chromatin, gene, and rna regulation · mol cancer res; 12(10); 1388–97. 2014 aacr. introduction...

Chromatin, Gene, and RNA Regulation

EZH2 Represses Target Genes through H3K27-Dependentand H3K27-Independent Mechanisms in HepatocellularCarcinoma

Shu-Bin Gao1,2, Qi-Fan Zheng1,2, Bin Xu1,2, Chang-Bao Pan1, Kang-Li Li1, Yue Zhao1, Qi-Lin Zheng1,Xiao Lin1,2, Li-Xiang Xue3, and Guang-Hui Jin1,2

AbstractAlterations of polycomb group (PcG) genes directlymodulate the trimethylation of histoneH3 lysine 27 (H3K27me3)

and may thus affect the epigenome of hepatocellular carcinoma (HCC), which is crucial for controlling the HCC cellphenotype. However, the extent of downstream regulation by PcGs in HCC is not well defined. Using cDNA micro-array analysis, we found that the target gene network of PcGs contains well-established genes, such as cyclin-dependentkinase inhibitors (CDKN2A), and genes that were previously undescribed for their regulation by PcG, including E2F1,NOTCH2, and TP53. Using chromatin immunoprecipitation assays, we demonstrated that EZH2 occupancy coincideswith H3K27me3 at E2F1 and NOTCH2 promoters. Interestingly, PcG repress the expression of the typical tumorsuppressor TP53 in human HCC cells, and an increased level of PcG was correlated with the downregulation of TP53in certainHCC specimens. Unexpectedly, we did not find obviousH3K27me3modification or an EZH2 binding signalat the TP53 promoters, suggesting that PcG regulates TP53 expression in an H3K27me3-independent manner. Finally,the reduced expression of PcGs effectively blocked the aggressive signature of liver cancer cells in vitro and in vivo.

Implications: Taken together, our results establish the functional and mechanistic significance of certain generegulatory networks that are regulated by PcGs in HCC.

Visual Overview: http://mcr.aacrjournals.org/content/12/10/1388/F1.large.jpg.Mol Cancer Res; 12(10); 1388–97. �2014 AACR.

IntroductionPolycomb group (PcG) proteins are highly conserved

epigenetic gene silencers that play important roles in themaintenance of cell fates, developmental programs, andtumorigenesis (1). PcG proteins bind to specific regions ofgene promoters and direct posttranslational modifications atcertain histone sites, thereby silencing gene expression (2–5).In mammals, PcG is made up of at least two multiproteincomplexes: PRC1 (polycomb repressive complex 1) andPRC2. These complexes work together to carry out theirrepressive effect (1). PRC2 is the core of PcG in humans,which consists of the proteins enhancer of zeste homolog 2(EZH2), suppressor of zeste 12 (SUZ12), and embryonicectoderm development (EED; ref. 1). PRC1, the mainte-

nance complex, includes B-lymphoma moloney murineleukemia virus insertion region-1 (BMI1), chromoboxhomolog 8 (CBX8), and others (1–3). The EZH2 SET(suppressor of variegation, enhancer of zeste, and trithorax)domain specifically catalyzes the trimethylation of Histone3 lysine 27 (H3K27me3). Methylated H3K27 is recognizedby other specific binding proteins, such as PRC1 compo-nent, which leads to the compression of chromatin structureand the repression of gene transcription (1–3). In addition,some PRC1 complexes can regulate gene expressionby monoubiquitylation at Lys 119 of histone H2A(H2AK119ub) via the ubiquitin ligases RING1A (ringfinger protein) and RING1B (3).Hepatocellular carcinoma (HCC) is the most common

type of liver cancer and the third most common cause ofcancer mortality worldwide (6). EZH2 is a useful biomarkerof the aggressive stages of HCC, and its overexpression isassociated with the malignant progression of HCC (7). Thesame study reported a high level of global H3K27me3 inHCC, which is closely correlated with the vascular invasionof HCC (8). LncRNA-HEIH is an oncogenic long-non-coding RNA that promotes HCC progression by upregulat-ing the expression of EZH2 in humans (9). Several micro-RNAs, such as the putative tumor suppressor microRNA-124, repress the expression of EZH2. Dysregulation of thesemicroRNAs could contribute to EZH2 overexpression incancer (10). Inversely, EZH2 exerts its oncogenic function

1Department of Basic Medical Sciences, Medical College, Xiamen Univer-sity, Xiamen, P.R. China. 2Fujian Provincial Key Laboratory of Chronic LiverDisease and Hepatocellular Carcinoma, Xiamen University, Xiamen, P.R.China. 3Department of Biochemistry and Molecular Biology, Health Sci-ence Center, Peking University, Beijing, P.R. China.

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

Corresponding Author: Guang-Hui Jin, Xiamen University, Xiang'anSouth Road, Xiamen, Fujian, P.R. China 361102. Phone: þ86-592-218-6173; Fax: þ86-592-218-1535; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-14-0034

�2014 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 12(10) October 20141388

on May 28, 2021. © 2014 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst June 10, 2014; DOI: 10.1158/1541-7786.MCR-14-0034

http://mcr.aacrjournals.org/

by way of epigenetic silencing of well-characterized tumorsuppressor miRNAs, such as miR139-5p, miR125b,miR101, let-7c, and miR200b (11). These findings providesignificant insights into the molecular mechanisms of PcGinvolvement in hepatocellular carcinogenesis.Tumor protein p53 (TP53), a typical tumor suppressor, is

a major genetic player in the development of various cancers,including HCC (12). The aberrant expression of EZH2 hasbeen associated with P53 alteration and contributed toprogression and pool prognosis in human squamous cellcarcinoma of the esophagus (13). Activation of TP53 sup-presses EZH2 expression through the repression of theEZH2 gene promoter (14). Inversely, the reduction ofEZH2 expression by RNA interference increases the expres-sion of TP53 (15). These features point to an interestingnegative feedback loop between oncogenic EZH2 andtumor suppressor TP53. Despite the importance of EZH2in controlling the aggressive nature of HCC, the regulationprofile of tumor suppressors by PcG in HCC is not welldefined.In this study, we demonstrate potential tumor-promoting

actions of PcG in HCC by the H3K27me3-dependent andH3K27me3-independent regulation of downstream geneexpression.

