EZH2-Mediated Concordant Repression of Wnt Antagonists Promotes B-Catenin–Dependent Hepatocarcinogenesis

Published OnlineFirst April 21, 2011; DOI: 10.1158/0008-5472.CAN-10-3342

Cancer Tumor and Stem Cell Biology Research

EZH2-Mediated Concordant Repression of Wnt Antagonists Promotes b-Catenin–Dependent Hepatocarcinogenesis

Alfred S.L. Cheng1, Suki S. Lau1, Yangchao Chen2, Yutaka Kondo5, May S. Li1, Hai Feng2, Arthur K. Ching3, Kin F. Cheung2, Hoi K. Wong4, Joanna H. Tong3, Hongchuan Jin1, Kwong W. Choy4, Jun Yu1,2,KaF.To1,3, Nathalie Wong3, Tim H.-M. Huang6, and Joseph J.Y. Sung1,2

Abstract Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the Polycomb-repressive complex 2 (PRC2) that represses gene transcription through histone H3 lysine 27 trimethylation (H3K27me3). Although EZH2 is abundantly present in various cancers, the molecular consequences leading to oncogenesis remain unclear. Here, we show that EZH2 concordantly silences the Wnt pathway antagonists operating at several subcellular compartments, which in turn activate Wnt/b-catenin signaling in hepatocellular carcinomas (HCC). Chromatin immunoprecipitation promoter array and gene expression analyses in HCCs revealed EZH2 occupancy and reduced expression of Wnt antagonists, including the growth-suppressive AXIN2, NKD1, PPP2R2B, PRICKLE1, and SFRP5. Knockdown of EZH2 reduced the promoter occupancy of PRC2, histone deacetylase 1 (HDAC1), and H3K27me3, whereas the activating histone marks were increased, leading to the transcriptional upregulation of the Wnt antagonists. Combinatorial EZH2 and HDAC inhibition dramatically reduced the levels of nuclear b-catenin, T-cell factor–dependent transcriptional activity, and downstream pro-proliferative targets CCND1 and EGFR. Functional analysis revealed that downregulation of EZH2 reduced HCC cell growth, partially through the inhibition of b-catenin signaling. Conversely, ectopic overexpression of EZH2 in immortalized hepatocytes activated Wnt/b-catenin signaling to promote cellular proliferation. In human HCCs, concomitant overexpression of EZH2 and b-catenin was observed in one-third (61/179) of cases and significantly correlated with tumor progression. Our data indicate that EZH2-mediated epigenetic silencing contributes to constitutive activation of Wnt/b-catenin signaling and consequential proliferation of HCC cells, thus representing a novel therapeutic target for this highly malignant tumor. Cancer Res; 71(11); 1–12. 2011 AACR.

Introduction accumulation of genetic and epigenetic defects that alter the transcriptional program. High-throughput "omics" analyses of Hepatocellular carcinoma (HCC) is the fifth most common the gene expression in large patient cohorts have led to the cancer and the third most frequent cause of tumor-related identification of the key signaling pathways in this multistep deaths worldwide (1, 2). Despite recent epidemiologic data in process (2, 4–6). A detailed understanding of the regulation of the United States, which indicates that the overall cancer these signal transduction pathways and their interconnectiv- incidence and death rates are declining, liver cancer has the ity should shed new insights into the development of ther- fastest-growing death rate among malignancies (2, 3). Hepa- apeutic interventions against HCC. tocarcinogenesis is a complex process associated with the The Wnt/b-catenin signaling pathway plays a unique and essential role both in liver physiology and pathology by

