normal control for all experiments. miR-101 and miR-26a levels are reduced in MPM specimens relative to normal pleura.

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Figure 4. Effects of stable knockdownofEZH2 andEED in culturedmesothelioma cells. �,P < 0.05; ��,P < 0.01; ��,P < 0.001. A, immunoblot analysis of EZH2,EED, andH3K27Me3 levels inMPM lines following transductionwith shRNA targetingEZH2,EED, or shamsequences.Knockdownof EZH2 (left) or EED (right)diminishes H3K27Me3 levels in these cells. B, left, effects of knockdown of EZH2 and EED on proliferation of MES1 and H28 cells. The effects of EEDknockdownseem to bemore pronounced in both of these cells. Similar resultswere observed forMES2 andH2452 cells. Right, effects of knockdownofEZH2andEEDonmigrationofMES1andH28cells.EZH2andEEDknockdown inhibitsmigration ofMES1andH28cells. Similar resultswereobserved forMES2andH2452 cells. C, effects of knockdown of EZH2 and EED on soft agar clonogenicity of MES1and H2452 cells. EZH2 and, to a greater extent, EED knockdownsignificantly inhibits soft agar clonogenicity of MPM cells. D, top, effects of EZH2 knockdown on tumorigenicity of MES1 cells in nude mice. Knockdown ofEZH2mediates a modest increase in time to tumor take, and a significant decrease in average tumor volume and tumor mass of these xenografts. Bottom,effects of knockdownofEEDon tumorigenicity ofMES1 xenografts in nudemice. KnockdownofEED increases time to tumor take and significantly decreasestumor volume and tumor mass. Results depicted are representative of 2 independent experiments for each condition.






expression of either of the EZH2 splice variants in MPMcorrelated significantly with shorter patient survival(Fig. 3B). The magnitude of EZH2 overexpression didnot seem to correlate with histology. The sample sizeprecluded multivariate analysis to determine whetherEZH2 expression was an independent prognosticator ofsurvival in MPM patients.

Association of EZH2 with miR-101 and miR-26expression in MPM cells and primary MPM specimens

Recent studies have shown that overexpression of EZH2in several human cancers coincides with loss of miR-101or miR-26, which normally target the 30-untranslatedregion of EZH2 (19, 20). As such, qPCR experimentswere conducted to examine miR-101 and miR-26 expres-sion levels relative to EZH2 expression in primary MPMspecimens and normal pleura (Fig. 3C). These experi-ments showed that miR-101 levels were significantlyreduced in MPM cell lines and pleural mesotheliomaspecimens relative to normal pleura. Furthermore, miR-26a levels were decreased in primary MPM specimensrelative to normal pleura. In contrast, levels of miR-26b(which does not target EZH2) in MPM and normal pleurawere similar, suggesting that the alterations in miR-101and miR-26a levels observed in this preliminary analysiswere not attributable to a global decrease in miRNAexpression (Fig. 3C).

Effects of EZH2 or EED knockdown in MPM cellsAdditional experiments were carried out to examine

whether aberrant PRC-2 activity directly contributes to themalignant phenotypeofmesothelioma cells. Briefly, shRNAtechniques were used to knockdown EZH2 in culturedMPM cells; the shRNA used for these experiments targetedboth splice variants of EZH2. Similar experiments wereundertaken to knockdown EED, which although not appar-ently overexpressed in primary MPM, is critical for main-taining stability of PRC-2 and histone methyltransferaseactivity of EZH2 (15). Immunoblot analysis confirmed thatrelative to control sequences, shRNAs depleted EZH2 andEED and decreased global levels of H3K27Me3 in MES1-2,H28, and H2452 (Fig. 4A). Furthermore, knockdown ofthese PRC-2 components resulted in 30% to 50% decreasesin proliferation and migration of these 4 MPM lines (Fig.4B). Interestingly, the effects of EED knockdown on globalH3K27Me3 levels, as well as proliferation and migration,seemed to be more pronounced than EZH2 knockdown inMPM cells. Experiments using different shRNAs targetingEZH2 and EED confirmed that knockdown of these PRC-2components inhibited proliferation of MPM cells, suggest-ing that the aforementioned results were not due tooff-target effects of the shRNAs; the extent of growth inhi-bition mediated by shRNAs targeting EZH2 or EED seemedto correlate with efficiency of knockdown (SupplementaryFig. S4).