Materials and MethodsCell cultureThe human HCC cell line HepG2 (wild-type P53) and

Hep3B (P53 null) were obtained from the Type CultureCollection of the Chinese Academy of Sciences (Shanghai,China). PLC/PRF5 (human HCC with P53 mutant),SMMC-7721 (human HCC with wild-type P53), andHO-8910 (human ovarian cancer cell) were obtained fromthe China Center for Type Culture Collection (Wuhan,China). Wilm's (human nephroblastoma), MCF-7 (humanmammary gland adenocarcinoma), and A549 (human lungadenocarcinoma) were obtained from the Cell CultureCenter of the School of Basic Medicine, Peking UnionMedical College (Beijing, China). The cell cultures wereperformed as previously described (16–19). Briefly, the cellswere cultured in DMEM (Gibco), RPMI-1640 medium(Gibco), or F12 (Gibco) supplemented with 10% FBS(Hyclone) and 1% penicillin–streptomycin.

Chromatin immunoprecipitation assaysChromatin immunoprecipitation (ChIP) assays were per-

formed as previously described (16). In brief, 1 � 106

HepG2 cells were treated with 1% formaldehyde for 10minutes at 37�C;, followed by pulsed ultrasonication toshear cellular DNA. ChIP assays were then carried out withthe indicated antibodies according to the protocol of theChIP Assay Kit (Millipore). After overnight incubation withthe antibodies, protein A- or protein G-agarose beads wereadded. The cross-links between nuclear proteins and geno-mic DNA were reversed, and DNA pulled down by theantibody was purified by phenol/chloroform extraction. Theprimer pair sequences and antibodies for the ChIP assays areshown in Supplementary Tables S1 and S2.

Western blotting and immunofluorescenceWestern blotting and immunofluorescence (IF) staining

were performed as previously described (16). Antibodies arelisted in the Supplementary Table S1.

Real-time qRT-PCRqRT-PCR was performed as previously described (16)

using an ABI Step One detection system with the primerslisted in Supplementary Table S2. qRT-PCR reactions wereperformed using SYBR Green (Roche Applied Science)technology and were repeated at least three times.

Cell proliferation assaysCell growth and viability were evaluated by using either

Bromodeoxyuridine (5-bromo-20-deoxyuridine, BrdUrd) or3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoliumbromide (MTT) cell proliferation assays. The cells wereplated at 2� 105 cell/mL in 100 mL/well of DMEMmedia,and the BrdUrd incorporation and detection were per-formed using a cell proliferation ELISA BrdU kit (Millipore)according to the provided protocol. For the MTT assay, thecells were seeded at a density of 1 � 103 cells/well into 96-well plates, and measured as according to the manufacturer'sprotocol (Merck). For BrdUrd or MTT detection, theoptical density at 450- or 570-nm absorbance was measuredby using an ELISA plate reader (Bio-Tek Instruments),respectively. For both assays, the mean absorbance valuesof at least three independent experiments were used forstatistical analysis.

Colony-forming assaysThe colony-forming assays were performed as previously

described (16). In brief, 1,000 cells were seeded in 6-wellplates for 10 days. Colonies were stained with 0.05% crystalviolet, photographed, and counted in triplicate using micro-scope (Nikon Corporation).

Cell migration assayMigration assays were performed, as previously described

(19), in 24-well transwell plates (Millipore) using uncoatedpolycarbonate membranes with 8-mm pores. Serum-starvedcells were harvested and resuspended at a concentration of1 � 104 cells per 0.2 mL in serum-free medium containing10%BSA, and added to the upper chamber. At 24 hour afterincubation, nonmigrated cells were scraped off the upperside of the filter, and filters were stained with 0.1% crystalviolet. The number ofmigrated cells on the reverse side of themembrane was quantified by average cell counts from sixrandom fields in each well, under a microscope at �100magnifications. Each condition was assayed in triplicatewells, and the resulting data were subjected to statisticalanalysis using the Student t test.

Terminal deoxynucleotidyl transferased UTP nick endlabeling assayTerminal deoxynucleotidyl transferased UTP nick end

labeling (TUNEL) assay (Roche Applied Science) was usedto detect apoptotic cells. Apoptotic cells were determined by

Downstream Regulation by PcG in HCC

www.aacrjournals.org Mol Cancer Res; 12(10) October 2014 1389




counting positive-stained cells in five randomly selectedfields in each tumor. At least three tumors in each mousewere randomly selected.

Statistical analysisAll the statistical analyses were performed using SPSS

software version 13.0. Data from cell growth, clonal assays,and cell migration experiments were analyzed by two-tailedStudent t tests. The results for parametric variables areexpressed as the mean � SD or the mean � SEM. In allcases, P < 0.05 was considered statistically significant.