1 regulating various basic cellular events, including differentia- Authors' Affiliations: Institute of Digestive Disease and Li Ka Shing – Institute of Health Sciences; Departments of 2Medicine and Therapeutics, tion, proliferation, and survival (7 9). In human HCC, the 3Anatomical and Cellular Pathology, and 4Obstetrics and Gynaecology, activation of the Wnt/b-catenin pathway can occur through 5 The Chinese University of Hong Kong, Hong Kong SAR, China; Division of somatic mutations in CTNNB1 (encoding b-catenin), AXIN1, Molecular Oncology, Aichi Cancer Center Research Institute, Nagoya, AXIN2 Japan; and 6Human Cancer Genetics Program, Department of Molecular and (7, 10). The common denominator is the stabiliza- Virology, Immunology and Medical Genetics, Comprehensive Cancer tion of b-catenin, which translocates into the nucleus and Center, The Ohio State University, Columbus, Ohio associates with the T-cell factor (TCF) family of transcription Note: Supplementary data for this article are available at Cancer Research factors for efficient activation of Wnt target genes. Apart from Online (http://cancerres.aacrjournals.org/). infrequent genetic mutations, it remains unclear how different Corresponding Author: Alfred S.L. Cheng, Institute of Digestive Disease, b The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, components of the Wnt/ -catenin pathway are deregulated in NT, Hong Kong SAR, China. Phone: 852-3763-6100; Fax: 852-2635-0075. HCC. E-mail: [email protected] Epigenetic aberrations, including DNA methylation, histone doi: 10.1158/0008-5472.CAN-10-3342 modifications, and microRNA dysregulation, are now estab- 2011 American Association for Cancer Research. lished in tumor initiation and progression (11). Polycomb

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proteins are highly conserved from Drosophila to humans and in the Gene Expression Omnibus (GEO) database (accession they form multiple Polycomb-repressive complexes (PRC1 and number: GSE17733). Statistical analysis was done with ChIP PRC2). Among the core components of PRC2, enhancer of Analytics Software (version 1.3), which utilizes the Whitehead zeste homolog 2 (EZH2) contains the histone methyltransfer- neighborhood model to make binding calls based on an ase activity, whereas SUZ12 and EED are also required for this intensity-based P value of each probe and its neighbors. This activity (12, 13). EZH2 catalyzes histone H3 lysine 27 trimethy- algorithm computes robust regions of increased probe signal lation (H3K27me3) necessary for PRC2-mediated gene repres- (peaks), where probes having P < 0.001 were regarded as sion (14, 15). Previous reports have shown that EZH2 significant EZH2-DNA–binding events. The compiled EZH2 overexpresses in various cancers (16–19), and it contributes target gene lists were further analyzed on the basis of KEGG to oncogenesis by promoting neoplastic transformation of pathway associations (pathway analysis) by using BioScript immortalized epithelial cells (16, 17), increasing cell prolifera- SG3b-1 from BioScript library 2.1 in GeneSpringTM 7.3.1 tion (16, 17), inhibition of senescence, and differentiation (18). (Agilent). For real-time ChIP-PCR validation, equal amounts Dysregulation of EZH2 has been previously found in human of ChIP and diluted input DNAs were used for SYBR Green- HCC (20), wherein we have shown that the lentiviral-mediated based detection (Applied Biosystems), as previously described EZH2 suppression dramatically inhibited cell growth and (22). diminished tumorigenicity in vivo (21). By using chromatin immunoprecipitation microarray (ChIP-chip; refs. 22, 23) and RNA interference and lentivirus transduction independent confirmation assays, we and others have recently The cells were transfected with 25 nmol/L siRNA by using shown that EZH2 promotes oncogenesis through repressing HiPerFect (Qiagen) for 72 