Additional experiments were carried out to ascertainwhether knockdown of EZH2 or EED inhibited clono-genicity and tumorigenicity of MPM cells. Knockdown of

EZH2 or EED diminished soft agar clonogenicity of MES1cells by 36% and 66%, respectively, relative to controls.Similarly, knockdown of EZH2 and EED decreased clo-nogenicity of H2452 cells by 40% and 57%, respectively(Fig. 4C). Subsequent experiments revealed that knock-down of EZH2 or EED modestly delayed tumor take andsignificantly decreased size and volume of subcutaneousMES1 xenografts in nude mice (Fig. 4D). Consistent withresults from immunoblot, proliferation, and migrationexperiments, the effects of knockdown of EED on clono-genicity and tumorigenicity were more pronounced thanthose observed following EZH2 knockdown in MPMcells.

Effects of DZNep in MPM cellsAdditional experiments were carried out to ascertain

whether pharmacologic agents in early clinical develop-ment could recapitulate the effects of EZH2 or EEDknockdown in MPM cells. Our analysis focused onDZNep, an S-adenosylhomocysteine hydrolase inhibitorthat has been shown to decrease DNA methylation,deplete PRC-2 components, and mediate cell-cycle arrestand apoptosis in cancer cells (21, 22). Preliminary experi-ments revealed that DZNep inhibited proliferation ofMPM cells, with the effects tending to plateau off at highdoses, suggestive of cytostasis rather than cell death(Supplementary Fig. S5A); consistent with these findings,Apo-BrdU analysis revealed that the growth inhibitoryeffects of DZNep (as well as EZH2 or EED knockdowns)were not associated with increased apoptosis (data notshown). Interestingly, cultured normal mesothelial cells(LP9), which exhibit very low level EZH2 expression,seemed relatively resistant to DZNep (SupplementaryFig. S2 and S5A). The growth inhibitory effects of DZNepdid not seem to coincide with global DNA demethylation,as evidenced by pyrosequencing of repetitive DNAsequences (representative results pertaining to NBL2 aresummarized in Supplementary Fig. S5B). Immunoblotexperiments revealed that 72-hour DZNep treatmentmediated dose-dependent depletion of EZH2, EED, andH3K27Me3 in MES1-2, H28, and H2452 cells (Fig. 5A;bottom panel); these effects coincided with significantlydecreased proliferation and migration of MPM cells (Fig.5A; top panel, and Fig. 5B). Furthermore, DZNep signif-icantly diminished soft agar clonogenicity of MES1 andH2452 cells (Fig. 5C).

Additional experiments were carried out to examinewhether DZNep could inhibit growth of establishedMPM xenografts. Preliminary experiments showed thatthe maximum tolerated dose of DZNep administeredintraperitoneally in nude mice with tumor xenograftswas 2.5 mg/kg twice a day (data not shown). In subse-quent experiments, nude mice were injected subcutane-ously with MES1 cells sufficient to produce 100% tumortake at 7 days. Commencing on day 7, mice received 3cycles of DZNep (2.5 mg/kg twice a day qd x3 q7d).This treatment regimen resulted in significant reductionof tumor size after each treatment cycle, with an

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approximate 50% reduction in tumor mass at the end ofthe treatment course (Fig. 5D) and no visible systemictoxicity.

Inhibition of PRC-2 activity modulates tumorsuppressor gene expressionBecause PcG proteins have been implicated in regulating

expression of genes modulating cell-cycle progression, dif-ferentiation, and stemness (15, 23), focused SuperArray

techniques were used to examine whether selective deple-tion of EZH2 and EED, or DZNep treatment modulatedexpression of oncogene and tumor suppressors as well asstem cell–related genes in MES1 and H28 cells. Thispreliminary analysis showed a variety of genes regulatingcell-cycle progression and apoptosis, which were modu-lated by knockdown and/or DZNep treatment, severalof which were selected for further analysis (data notshown). Subsequent qRT-PCR experiments confirmed