ResultsA role for EZH2 in hepatic cancer cell growth in vitro andin vivoA recent report indicates that knockdown (KD) of EZH2

suppresses HCC tumorigenicity and extrahepatic metastasisin vivo (11); nonetheless, few studies have explored whetherother PcG components are involved in the control of theaggressive phenotype of HCC cells. To explore a potentialfunctional implication of PcG components in HCC, westably transfectedHepG2 cells, which are often used to studythe behavior of HCC cells (16), with either vectors orconstructs expressing one of the 2 to 3 distinct shRNAs

that specifically target EZH2, SUZ12, BMI1, or CBX8.Western blotting results indicate that correlated with thereduction level of EZH2, SUZ12, BMI1, and CBX8, theglobal level of H3K27me3 was remarkably reduced in bothPcG KD HepG2 cells (Supplementary Fig. S1A). TheCBX8–shRNA3 failed to reduce the expression of CBX8and was unable to reduce the global H3K27me3 levels(Supplementary Fig. S1A). IF staining results indicate thatEZH2 was colocalized with SUZ12 and H3K27me3 at thenucleus (Supplementary Fig. S1B). In addition, the stablyectopic expression of EZH2 substantially increased the levelsof SUZ12 and H3K27me3 in HepG2 (Supplementary Fig.S1B). These data indicate that the components of PcGeffectively catalyze the global trimethylation of H3K27 inliver cancer cells. Next, the shRNA-mediated KD of EZH2,SUZ12, CBX8, or BMI1 dramatically suppressed theproliferation of HepG2 cells in vitro (Fig. 1A). Controland EZH2 shRNA cells were synchronized in the G0–G1phase by serum starvation and reentered cell cycle with therestoration of serum culture. Cyclin B1 (CCNB1) proteinexpression was analyzed by Western blotting, which cor-responded to the G2–M checkpoint. We found that bothcontrol and EZH2 shRNA HepG2 cells cultured in aserum-free medium had a relatively low level of CCNB1expression. Following the addition of serum into the

Figure 1. A role for EZH2 in livercancer cell proliferation andmigration. A, the proliferation ratesof HepG2 cells that were stablytransfected with shEZH2,shSUZ12, shBMI1, or shCBX8were estimated using an MTTassay. B, the expression of CCNB1and pCCNB1 was analyzed byWestern blotting in shRNA-mediated KD of EZH2 in HepG2cells at the indicated time points.C and D, the proliferation of HepG2cells transiently transfected withLuc, EZH2, or SUZ12 siRNA wasestimated by BrdUrd (OD450) orMTT (OD570) assay. E, HepG2cells transiently transfected withLuc or EZH2 siRNA were added tothe upper-filter, and 1 � 104 cellmigration was determined bytranswell chamber assay. F,colony-forming assays wereperformed using HepG2 cellstransfected with Luc or EZH2siRNA.

Gao et al.

Mol Cancer Res; 12(10) October 2014 Molecular Cancer Research1390




medium, the protein expression of phospho-CCNB1 dra-matically increased at 4 to 24 hours after serum stimulationin control HepG2 cells (Fig. 1B, lanes 3–7). However, inshEZH2 HepG2 cells, the protein expression levels startedto increase at 6 hours and returned to a level less thanbaseline after 12 hours (Fig. 1B, lanes 11 and 12). Thesedata indicate that EZH2 promotes HepG2 cell prolifera-tion at least partly by accelerating G2–M progress. Con-sistent with this hypothesis, the siRNA-mediated KD ofeither EZH2 or SUZ12 dramatically suppressed the pro-liferation of HepG2 cells at the indicated time point, asdetermined by BrdUrd or MTT incorporation, respectively(Supplementary Fig. S1C; Fig. 1C and D). Because cancermetastasis usually involves enhanced cell migration, wesought to determine whether one of the functions of EZH2is involved in controlling hepatic cancer cell migration. Tothis end, we performed a modified transwell chamberassay to evaluate the impact of altered EZH2 expressionon HCC cell migration. The results indicate that EZH2siRNA KD significantly reduced the extent of migration(Fig. 2E, P < 0.001) and the colony-forming ability ofHepG2 cells (Fig. 2F, P < 0.001).We next set out to explore whether PcGs promote the

growth of HepG2 cell–derived tumors in nude mice. Thesize of the solid tumor was measured after various periodsfollowing transplantation. The shRNA-mediated KD ofEZH2, SUZ12, CBX8, or BMI1 significantly suppressed

tumor growth and weight in subcutaneous xenografts (Fig.2A and B). In addition, the incidence of apoptotic tumorcells, as assessed by the TUNEL assay, was significantlyincreased in EZH2, SUZ12, and CBX8 shRNA HepG2xenografts, with no obvious effect observed for BMI1shRNA (Fig. 2C and D). Collectively, these results stronglyargue that both components of PcG play a crucial role inpromoting liver cancer in vitro and in vivo.

Identification of downstream pathway of PcG in HCCAlthough recent evidence suggests that the abnormal

expression of EZH2 and a high level of global H3K27me3can be attributed to the aggressive nature of HCC (7, 8), theextent of downstream regulation by PcGs inHCC is not welldefined. To obtain a broader understanding of themolecularnetwork of PcG in HCC, cDNA microarray analysis wasperformed on shRNA KDs of EZH2, SUZ12, BMI1, orCBX8 HepG2 cells. Interestingly, the results indicated thatPRC1 (CBX8 and BMI1) and PRC2 (EZH2 and SUZ12)display very different characteristics of gene expressionregulation in HCC. We found genes that are targeted byPRC2 without PRC1 and vice versa (Fig. 3A and Supple-mentary Table S3, GSE 54108). The microarray resultsindicated that EZH2, SUZ12, BMI1, and CBX8 shRNArevealed a significant coregulation of only 61 or 159 genes inHepG2 cells up or down, respectively (Fig. 3B). Figure 3Chighlights the major molecular functions (MF) from the list

Figure 2. The KD of PcGs repressestumor growth inHepG2xenografts.A, the control, shEZH2, shSUZ12,shBMI1, or shCBX8 HepG2 cellswere injected subcutaneously intonude mice. Tumor formation wasexamined on day 6 aftertransplantation. The tumor volumeof recipientmice (N¼ 10 per group)is shown. B, recipient mice weresacrificed on day 30 aftertransplantation; the tumor weightswere measured (N ¼ 10). C and D,the recipient mice were sacrificed30 days after transplantation, andthe apoptotic cells were detectedby TUNEL assay (green) withPropidium Iodide (PI) staining (red)in these tumors on the same day.The number of apoptotic cells wasquantified.






of limbal genes based on the significance of the increase ineither CBX8 or EZH2 shRNA KD (P < 0.05). ATPaseregulator activity, ribonuclease activity, and NADH dehy-drogenase activity–associated genes have the highest signif-icance in both the EZH2 and SUZ12–shRNA microarrayGene Ontology (GO) term analysis. MAP kinase phospha-tase activity, oxygen transporter activity, and oxidoreductaseactivity were involved in both CBX8 and BMI1-shRNAGOterm analysis.