hours, according to the man- the tumor-suppressor genes (19, 24). However, the mechan- ufacturer's instructions. The cells were also treated with isms whereby EZH2 promotes hepatocarcinogenesis are 300 nmol/L trichostatin A (TSA; Sigma) or ethanol vehicle unclear; this is because EZH2-altered downstream targets control for the last 20 hours in indicated experiments. The and pathways in HCC are largely unknown. In this study, Myc-EZH2 and enhanced green fluorescent protein (EGFP) by using ChIP-chip analysis in HCC cells, we uncovered 12 lentiviral vectors were previously described (25). The MIHA Wnt/b-catenin signal antagonists, whose promoters were cells seeded on 24-well plate were transduced with lentivirus concordantly occupied by EZH2. Further molecular and func- in the presence of 8 mg/mL polybrene (Sigma). Real-time tional analyses illustrated that the EZH2-mediated transcrip- reverse transcriptase PCR (RT-PCR) and Western blot ana- tional repression of these Wnt pathway inhibitors allows lyses were done, as previously described (21, 22). constitutive Wnt/b-catenin signaling, which likely plays a critical role in EZH2-stimulated cellular proliferation. These Luciferase reporter assays in vitro findings concurred with immunohistochemical ana- The effects of EZH2 RNA interference and naked cuticle-1 lysis in human HCC tissue microarray (TMA), showing that (NKD1) on the Wnt/b-catenin activity were measured by EZH2 and b-catenin were concordantly overexpressed and TOPflash reporter assay (28). Two days after the transfection were significantly associated with tumor progression. Collec- of plasmids, the cells were harvested and assayed by the Dual tively, our results provide mechanistic and functional links Luciferase Reporter Assay System (Promega) by using GloMax between EZH2 and Wnt/b-catenin signaling in the develop- microplate luminometer (Promega). ment of HCC. Cell growth assays Materials and Methods The HKCI-10 cells were transfected with control or domi- nant positive (DP)-b–catenin expression plasmids, followed by Cell lines and plasmids siControl or siEZH2 on the following day. The cells were The human HCC cell lines Huh7, PLC5, the embryonic counted in Trypan Blue dye exclusion medium on 4-day kidney epithelial cell line Hek293FT (ATCC), and immorta- post-siRNA transfection by using a hemacytometer chamber. lized human hepatocyte MIHA cells (25) were maintained in The EGFP- and EZH2-lentivirus–infected MIHA cells seeded on Dulbecco's modiﬁed Eagle's medium (Invitrogen) supplemen- 12-well plates were transfected with sib-catenin or siControl ted with 10% FBS (HyClone). The HCC cell line HKCI-10 (26) for 4 days. The cells were counted on the day of transfection was maintained in RPMI 1640 medium (Invitrogen) supple- (Day 0), 2- and 4-day posttransfection. mented with 10% FBS. The cell lines were authenticated by short tandem repeat genotyping (AmpF‘STR Identifiler; Clinical samples and immunohistochemistry Applied Biosystems) in September, 2009. The construction Tissue samples were collected with the prior informed of plasmid and detailed methodology are described in the consent of the patients, and the prior Joint CUHK-NTEC Supplemental Material. Clinical Research Ethics Committee approval. Formalin-fixed paraffin-embedded archive tissues of 179 paired HCC and ChIP-chip and bioinformatics analysis matched nontumorous liver tissues were arranged in tissue ChIP followed by microarray hybridization was done as array blocks and stained with antibodies against EZH2 and described previously (22, 23, 27). Replicate dye-swap experi- b-catenin. The specificity of antibodies was validated in the ments were conducted on human promoter ChIP-chip micro- present and previous studies (16, 17, 29). Immunohistochemical array set (Agilent). The microarray data have been deposited staining and scoring were done, as previously described (26).