Figure 5. Effects of DZNep in MPM cells. A, DZNep mediates dose-dependent reductions in EED, EZH2, and global H3K27Me3 levels, which coincide withinhibition of proliferation of MES1 and H28 cells. Similar results were observed for MES2 and H2452 cells. B, effects of DZNep on migration of MPM cells.Similar resultswere observed forMES2 andH2452 cells. C, effects of DZNep on soft agar clonogenicity ofMES1 andH2452 cells. D, effects of intraperitonealadministration of DZNep on growth of established MES1 xenografts in nude mice.






upregulation of NF1, FOXD3, FHIT, HIC1, p21, andRASSF1A in MES1 and/or H28 cells following knock-down of EZH2 or EED (Supplementary Table S3). Ingeneral, the magnitude of induction of these tumorsuppressor genes was more pronounced in EED knock-down cells relative to EZH2 knockdowns. DZNep upre-gulated FHIT, HIC1, p21, and RASSF1A, but not NF1 orFOXD3 in MPM cells, suggesting that DZNep mediatesoverlapping as well as differential effects relative to EZH2or EED knockdown in these cells. Immunoblot analysisconfirmed results of qRT-PCR experiments (representa-tive data pertaining to p21 and RASSF1A are depicted inSupplementary Fig. S6). Collectively, these data suggestthat the growth inhibitory effects of targeted disruption ofPRC-2 or DZNep treatment in MPM cells are mediated, atleast in part, by upregulation of several tumor suppressorsthat are known to modulate pluripotency, cell-cycle pro-gression, and apoptosis in cancer cells.

Chromatin immunoprecipitation (ChIP) experimentswere undertaken to ascertain whether activation of tumorsuppressor genes in MES1 and H28 cells by DZNep wasattributable to depletion of PRC-2, as evidenced bydecreased H3K27Me3 levels within the respective promo-ters. As shown in Fig. 6A, H3K27Me3 levels within thepromoters of RASSFIA and HIC-1 (putative stem cellpolycomb target genes; ref. 24) were markedly decreasedin cells exhibiting knockdown of EZH2 or EED, relative tocontrol cells. Similarly, treatment with DZNep decreasedH3K27Me3 levels within the RASSFIA and HIC-1 promo-ters. A similar phenomenon was observed following anal-

ysis of p21, suggesting that the growth inhibitory effects ofDZNep were not restricted to epigenetic activation of stemcell polycomb targets.

Illumina array experiments were carried out to examineeffects of DZNep on global gene expression in MES1 andH28 cells. Using criteria of more than 1.5-fold change andP < 0.05 for drug treatment relative to untreated controls,940 and 513 genes weremodulated byDZNep inMES1 andH28, respectively, of which 122 overlapped. Using criteriaof more than 1.5-fold change and more stringent adjustedP values (25), 830 and 435 genes were differentially mod-ulated by DZNep in MES1 and H28, respectively (Fig. 6B);55% of differentially expressed genes were upregulated byDZNep. Only 11 genes were significantly modulated inboth cell lines by DZNep, of which only 3 were simulta-neously up- or downregulated (Supplementary Table S4),suggesting significant differences in underlying histology orgenotype. Gene set enrichment analysis revealed significantenrichment of EZH2 gene set in MES1, but not H28 cellsfollowing DZNep treatment (Table 1). A variety of addi-tional gene sets and ontologies pertaining to proliferationand signal transduction were modulated in these cells(Supplementary Tables S5 and S6).

Discussion

PcG proteins have emerged as critical epigenetic med-iators of pluripotency and differentiation of stem cells(26, 27), as well as aberrant gene repression duringmalignant transformation (15, 28). Two major PRCs,

Figure 6. Effects of DZNep on geneexpression in MPM cells. A, ChIPanalysis of H3K27Me3 levels inRASSF1A, HIC-1, and p21promoters in MES1 and H28 cellsfollowing knockdown of EZH2 orEED, or DZNep treatment. B, Venndiagram of DZNep-modulatedgenes inMES1 and H28 cells usingmore than 1.5-fold and adjusted P< 0.05. Very little overlap wasobserved between these lines.