PcG indirectly regulates TP53 expression in HCCInterestingly, the TP53 gene, a tumor suppressor, was

strikingly regulated by both PRC1 and PRC2. Althoughextensive studies have demonstrated that theTP53mutationis a major genetic event for HCC, the transcriptionalregulation of TP53 by critical effectors in HCC has notbeen well defined. It has been reported that the expression ofEZH2 is negatively correlated with the p53 pathway inhuman cancers, suggesting that EZH2 regulates p53 activityor expression (15). We thus turned our attention to theimpact that PcG has on the regulation of TP53 expression.To rule out the effect of the TP53 mutation on its own

expression, we first performed amutation screen of the entireTP53 locus in HepG2 cells. We focused our analysis onnovel changes that were predicted to affect the protein-coding sequence. We discovered only one variation, P72R,which has been described in a previous report. P72R affectsexon 4 of the TP53 gene in HepG2 and is considered aneutral SNP (20). Thus, we investigated the regulatory

relationship between PcG and TP53 using HepG2 cells.The reduction of EZH2 levels by shRNA increased theexpression ofTP53 and its downstream effector P21 levels inHepG2 cells, as determined by qRT-PCR and Westernblotting (Fig. 4A). Similarly, the KD of EZH2 by threedistinct siRNA clearly increased TP53 and P21 mRNAexpression (Fig. 4B). Next, the shRNA-mediated KD ofCBX8 increased the mRNA expression of TP53 and P21 inHepG2 cells (Fig. 4C). TheCBX8–shRNA3 failed to reduceCBX8 expression and was unable to increase the level ofTP53 orP21mRNA (Fig. 4C). In time-course detection, themRNA expression of TP53 was elevated by the substantialKD of EZH2 or CBX8 siRNA at days 2 and 4 in bothHepG2 and SMMC-7721 cells (Supplementary Fig. S2Aand S2B). Furthermore, the KDof SUZ12 also increased theprotein levels of TP53 and P21 in HepG2 cells (Fig. 4D).EZH2 siRNA KD moderately increased the mRNA level ofTP53 in another type of human cancer cell line, lungadenocarcinoma A549 (Fig. 4E). However, we did notobserve an obvious effect of EZH2 on repressing TP53mRNA level in other type tumor cell lines,HO-8910,MCF-7, and Wilms (Supplementary Fig. S2C). This result sug-gests that EZH2 regulates TP53 expression in a tissue-specific manner. Furthermore, inversely correlated with thereduction of PcGs, the mRNA levels of TP53 and CDKN2Awere increased in PcG KD HepG2 xenografts (Fig. 4F).Interestingly, we further found that EZH2 did not regulatethe TP53 mRNA expression in Hep3B with p53-nullbackground and PLC/PRF/5 with p53-mutant background

shLu

c

shEZH2

shSUZ12

shCBX8

shBM

I1

shLu

c

shEZH2

shEZH2

shSUZ12

shSUZ12

A B Csh

CBX8

shCBX8

shBM

I1

shBMI1

shEZH2shSUZ12 shCBX8

shBMI1

WDR75RWDD1

BNIP2CYB5D1

MAL2LYRM4

ATP6V1C1TYW1

PPP2R2BGTPBP4

UBL5ACAT1

IPO5AKAP1GART

RPL26L1RDH14

SRSF10BLOC1S3

VAPBTJP2

HP1BP3NGFRAP1

MKI67IPC13orf27MRPL45

BAMBIFNBP4RAB5BUBE2K

0 5 8Top 200

upregulated genes

Upregulated genes

Downregulated genes

25181

802

1963076354

562 10 739

2013912

22

72

1861

238

148

2157

2048742

305 39 810

247053

26

113159

61

ATPase regulator activitytRNA-specific ribonuclease activity

Structural constituent of ribosomeRNA helicase activityRibonuclease activity

Hydrogen ion transmembrane transporter activity

rRNA bindingRNA-dependent ATPase activity

NADH dehydrogenase (quinone) activity

NADH dehydrogenase (ubiquinone) activityNADH dehydrogenase activity

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

MAP kinase tyrosine/serine/threoninePhosphatase activity

Oxygen transporter activityMAP kinase phosphatase activity

Aromatase activity

Oxidoreductase activityCoreceptor activity

Rab GTPase bindingProtein tyrosine/serine/threonine phosphatase activity

Acting on the aldehyde or oxo group of donorsPhospholipid transporter activity

Insulin receptor binding

Oxygen binding

0 2 4 6 8 10 12 14

Fold enrichment ((Count/Pop.Hits)/(List.Total/Pop.Total))

Dow

nstream of P

RC

1D

ownstream

of PR

C2

Figure 3. Screen of target network of PcG in HepG2. A, expression microarray experiments were performed with control, shEZH2, shSUZ12, shCBX8,and shBMI1 HepG2 cells, and the Venn diagram analysis of target genes regulated by EZH2, SUZ12, BMI1, and CBX8 shRNA in the indicatedcategories. Hierarchical clustering analysis was performed for the top 200 most affected target genes in microarray assays. Each row, a single gene.The microarray data have been deposited in NCBI-GEO under accession numbers GSE 54108 B, Venn diagram analysis of target genes regulatedby EZH2, SUZ12, CBX8, and BMI1 in the indicated categories. C, GO enrichment analysis of the expression microarray assays of PRC2 or PRC1 inHepG2 cells, categorized by MF.