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Statistical analysis both cell lines (Fig. 1C). We next determined the association For real-time PCR and luciferase assays, data were pre- between EZH2 occupancy and histone modifications, includ- sented in mean SD for triplicate experiments. The 2-tailed ing the EZH2-catalyzed H3K27me3, H3K9me2-repressive Student's t test was used for statistical analysis. The Wilcoxon marks, acetylated H3K9 (H3K9ac), and H3K4me3 activating signed rank test was used to compare the gene expression marks (22). As a control for histone modifications, similar between the tumor and the nontumorous tissues. For immu- levels of histone H3 among the Wnt antagonists and b-actin nohistochemical analysis, the Pearson c2 and Mann–Whitney were shown by real-time ChIP-PCR (Fig. 1D). Notably, the tests were used to compare protein expressions in HCC and H3K27me3, but not the H3K9me2-repressive mark, was their relationships with different differentiation types, respec- enriched in the majority of the Wnt antagonists, whereas only tively. The values of P < 0.05 were considered significant. low levels of the H3K9ac and the H3K4me3-active marks were detected (Fig. 1D). For example, the NKD1 and PPP2R2B loci Results were both marked by H3K27me3, but lacked the active marks in both cell lines (Fig. 1D). Integrative ChIP-chip and pathway analysis reveals a To determine whether the EZH2 occupancy on the Wnt significant association between EZH2 and Wnt/ antagonists was a cancer-specific silencing event, we con- b-catenin signaling in human HCC ducted ChIP and expression analysis by using normal To characterize the direct targets of EZH2 that confer human liver and Huh7 cells. ChIP-PCR showed no EZH2 oncogenic properties in HCC, we first carried out ChIP-chip occupancy on the NKD1 and PPP2R2B promoters in the in the 2 HCC cell lines Huh7 and PLC5, which express high normal liver (Fig. 2A) which had no detectable EZH2 levels of EZH2 protein (21). By using promoter arrays of expression (Fig. 2B). In the Huh7 cells, EZH2 occupancy approximately 17,000 best-defined human transcripts, we wasassociatedwiththereducedexpressionofNKD1, found that 785 and 807 promoters (4%–5%) were significantly PPP2R2B, and most of the other Wnt antagonists compared bound by EZH2 in Huh7 and PLC5 cells, respectively (Supple- with normal liver tissues (Fig. 2C and Supplementary mentary Table S1). The proportion of EZH2-bound promoters Fig. S3). More importantly, in a cohort of 23 human HCC in the HCC cells showed concordance with our previous global specimens, the expressions of 11 Wnt antagonists were assessment of the EZH2-catalyzed H3K27me3 modification in significantly reduced in HCC tissues when compared with prostate cancer cells (24). Independent analysis by using site- matched nontumorous liver tissues (P < 0.05 to < 0.0001; specific real-time PCR indicated that the frequency of false Fig. 2D and Supplementary Fig. S3). We then determined positives was approximately 2% (Supplementary Fig. S1). the DNA methylation status of the 12 Wnt antagonists in 10 Thirty percent of the identified promoters were known targets pairs of HCC specimens by using methylated DNA immu- of PRC2 and/or H3K27me3, which in turn confirms the validity noprecipitation assay, as previously described (27). Except of our array finding (Supplementary Table S1). In accordance AXIN2, of which the DNA methylation level was higher in with previous studies (19, 24), some direct targets were known HCCs compared with the matched nontumorous liver tis- to be tumor-suppressor genes and they were repressed by sues, no obvious DNA methylation enrichment was detected EZH2 in HCC cells (Supplementary Fig. S1). in other Wnt antagonists (Supplementary Fig. S4). These Next, we integrated the novel direct EZH2 targets to path- results were further validated by methyl-CpG–binding way analysis on the basis of the Kyoto Encyclopedia of Genes domain-based capture assay (Supplementary Fig. S4). These and Genomes classification. We found that the EZH2 targets data are consistent with the hypothesis that some Wnt identified in the Huh7 and PLC5 cells were concordantly antagonists which are actively transcribed in a normal liver involved in a few important cancer-related pathways (P < are repressed primarily through EZH2 in HCC cells. Taken 0.05; Fig. 1A; Supplementary Table S2). Among the direct EZH2 together, our integrated analysis shows a significant asso- targets involved in the Wnt/b-catenin signaling pathway, 12 ciation between EZH2 and Wnt/b-catenin signaling in are well-characterized Wnt/b-catenin signal antagonists that human HCC. function in various physiologic and pathologic conditions (Supplementary Table S3). Besides, a number of Wnt/Frizzled Direct transcriptional repression of Wnt antagonists by receptor elements were also targeted by EZH2 (Supplemen- EZH2 in HCC cells tary Table S4; ref. 30), and they were repressed in HCC cells in We next elucidated the mechanism whereby EZH2 regu- contrast to other actively expressing non-EZH2–bound Wnt/ lates the expression of the bound Wnt antagonists. NKD1 is an Frizzled genes (Supplementary Fig. S2). inducible antagonist via physically interfering Dishevelled, a For the Wnt/b-catenin signal antagonists, a significant central component of Wnt/b-catenin signaling (31). Because enrichment of EZH2 was detected proximal to their transcrip- EZH2 functions in a multiprotein PRC2 complex in the tion start sites, both in the Huh7 and PLC5 cells (Fig. 1B and transcriptional repression, we determined the occupancy of data not shown). To validate the microarray findings, we PRC2 components in the NKD1 promoter. In the Huh7 cells, conducted conventional and real-time ChIP-PCR by using conventional ChIP-PCR using 2 sets of primers targeting the primers specific to the bound promoter regions. In all of EZH2-binding region (see Fig. 1B) showed that EZH2, EED, the 12 Wnt antagonists, we observed strong enrichment by and SUZ12 were enriched (Fig. 3A). No significant binding an EZH2 antibody relative to the control antibody against could be observed in the flanking fragments 2 kb from the irrelevant IgG (Supplementary Fig. S2) and b-actin control in bound region (Fig. 3A), thus suggesting that the PRC2