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each of which contains a variety of core subunits havebeen identified in mammals. The initiation complex,PRC-2, containing EZH2, SUZ12, PCL, and EED subunits,mediates trimethylation of H3K27. PRC-2 recruits themaintenance complex PRC-1, containing core subunitsof PCAF, PHC, RING1, CBX, and SML, as well as SFMBTand L3MBTL proteins that mediate ubiquitination ofH2AK119 (15). These histone marks coincide withrecruitment of chromatin remodeling complexes, forma-tion of heterochromatin, and repression of gene expres-sion by mechanisms, which to date have not been fullyelucidated (29, 30). Of particular relevance with regard tothe role of PcG proteins in tumorigenesis are recentobservations that PRC-2 inhibits differentiation of nor-mal pluripotent stem cells (31, 32) and that EZH2 isincreased and essential for maintenance of cancer stemcells (33, 34).In this study, we observed overexpression of EZH2 in the

majority of cultured MPM lines and primary MPM relativeto normal mesothelial cells or pleura. Interestingly, TMA

experiments also showed overexpression of EZH2 in PM,suggesting that common epigenetic pathways mediate thepathogenesis of pleural and PMs. Overexpression of EZH2tended to coincide with tumor burden and correlated withdecreased survival of patients with these neoplasms; thesefindings are consistent with recent studies showing thatoverexpression of EZH2 in a variety of humanmalignanciesincluding breast, lung, and esophageal cancers frequentlycorrelates with aggressive tumor phenotypes, advancedstage of disease, and decreased patient survival (35–37).Knockdown of EZH2 or EED significantly inhibited themalignant phenotype of mesothelioma cells in vitro and invivo. To the best of our knowledge, these studies are the firstto show aberrant expression of PcG proteins in mesotheli-omas, and the potential relevance of targeting PRC-2 for thetreatment of these malignancies.

Our analysis revealed a spectrumof EZH2 overexpressionin pleuralmesotheliomas. Variability in EZH2mRNA levelsinMPMcould be attributed to stromal elements, which lackEZH2 expression, as these tumors were not microdissected.

Table 1. EZH2 targets upregulated in MES1 following DZNep treatment

Symbol logFC adj.P.Val Symbol logFC adj.P.Val

ADM 3.15 0BDKRB1 0.752 0 MT1A 1.33 0CCL20 2.24 0 MT1X 1.57 0CD44 0.711 0 MT2A 1.88 0CLEC2B 0.959 0 NLRP3 0.587 0CORO1C 0.668 0 NOV 1.34 0CXCL2 1.14 0 PI3 2.09 0DCUN1D3 0.684 0 PLAT 0.952 0DNER 0.845 0 PRNP 0.79 0ENTPD7 0.631 0 PTGS2 1.59 0FAM46A 0.843 0 PTPRR 1.11 0FRMD6 0.724 0 RGS4 1.08 0FTHL7 1.19 0 SAA1 1.494 0GDF15 2.9 0 SAMD9 0.826 0HMOX1 1.11 0 SERPINE1 1.44 0IGFBP3 1.98 0 SERPINE2 1.1 0IL-1B 1.44 0 SERTAD4 0.98 0IL-24 2.1 0 SLIT2 1.04 0IL-6 3.01 0 SP140 0.975 0IL-8 3.89 0 SQSTM1 1.18 0KRT34 4.42 0 THBS1 0.91 0LAMC2 1.18 0 TMEM45A 0.684 0MGLL 0.589 0 TP53BP2 0.597 0

Dataset Ttest_MES1_nm_Dznep_1p5_adjp05Upregulated in class na_posGeneSet NUYTTEN_EZH2_TARGETS_UPEnrichment Score (ES) 0.4477096Normalized Enrichment Score (NES) 2.3391728Nominal P 0FDR Q 0.001003521FWER P 0.007