Gao et al.





(Supplementary Fig. S2D). This result suggests that EZH2represses TP53 expression depending on wild-type TP53genomic status.To gain insight into the role of the PcG in regulatingTP53

expression, we performed qRT-PCR analysis on total RNAextracted from HCC liver samples and their matched non-neoplastic counterparts. First, the whole-exome sequencingof TP53 in the primary HCC tissues of 48 patients excludedthe typical TP53 mutation, and we further analyzed themRNA expression of EZH2, SUZ12, CBX8, BMI1, andTP53. These HCCs included cases with wild-typeTP53 (19cases), TP53 with synonymous mutations (three cases),TP53 with the neutral SNP variation (P72R, 21 cases), andTP53 with four previously unreported SNP variations (atotal of five cases; data not shown). Using a large cohort ofHCC specimens (n ¼ 48), we confirm here that theexpression of EZH2, CBX8, BMI1, and SUZ12 were sig-nificantly overexpressed in HCC samples compared withtheir nontumoral counterparts (Fig. 5). In addition, wefound that the TP53mRNA expression levels are decreasedin HCC specimens compared with adjacent tissues (Fig. 5).Spearman analysis performed using qRT-PCR expressiondata indicates that the mRNA expression of TP53 was

negatively correlated with the upregulation of EZH2,CBX8, and BMI1, but not SUZ12 (Fig. 5). This observationfurther suggests a potential role for PcGs in the regulation ofTP53 in HCC.We were interested in whether PcG regulated TP53

transcription through an epigenetic mechanism. Thus, wedesigned ChIP primers for the regions spanning 25-kbupstream and downstream of the transcriptional start siteof the TP53 promoter (Supplementary Fig. S3A) and per-formedChIP assays using control and shEZH2HepG2 cells.IgG and H3-ChIP were used as negative and positivecontrols, respectively (Supplementary Fig. S3B). Unexpect-edly, we did not find any obvious H3K27me3 modificationor an EZH2-binding signal at the promoter loci usingH3K27me3 and EZH2 antibody ChIP assays, respectively(Supplementary Fig. S3C and S3D), suggesting that EZH2regulates TP53 expression through an H3K27me3-inde-pendent mechanism.

PcG directly controls target gene transcription throughH3K27me3In our previous ChIP-on-chip assay (GSE52300), we

screened out the H3K27me3-related target gene network

Figure 4. PcG indirectly regulatesTP53 expression in HCC. A, themRNA and protein levels of EZH2,TP53, and P21 were determined inshLuc and shEZH2HepG2 cells. B,control (siLuc) andeachof the threedistinct siRNAs that specificallytarget EZH2 were transientlytransfected into HepG2, and TP53and P21 mRNA expression wasdetermined by qRT-PCR at 4 daysafter transfection. C, the mRNAexpression of CBX8, TP53, andP21 was detected by qRT-PCR incontrol and shCBX8 HepG2 cells.D, the expression of TP53 and P21proteins was detected by Westernblotting assays in control andshSUZ12 HepG2 cells. E, themRNA levels of EZH2 and TP53were determined by qRT-PCR inA549 cells transfected with controlor EZH2 siRNA at 2 days aftertransfection. F, qRT-PCRdetection of PcGs, TP53, andCDKN2A mRNA levels in PcG KDxenograft (N ¼ 9 or 10).






of PcG, which contains well-established genes, such asCDKN2A, and other previously unstudied genes in livercancer cells, including E2F1 and NOTCH2 (21). Thefunctional importance of these genes in controlling liverfunction has been established (22, 23). This finding suggestsa preferential epigenetic action of EZH2 on the regulation ofthe transcription of E2F1 and NOTCH2 in HCC. To testthis hypothesis, we performed ChIP assays using threedistinct pairs of ChIP primers at E2F1 promoter loci (Fig.6A). Using an EZH2-specific antibody, we demonstrated

that EZH2was present at theE2F1 promoter inHepG2 cells(Fig. 6B). As expected, the KD of EZH2 by shRNAdecreased the binding of EZH2 to the E2F1 loci (Fig.6B). EZH2–shRNA also led to a significant decrease inthe H3K27me3 level at the promoter of the E2F1 locus(Fig. 6C). In addition, we demonstrated that EZH2 andH3K27me3 were present at the promoter of NOTCH2 andthat the KD of EZH2 by shRNA led to a severe decrease inthe binding of EZH2 and the H3K27me3 levels at thepromoters in HCC (Fig. 6D–F). Therefore, we conclude

Figure 5. A negative correlation between PcGs and TP53 expression in HCC specimens. The mRNA levels of TP53, EZH2, SUZ12, CBX8, and BMI1 weredetermined by qRT-PCR in 48 cases of HCC specimens and adjacent tissues, and the correlation between PcGs and TP53 expression is shown (linearregression analysis, Pearson correlation). The mRNA levels were normalized to those of b-actin.

Figure 6. PcG directly regulatesE2F1 and NOTCH2 expressionthrough H3K27me3 in HCC. A andD, schematic representation ofhuman E2F1 and NOTCH2 primerpairs (PP) used for ChIP assays. Band E, ChIP assays using antibodyagainst EZH2 were performedusing the indicated primer pairs ofthe E2F1 or NOTCH2 promoters incontrol and shEZH2 HepG2 cells.Rabbit-IgG was used as a negativecontrol. C and F, H3K27me3 ChIPassays were performed using theindicated primer pairs of the E2F1or NOTCH2 promoter in controland shEZH2 HepG2 cells. Rabbit-IgGwas used as anegative control.

Gao et al.





that the transcription of E2F1 and NOTCH2 were directlyrepressed by EZH2-mediated H3K27me3 in HCC cells.