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A Huh7 PLC5

Axon guidance Antigen processing

Calcium signaling MAPK Cell cycle

Neuroactive ligand- Cytokine interaction receptor interaction Focal adhesion

TGF-β Long-term depression Gap junction Wnt/β-catenin Phosphatidylinositol Glycerolipid metabolism Figure 1. Integrative genomics signaling analysis reveals a significant Hedgehog signaling Tight junction association between EZH2 direct target genes and Wnt/b-catenin signaling in HCC cells. A, a Venn diagram showing the signaling B pathways significantly associated NKD1 PPP2R2B Huh7 with EZH2 in both HCC cell lines. 2.5 2.5 PLC5 B, EZH2-binding maps in the 2 2 proximal promoters of Wnt 1.5 1.5 antagonists. The X-axis and 1 1 Y-axis represent the probe 0.5 0.5 CDBA

Enrichment ratio location relative to the 0 0 –4,000 –3,000 –2,000–1,0000 1,000 2,000 –5000 500 1,000 1,500 2,000 2,500 3,000 transcription start site (TSS) and Distance (bp) to TSS the EZH2 enrichment ratio (IP/ input), respectively. Dotted lines indicative of no enrichment are CD shown. C, real-time ChIP-PCR H3K27me3 confirms the Wnt antagonists 170 15 H3K9me2 Huh7 Huh7 H3K9ac identified by microarray as direct H3K4me3 150 H3 EZH2 targets. Fold enrichment 100 10 was determined as a percentage 75 of input DNAs of EZH2 target divided by that of b-actin, and 50 5 each error bar represents SD 25 calculated from triplicates. D, real-time ChIP-PCR shows the 0 0 5 α 1 occupancy of histone H3 and P NLK NLK 2CB CER1CK1αDKK1 NKD1 TP53 CER1 CK1 DKK1 NKD1 P TP53 various histone marks in the Wnt AXIN2 EP300 SFR AXIN2 EP300 SFRP5 PPP2CB PP PPP2R2BPRICKLE1 PPP2R2BPRICKLE antagonists. Fold enrichment was 60 PLC5 20 PLC5 determined as in (C). Dotted lines 40 indicative of no enrichment Fold enrichment Fold enrichment Fold 15 compared with b-actin are shown. 20 10

10 5

0 0 α 5 2B 2B P NLK NLK 2CB CER1CK1DKK1 NKD1 TP53 CER1 CK1α DKK1 NKD1 P 2R TP53 AXIN2 EP300 SFRP5 AXIN2 EP300 SFR PPP2CB PP PPP2RPRICKLE1 PPP PRICKLE1

components were colocalized on the specific NKD1 promoter H3K27me3 promoter occupancy was reduced in association region. PRC2-mediated repression of gene activity involves with a global loss of H3K27me3 (Fig. 3B and Supplementary histone deacetylation (32). Thus, we examined the binding of Fig. S5). In contrast, the levels of the H3K9ac- and H3K4me3- histone deacetylase 1 (HDAC1) and found that HDAC1 co- active marks were increased (Fig. 3B and Supplementary occupied the NKD1 promoter with the PRC2 complex (Fig. 3A Fig. S5). and Supplementary Fig. S5). By combining real-time ChIP-PCR To examine whether this chromatin remodeling was asso- with RNA interference, we found that knockdown of EZH2 not ciated with an alteration of the NKD1 promoter activity, we only decreased the occupancy of its own, but also the PRC2 cloned an endogenous 1 kb regulatory region (845 to þ155 components and the HDAC1 on the promoters of NKD1 and bp) of human NKD1, that contained the EZH2-binding region several other Wnt antagonists (Fig. 3B and Supplementary (Fig. 1B), to a luciferase reporter construct for promoter assay. Fig. S5). In agreement with the loss of these binding activities, Knockdown of EZH2 significantly reduced the H3K27me3

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A Huh7 NL C AXIN2 NKD1 25 600 IP EZH2 EZH2 IgG TBP Input H2O 20 NFD1 400 15

10 200 PPP2R2B 5 0 0 Huh7 NL1 NL2 NL3 Huh7 NL1 NL2 NL3

Figure 2. EZH2 occupancy on the B PPP2R2B SFRP5 Wnt antagonists as a cancer- Huh7 NL1 NL2 60 800 specific silencing event. IP, 400 60 EZH2 40 immunoprecipitation. A, ChIP- 40