However, pathologic evaluation confirmedmore than 70%tumor cells in specimens submitted for analysis. Moreimportantly, immunostaining showed a range of percentand intensity of EZH2 expression in mesothelioma cells invivo. Furthermore, patterns of EZH2 expression in primaryMPMswere retained in cell lines derived from these tumors,suggesting that differences in EZH2 expression are due tospecific genetic/epigenetic factors, which vary amongmeso-theliomas of similar histologies. Presently, themechanismsunderlying this phenomenon remain unclear. Themajorityof MPM exhibit disruption of Rb as well as p53-mediatedpathways because of allelic loss or epigenetic silencing ofp16/p14 (4, 5). A number of PcG genes including EZH2 arepotential E2F targets (38). Furthermore, EZH2 expression isnegatively regulated by p53 (39), as well as several miRNAsincluding miR-101 and miR-26a (19, 20). Our preliminaryanalysis indicated that miR-101 and miR-26a are down-regulated in primary MPM relative to normal pleura, sug-gesting that dysregulation of miRNAs also contributes tooverexpression of EZH2 in these neoplasms. Studies areunderway to delineate genetic and epigenetic mechanismsregulating EZH2 expression in normal mesothelia andMPM and to further define the prognostic significance ofEZH2 overexpression in these malignancies.

Although EED has been extensively studied in stem cells(40, 41), the role of this PRC-2 component in cancer has notbeen fully defined. EED is necessary for di- and trimethyla-tion of H3K27 and PRC-2–mediated gene silencing inmurine embryonic stem cells (42). Direct binding of theWD40 domain of EED is critical for histonemethyltransfer-ase activity of the PRC-2 complex and placement of therepressive mark H3K27Me3 in murine embryonic fibro-blasts and Drosophila melanogaster embryos (43, 44). EEDspecifically binds to H3K27Me3 and is necessary for prop-agation of this repressive mark during cell division. Inter-estingly, the effects of EED depletion seemed to be moreprofound than those seen following EZH2 depletion inMPM cells. These results may simply be due to relativeknockdown efficiencies; alternatively, theymay reflect com-pensation of EZH2 depletion by EZH1 (45) andmagnitudeof destabilization of PRC-2 by loss of EED.

Originally developed as an antiviral drug, DZNep hasemerged as a novel cancer therapeutic agent (21). Thisnucleoside analogue induces proteolytic degradation ofEZH2 and other PRC-2 components and modest globalDNA demethylation (22, 46). Tumor-initiating cells seemexquisitively sensitive to DZNep because of the critical role

of PcG proteins in maintenance of cancer stem cells (34).Furthermore, DZNep induces differentiation of cancerstem cells by caspase-dependent degradation of NANOGandOCT4 (47). Our analysis revealed that DZNep depletedEZH2, EED, and H3K27Me3 and upregulated severaltumor suppressor genes, including p21 and RASSF1A,which were also activated following knockdown of EZH2or EED in MPM cells, suggesting that the cytotoxic effects ofDZNep are attributable, at least in part, to inhibition ofPRC-2 activity. Consistent with this notion, DZNep medi-ated growth inhibition without apoptosis in MPM cells;similar results were observed following knockdown ofEZH2 and EED in these cells. These findings are in accor-dance with recent observations that knockdown of EZH2induces cellular senescence in melanoma cells in partby upregulation of p21 (48) and delays G2/M transitionwithout inducing apoptosis in ER-negative breast cancercells (49).

On the other hand, our current findings do not conclu-sively implicate depletion of PRC-2 as the major mecha-nism bywhichDZNep inhibits themalignant phenotype ofpleural mesothelioma cells. Indeed, our highly stringentmicroarray analysis suggests that DZNep mediates pleio-tropic effects inmesotheliomas, whichmay vary dependingon histologic subtype or genotype of these neoplasms.Studies are in progress to further evaluate genomicresponses to DZNep in additional cell lines recently estab-lished from mesotheliomas of confirmed histologies. Fur-thermore, experiments are underway to ascertain whetherconstitutive expression of EZH2 and/or EED abrogatesDZNep-mediated cytotoxicity in MPM cells. Whereas theprecise mechanisms by which DZNep mediates growtharrest in mesothelioma cells have not as yet been fullyelucidated, our findings warrant further analysis of PcGprotein expression in mesotheliomas and the developmentof agents targeting PRC-2 expression/activity for treatmentof these malignancies.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedApril 13, 2011; revised September 23, 2011; acceptedOctober 12,2011; published OnlineFirst October 25, 2011.

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2012;18:77-90. Published OnlineFirst October 25, 2011.Clin Cancer Res Clinton D. Kemp, Mahadev Rao, Sichuan Xi, et al. TherapyPolycomb Repressor Complex-2 Is a Novel Target for Mesothelioma

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