DiscussionEZH2 promotes HCC cell motility and metastasis by

repressing tumor suppressor miRNAs (11), but the specificgene network and epigenetic characteristics are not wellestablished. The CDKN2A locus encodes two distinct pro-teins, p16INK4A and p14ARF, both of which have beenimplicated in a variety of cancers. In acute myeloid leukemiacells, H3K27me3 and EZH2 seem to be distributed pri-marily at the promoters of CDKN2A (24). It has also beendemonstrated that H3K27me3 is an early-epigenetic eventof p16INK4A silencing in hepatoma cells (25). Our previousresults indicated that EZH2 and H3K27me3 cooccupi-ed the promoter of CDKN2A (21), indicating thatH3K27me3-dependent repression is a common feature atINK4-ARF in tumors, including HCC. E2F1, a tumorsuppressor, binds to the promoter of EZH2 and EED andfunctionally controls their expression in prostate cancer (26).An interesting negative-feedback loop was found in thepresent study, in which EZH2 repressed E2F1 expressionby binding to the promoter of E2F1 in HCC. Our findingshighlight H3K27me3 remodeling as an interesting newdirection in the elucidation of the E2F1 transcriptionalregulation mechanism.In agreement with a previous report (27), we identified

genes that are targeted by PRC2 without PRC1 and viceversa. Although EZH2 and SUZ12 occupy the promoterregions of thousands of human genes, only 340 genesshare H3K27me3 in HepG2 (21). This result suggeststhat PcG regulates gene expression in both anH3K27me3-dependent and an H3K27me3-independentmanner. The mutation-induced inactivation of TP53,which is one of the important tumor suppressors, hasfrequently been associated with a variety of human can-cers, including HCC. Nevertheless, the transcriptionalrepression of TP53 by critical effectors in HCC has notbeen well defined. In the present study, we found that theexpression of TP53 was dramatically repressed by bothPRC1 and PRC2, and a significant negative correlationbetween the expression of PcGs and TP53 was found inprimary HCC specimens. However, we failed to observean obvious impact of H3K27me3 on TP53 expression,suggesting that PcG regulates TP53 expression in anH3K27me3-independent manner in HCC. This activityis presumably dependent on the direct regulation ofCDKN2A or E2F1, which are known upstream regulatorsof the TP53 pathway (28, 29). The ARF product func-tions as a stabilizer of the p53 protein, which was respon-sible for the degradation of p53 (28). CDKN2A productinteracts with and inhibits the oncogenic action ofMDM2 by blocking MDM2-induced degradation ofp53 and enhancing p53-dependent transactivation andapoptosis (28). Confusingly, we found that both proteinand mRNA levels of TP53 were substantially repressed byPcG in HCC cells. A previous report found a CCCTC-

binding factor (CTCF) that binds at the P53 promoterand protects the P53 gene promoter against repressivehistone marks (30). Interesting findings have shown thatEZH2 and SUZ12 were directly bound at CTCF pro-moter loci with H3K27me3 modification and that theincreased accumulation of H3K27me3 at the CTCFpromoter is associated with the reduction of CTCF inHCC specimens (21). These results suggest that PcGregulates P53 expression in HCC at least partly throughCTCF in HCC. Functionally and mechanistically signif-icant work remains to be done to identify the competitivefunctions between PcG and CTCF in HCC. TP53 wasfrequently mutated in HCC and is a major genetic eventfor the development of HCC. Our results emphasize theinteresting fact that, independent of genetic mutation, thetranscriptional silencing of TP53 by PcG contributes tothe aggressive nature of HCC. Recently, Lu and colleagues(31) reported that p53 is not the target of EZH2 innasopharyngeal carcinoma. Similar to this, we did notobserve an obvious regulation effect of EZH2 on repres-sing TP53 mRNA level in other type tumor cell lines,including HO-8910, MCF-7, and Wilm. This resultsuggests an interesting tissue-specific regulation profile ofEZH2 on TP53 expression. Conversely, Tang and col-leagues (14) reported that P53 repress EZH2 transcriptionthrough binding to its promoter locus in primary humanfibroblasts. These findings suggest a potential feedbackloop of tumor suppressor P53 and tumor promoter geneEZH2, an important interplay governing tumor activationand suppression in the development of HCC. This processis similar to our previous reports that the interplaybetween oncogene K-Ras and tumor suppressor meninplays an important role in regulating the development oflung cancer (17). However, it is unclear whether theactivated P53 suppresses the expression of EZH2 in HCC.The link between p53 and EZH2 in controlling aggressivephenotype of HCC needs further investigation.Alteration of PcG components was observed in various

cancers such as melanoma, lymphoma, and breast andprostate cancer, leading to the hypothesis that PcG hastumor-promoting functions (32). The overexpression ofEZH2 promotes the growth and cell invasion of immor-talized human mammary epithelial cells (33), and thereduction of EZH2 expression inhibits prostate cancer cellproliferation in vitro (34). Further studies demonstratethat the oncogenic effect of EZH2 is linked to theH3K27me3-dependent repression of the tumor suppres-sor genes, including E-cadherin, RUNX3, INK4/ARF (alsoknown as CDKN2A; refs. 24, 35, 36), and others. Here,we found that the alterations of PcG proteins directlymodulate H3K27me3 and may thus affect the HCCepigenome, which is crucial for HCC cell growth bothin vitro and in vivo. We also observed that the reduction ofEZH2 expression induces G2–M cell-cycle arrest in HCCcells. This finding is consistent with a previous report thatCDKN2A induces G2 arrest in a p53-independent mannerby preventing the activation of cyclin B1/CDC2 com-plexes (37). Our findings demonstrate a potential tumor-






promoting action of EZH2 by directly or indirectlyregulating the expression of tumor suppressors with crit-ical functions in HCC, at least partly through theCDKN2A–TP53–P21 pathway. These findings alsoreveal the EZH2 pathway as a potential target for thetreatment of EZH2-positive human HCC with EZH2inhibitors, such as GSK126, which is a specific smallmolecular inhibitor of the catalytic SET domain of EZH2(38). Significant work remains to identify the major targetof PcGs in HCC.