PCR analysis of EZH2 and TATA mRNA level Relative β 20 box–binding protein (TBP, positive -Actin 20 control) occupancy in the NKD1 0 0 Huh7 NL1 NL2 NL3 Huh7 NL1 NL2 NL3 and PPP2R2B promoters of Huh7 cells and human normal liver (NL). D B, Western blot analysis of EZH2 AXIN2 CER1 NKD1 expression in Huh7 cells and 15 20 20 human NL tissues. b-Actin was 15 15 used as loading control. C and D, 10 real-time RT-PCR analysis of Wnt 10 10 antagonists in Huh7 cells, human 5 NL tissues, HCC primary tumor 5 5 (T), and matched nontumorous P = 0.0030 P = 0.0002 P = 0.0111 0 0 0 liver (N) tissues. The NT NT NT housekeeping gene GAPDH served as internal control. PPP2CB PPP2R2B SFRP5 8 16 15

6 12 10 Relative mRNA level Relative 4 8 5 2 4 P < 0.0001 P < 0.0001 P < 0.0001 0 0 0 NT NT NT

occupancy (P < 0.001; Supplementary Fig. S6), and it increased and total b-catenin. In the HKCI-10 HCC cell line with high the activity of the NKD1-reporter construct in the PLC5 and activation of Wnt/b-catenin signaling (26), we found that the Hek293 cells (P < 0.005; Fig. 3C). Consistently, real-time RT- knockdown of EZH2 increased the NKD1 protein expression, PCR showed that downregulation of EZH2 significantly with a concurrent reduction in the level of active b-catenin increased the NKD1 mRNA expression in the Huh7 and (Fig. 4A). Although there was no obvious change in the level of PLC5 cells (P < 0.05; Fig. 3D). Because HDAC1 occupancy total b-catenin in the whole cell lysate, the level of b-catenin in was observed on the NKD1 promoter (Fig. 3A), we determined the nucleus was decreased by siEZH2 (Fig. 4B). Furthermore, the NKD1 mRNA level on TSA treatment, which inhibits HDAC the levels of active b-catenin (data not shown) and b-catenin activity and blocks histone deacetylation. As expected, the in the nucleus were markedly decreased by the concomitant NKD1 mRNA level was significantly increased by TSA treat- inhibition of EZH2 and HDAC (Fig. 4B). Moreover, the total ment (P < 0.005; Fig. 3D) and it was further enhanced in b-catenin level in the whole cell lysate was also diminished by combination with EZH2 knockdown (P < 0.005; Fig. 3D). the combination treatment (Fig. 4B). Indeed, most of the EZH2-bound Wnt antagonists were We next examined the effect of EZH2 on the b-catenin/ transcriptionally upregulated after blockage of EZH2 and TCF-dependent transcriptional activity by using TOPflash HDAC in the HCC cells (Fig. 3D). Taken together, our findings luciferase reporter assay (28). Downregulation of EZH2 in suggest that the PRC2 complex cooperates with HDAC to HKCI-10 cells significantly decreased the b-catenin tran- transcriptionally repress Wnt antagonists via histone methy- scriptional activity by more than 40% (P < 0.01), and the lation and deacetylation. suppression was further enhanced in the presence of TSA (P < 0.005; Fig. 4C). To exclude the potential off-target EZH2 allows constitutive Wnt/b-catenin signaling in effects of siRNA, we used an additional siRNA sequence HCC cells against EZH2 (Supplementary Fig. S1) and observed a To determine the effect of EZH2 on Wnt/b-catenin signal- similar suppressive effect on the TCF transcriptional activ- ing in HCC cells, we first conducted Western blot analysis by ity (Supplementary Fig. S7). We further confirmed the using antibodies specific for active (dephosphorylated; ref. 33) effect of EZH2 in Wnt-1/b-catenin–responsive Hek293 cells

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AB O IP H 2 EZH2 EED SUZ12 HDAC1 IgG Input siControl siEZH2 A EZH2

B H3K27me3

C β-Actin siControl D siEZH2

1.4 NKD1 12.0 NKD1 1.2 10.0 1.0 8.0 C 0.8 6.0 2.0 siControl 0.6 1.5 siEZH2 0.4 1.0 250 0.2 0.5 ** ** 0.0 0.0 200 IP:EZH2EED SUZ12 HDAC1 IP: H3K27me3H3K9ac H3K4me3 1.4 PPP2R2B PPP2R2B 150 2.5 1.2 100 1.0 2.0 Relative 0.8 1.5 Relative enrichment level Relative

luciferase activity luciferase 50 0.6 1.0 0.4 0 0.5 PLC5 Hek293 0.2 0.0 0.0 IP: EZH2 EED SUZ12 HDAC1 IP: H3K27me3H3K9ac H3K4me3

Huh7 PLC5 siControl siEZH2 50 *** 8 *** 7 40 6 ** 30 ** 5 20 4 * 10 * 3 2 2 mRNA level 1 1 Relative NKD1Relative 0 0 TSA – + – +

250 Huh7 10 PLC5 200 *** EtOH siControl 150 8 *** EtOH siEZH2 100 *** TSA siControl 50 *** 6 TSA siEZH2 25 20 4 *** ** 15 *** 10 *** 2 Relative mRNA level Relative *** 5 * *** *** N.E. N.E. 0 *** 0 0 3 P5 NLK NLK CER1 CK1aDKK1 NKD1 TP53 CER1 CK1aDKK1 NKD1 TP5 AXIN2 EP300 SFR AXIN2 EP30 P2R2B SFRP5 PPP2CB PPP2CB PPP2R2BPRICKLE1 PP PRICKLE1

Figure 3. Direct transcriptional repression of Wnt antagonists by EZH2 in HCC cells. A, ChIP-PCR analysis of EZH2, EED, SUZ12, and HDAC1 occupancy in the NKD1 promoter of Huh7 cells. Precipitated DNA was amplified with primers spanning sites A to D (Fig. 1B). B, Western blot analysis (above) of EZH2 and H3K27me3 in Huh7 cells following RNA interference. Real-time ChIP-PCR analysis (below) of the PRC2 complex and histone marks occupancy in the NKD1 and PPP2R2B promoters of Huh7 cells following RNA interference. Quantitation of binding was determined as a percentage of input DNAs. N.B., no binding could be detected. C, NKD1 promoter activity of PLC5 and Hek293 cells following RNA interference. Luciferase activity relative to Renilla control was measured. D, real-time RT-PCR analysis of Wnt antagonists expression in Huh7 and PLC5 cells following siRNA and/or TSA treatment. Each error bar represents SD calculated from triplicates. N.E., no expression could be detected. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

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Figure 4. EZH2 allows constitutive Wnt/b-catenin signaling. A, AB Western blot analysis of EZH2, siControl siEZH2 NKD1, and active b-catenin EZH2 EZH2 (b-catenin*) expression in HKCI- 10 HCC cells following RNA NKD1 β-Catenin Whole cell lysate interference. B, Western blot β-Catenin* β analysis of EZH2 and b-catenin -Actin expression in whole cell lysate and β-Actin β-Catenin nucleus of HKCI-10 cells following Nuclear lysate RNA interference and TSA Histone H3 treatment. b-Actin and histone H3 were used as loading controls. C, siEZH2 ––+ + TCF-dependent luciferase activity TSA ––+ + in HKCI-10 cells following siRNA and/or TSA treatment. The relative luciferase activities were the ratios CD of TOP/FOP normalized to the siControl ** siEZH2 120 *** Renilla luciferase activities. Each 120 *** 100 100 siControl siEZH2 error bar represents SD calculated 80 from 3 independent transfections. 80 EZH2 60 60 D, Western blot analysis of EZH2 Relative 40 β-Actin Relative 40 (left) and TCF-dependent 20 20 luciferase activity luciferase luciferase activity (right) in Hek293 0 activity luciferase 0 cells following RNA interference. TSA –+ siControl siEZH2 **, P < 0.01; ***, P < 0.005.