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

Authors' ContributionsConception and design: S.B. GaoDevelopment of methodology: B. Xu, Q.-L. ZhengAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): S.B. Gao, Q.-L. Zheng, G.-H. Jin

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Q.-F. Zheng, C.-B. Pan, Q.-L. Zheng, X. Lin,L.-X. Xue, G.-H. JinWriting, review, and/or revision of the manuscript: S.B. Gao, Q.-F. Zheng,L.-X. Xue, G.-H. JinAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): S.B. Gao, K.-L. Li

AcknowledgmentsWe appreciate the valuable comments from other members of our laboratories.

Grant SupportThis work was supported by the grants from the Natural Science Foundation of

China (81101763, to S.-B. Gao; 91229111 and 81272719, to G.-H. Jin; 81301754,to B. Xu) and the Natural Science Foundation of Fujian Province (2011J06016 toG.-H. Jin).

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

Received January 20, 2014; revised April 30, 2014; accepted May 19, 2014;published OnlineFirst June 10, 2014.

References1. Sparmann A, van Lohuizen M. Polycomb silencers control cell fate,

development and cancer. Nat Rev Cancer 2006;6:846–56.2. CaoR,Wang L,WangH, Xia L, Erdjument-BromageH, Tempst P, et al.

Role of histone H3 lysine 27methylation in polycomb-group silencing.Science 2002;298:1039–43.

3. Cao R, Tsukada Y, Zhang Y. Role of Bmi-1 and Ring1A in H2Aubiquitylation and Hox gene silencing. Mol Cell 2005;20:845–54.

4. Milne TA, Briggs SD, Brock HW, Martin ME, Gibbs D, Allis CD, et al.MLL targets SET domain methyltransferase activity to Hox genepromoters. Mol Cell 2002;10:1107–17.

5. Yokoyama A, Somervaille TC, Smith KS, Rozenblatt-Rosen O, Meyer-sonM, Cleary ML. The menin tumor suppressor protein is an essentialoncogenic cofactor for MLL-associated leukemogenesis. Cell 2005;123:207–18.

6. Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet 2012;379:1245–55.

7. Cai MY, Tong ZT, Zheng F, Liao YJ, Wang Y, Rao HL, et al. EZH2protein: a promising immunomarker for the detection of hepatocellularcarcinomas in liver needle biopsies. Gut 2011;60:967–76.

8. CaiMY,Hou JH,RaoHL, LuoRZ, LiM, Pei XQ, et al. High expressionofH3K27me3 in human hepatocellular carcinomas correlates closelywith vascular invasion and predicts worse prognosis in patients. MolMed 2011;17:12–20.

9. Yang F, Zhang L, Huo XS, Yuan JH, Xu D, Yuan SX, et al. Longnoncoding RNA high expression in hepatocellular carcinoma facil-itates tumor growth through enhancer of zeste homolog 2 in humans.Hepatology 2011;54:1679–89.

10. Zheng F, Liao YJ, Cai MY, Liu YH, Liu TH, Chen SP, et al. The putativetumour suppressor microRNA-124 modulates hepatocellular carcino-ma cell aggressiveness by repressing ROCK2 and EZH2. Gut 2012;61:278–89.

11. Au SL,Wong CC, Lee JM, Fan DN, Tsang FH, Ng IO, et al. Enhancer ofzeste homolog 2 epigenetically silences multiple tumor suppressormicroRNAs to promote liver cancer metastasis. Hepatology 2012;56:622–31.

12. Fujimoto A, Totoki Y, Abe T, Boroevich KA, Hosoda F, Nguyen HH,et al. Whole-genome sequencing of liver cancers identifies etiologicalinfluences on mutation patterns and recurrent mutations in chromatinregulators. Nat Genet 2012;44:760–4.

13. Yamada A, Fujii S, Daiko H, Nishimura M, Chiba T, Ochiai A. Aberrantexpression of EZH2 is associated with a poor outcome and P53alteration in squamous cell carcinoma of the esophagus. Int J Oncol2011;38:345–53.

14. Tang X, MilyavskyM, Shats I, Erez N, Goldfinger N, Rotter V. Activatedp53 suppresses the histone methyltransferase EZH2 gene. Oncogene2004;23:5759–69.

15. Choi JH, Song YS, Yoon JS, Song KW, Lee YY. Enhancer ofzeste homolog 2 expression is associated with tumor cell pro-liferation and metastasis in gastric cancer. APMIS 2010;118:196–202.

16. Xu B, Li SH, Zheng R, Gao SB, Ding LH, Yin ZY, et al. Menin promoteshepatocellular carcinogenesis and epigenetically up-regulates Yap1transcription. Proc Natl Acad Sci U S A 2013;110:17480–5.

17. Wu Y, Feng ZJ, Gao SB, Matkar S, Xu B, Duan HB, et al. Interplaybetween menin and K-Ras in regulating lung adenocarcinoma. J BiolChem 2012;287:40003–11.

18. Xu B, Zeng DQ, Wu Y, Zheng R, Gu L, Lin X, et al. Tumor suppressormenin represses paired box gene 2 expression via Wilms tumorsuppressor protein-polycomb group complex. J Biol Chem 2011;286:13937–44.

19. Feng ZJ, Gao SB, Wu Y, Xu XF, Hua X, Jin GH. Lung cancer cellmigration is regulated via repressing growth factor PTN/RPTP beta/zeta signaling by menin. Oncogene 2010;29:5416–26.

20. Murata M, Tagawa M, Kimura M, Kimura H, Watanabe S, Saisho H.Analysis of a germ line polymorphism of the p53 gene in lung cancerpatients; discrete results with smoking history. Carcinogenesis 1996;17:261–4.

21. Gao SB, Xu B, Ding LH, Zheng Q, Zhang L, Zheng QF, et al. Thefunctional and mechanistic relatedness of the EZH2 and menin inhepatocellular carcinoma. J Hepatol 2014 May 15. [Epub ahead ofprint].

22. Dill MT, Tornillo L, Fritzius T, Terracciano L, Semela D, Bettler B, et al.Constitutive Notch2 signaling induces hepatic tumors in mice. Hepa-tology 2013;57:1607–19.

23. Ladu S, Calvisi DF, Conner EA, Farina M, Factor VM, Thorgeirsson SS.E2F1 inhibits c-Myc-driven apoptosis via PIK3CA/Akt/mTOR andCOX-2 in a mouse model of human liver cancer. Gastroenterology2008;135:1322–32.

24. Bracken AP, Kleine-Kohlbrecher D, Dietrich N, Pasini D, Gargiulo G,Beekman C, et al. The polycomb group proteins bind throughout theINK4A-ARF locus and are disassociated in senescent cells. GenesDev2007;21:525–30.

25. Yao JY, Zhang L, Zhang X, He ZY, Ma Y, Hui LJ, et al. H3K27trimethylation is an early epigenetic event of p16INK4a silencing forregaining tumorigenesis in fusion reprogrammed hepatoma cells.J Biol Chem 2010;285:18828–37.

Gao et al.





26. Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K. EZH2 isdownstream of the pRB-E2F pathway, essential for proliferation andamplified in cancer. EMBO J 2003;22:5323–35.

27. Tavares L, Dimitrova E, Oxley D, Webster J, Poot R, Demmers J, et al.RYBP-PRC1 complexes mediate H2A ubiquitylation at polycombtarget sites independently of PRC2 and H3K27me3. Cell 2012;148:664–78.

28. Zhang Y, Xiong Y, Yarbrough WG. ARF promotes MDM2 degra-dation and stabilizes p53: ARF-INK4a locus deletion impairsboth the Rb and p53 tumor suppression pathways. Cell 1998;92:725–34.

29. Sherr CJ, McCormick F. The RB and p53 pathways in cancer. CancerCell 2002;2:103–12.

30. Soto-Reyes E, Recillas-Targa F. Epigenetic regulation of the humanp53genepromoter by theCTCF transcription factor in transformed celllines. Oncogene 2010;29:2217–27.

31. Lu J, HeML,Wang L, Chen Y, Liu X, DongQ, et al. MiR-26a inhibits cellgrowth and tumorigenesis of nasopharyngeal carcinoma throughrepression of EZH2. Cancer Res 2011;71:225–33.

32. Bachmann IM, Halvorsen OJ, Collett K, Stefansson IM, Straume O,Haukaas SA, et al. EZH2 expression is associated with high prolifer-ation rate and aggressive tumor subgroups in cutaneous melanoma

and cancers of the endometrium, prostate, and breast. J Clin Oncol2006;24:268–73.

33. Kleer CG, Cao Q, Varambally S, Shen R, Ota I, Tomlins SA, et al. EZH2is a marker of aggressive breast cancer and promotes neoplastictransformation of breast epithelial cells. Proc Natl Acad Sci U S A2003;100:11606–11.

34. Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-SinhaC, Sanda MG, et al. The polycomb group protein EZH2 is involved inprogression of prostate cancer. Nature 2002;419:624–9.

35. Cao Q, Yu J, Dhanasekaran SM, Kim JH, Mani RS, Tomlins SA, et al.Repression of E-cadherin by the polycomb group protein EZH2 incancer. Oncogene 2008;27:7274–84.

36. Fujii S, Ito K, Ito Y, Ochiai A. Enhancer of zeste homologue 2 (EZH2)down-regulates RUNX3 by increasing histone H3 methylation. J BiolChem 2008;283:17324–32.

37. Normand G, Hemmati PG, Verdoodt B, von Haefen C, Wendt J, GunerD, et al. p14ARF induces G2 cell cycle arrest in p53- and p21-deficientcells by down-regulating p34cdc2 kinase activity. J Biol Chem2005;280:7118–30.

38. McCabe MT, Ott HM, Ganji G, Korenchuk S, Thompson C, Van AllerGS, et al. EZH2 inhibition as a therapeutic strategy for lymphoma withEZH2-activating mutations. Nature 2012;492:108–12.






2014;12:1388-1397. Published OnlineFirst June 10, 2014.Mol Cancer Res Shu-Bin Gao, Qi-Fan Zheng, Bin Xu, et al. H3K27-Independent Mechanisms in Hepatocellular CarcinomaEZH2 Represses Target Genes through H3K27-Dependent and

Updated version

10.1158/1541-7786.MCR-14-0034doi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2014/06/11/1541-7786.MCR-14-0034.DC1

Access the most recent supplemental material at:

Overview

Visual

http://mcr.aacrjournals.org/content/12/10/1388/F1.large.jpgA diagrammatic summary of the major findings and biological implications:

Cited articles

http://mcr.aacrjournals.org/content/12/10/1388.full#ref-list-1

This article cites 37 articles, 14 of which you can access for free at:

Citing articles

http://mcr.aacrjournals.org/content/12/10/1388.full#related-urls

This article has been cited by 7 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/12/10/1388To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-14-0034

http://mcr.aacrjournals.org/content/suppl/2014/06/11/1541-7786.MCR-14-0034.DC1

http://mcr.aacrjournals.org/content/12/10/1388/F1.large.jpg

http://mcr.aacrjournals.org/content/12/10/1388.full#ref-list-1

http://mcr.aacrjournals.org/content/12/10/1388.full#related-urls

http://mcr.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mcr.aacrjournals.org/content/12/10/1388


chromatin, gene, and rna regulation · mol cancer res; 12(10); 1388–97. 2014 aacr. introduction...

Documents