showing the hyperactive b-catenin transcriptional activity Ectopic overexpression of EZH2 activates (Supplementary Fig. S8). As expected, downregulation of Wnt/b-catenin signaling to promote cellular EZH2 by siRNA significantly inhibited the b-catenin tran- proliferation scriptionalactivitybynearly50%(P < 0.001; Fig. 4D), which To further prove the causal relationship between EZH2 and was comparable with the effect of ectopic NKD1 over- Wnt/b-catenin signaling, we used an immortalized human expression (Supplementary Fig. S8). Collectively, these data hepatocyte cell line, MIHA (25), which responded to the Wnt show that EZH2 represses the Wnt antagonist expression ligands (Wnt3a) with an increase in active and total b-catenin and allows constitutive Wnt/b-catenin signaling in HCC levels (Fig. 6A) in concordance with primary liver cells (36). cells. Lentiviral-mediated overexpression of EZH2 in MIHA cells caused an elevation of the active and total b-catenin levels Downregulation of EZH2 inhibits HCC cell growth compared with the EGFP-lentivirus-infected cells (Fig. 6B). partially through inhibition of b-catenin signaling Moreover, EZH2 overexpression significantly induced the We next examined whether Wnt/b-catenin signaling med- expressions of CCND1 and EGFR (Fig. 6B). These data show that iates the pro-proliferative effect of EZH2. Downregulation of EZH2 activates Wnt/b-catenin signaling in MIHA hepatocytes. EZH2 significantly decreased the expression of b-catenin We then tested the effects of b-catenin silencing on EZH2- downstream pro-proliferative target, cyclin D1 (CCND1), in stimulated hepatocellular proliferation. The effectiveness of the HKCI-10 cells (Fig. 5A). In the presence of TSA, the sib-catenin was confirmed by Western blot analysis of b-cate- inhibition of CCND1 expression was further exaggerated nin in EZH2-MIHA cells (Fig. 6C). Moreover, knockdown of (Fig. 5A). Similarly, EZH2 knockdown resulted in a significant b-catenin significantly reduced CCND1 and EGFR expressions decrease of epidermal growth factor receptor (EGFR) expres- (Fig. 6C). Ectopic overexpression of EZH2 significantly sion (Fig. 5B), a liver-specific b-catenin downstream target increased MIHA cell growth (P < 0.05; Fig. 6D). Remarkably, involved in hepatocellular proliferation (34), and the suppres- sib-catenin could significantly attenuate the EZH2-induced sion was further enhanced on the siEZH2 and TSA combina- cellular proliferation (P < 0.05) to the same level as the control tion treatment (Fig. 5B). EGFP-MIHA cells on day 4 posttransfection (Fig. 6D). The We then conducted cell growth assay in HKCI-10 cells with growth-suppressive effect was further validated by using an or without transfection of DP-b-catenin (Fig. 5C), which additional siRNA sequence against b-catenin (Supplementary contains mutations in the serine/threonine residues that have Fig. S7). Collectively, these data show that EZH2 activates Wnt/ been previously shown to affect protein stability (35). The b-catenin signaling to promote cellular proliferation. inactivation of EZH2 inhibited the HCC cell growth by 35% (P < 0.0005), an effect which was significantly reduced in the EZH2 overexpression significantly associates with DP-b-catenin–expressing HCC cells (35% vs. 18%; P ¼ 0.017; b-catenin accumulation and tumor progression in Fig. 5D). Collectively, these data show that EZH2 downregula- human HCCs tion inhibits HCC cell proliferation at least partially via the To validate the linkage between EZH2 and Wnt/b-catenin inhibition of b-catenin signaling. signaling in clinical specimens, we examined the protein

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A B *** siControl *** siControl 1.2 ** siEZH2 1.2 * siEZH2 1.0 1.0 * 0.8 0.8 0.6 0.6 ** 0.4 0.4 mRNA level mRNA 0.2 level mRNA 0.2 Relative CCND1 Relative CCND1 0.0 0.0 CCND1 EGFR TSA – + TSA – +

CD in siControl A3.1 aten 1.5 -c * siEZH2 P-β pcDN D 1.0 β-Catenin

0.5 β-Actin Relative cell number 0.0 pcDNA3.1 DP-β-cat

Figure 5. Downregulation of EZH2 inhibits HCC cell growth at least partially through the inhibition of b-catenin signaling. Real-time RT-PCR and Western blot analysis of (A) CCND1 and (B) EGFR expression in HKCI-10 cells following siRNA and/or TSA treatment. Each error bar represents SD calculated from triplicates. C, Western blot analysis of DP-b-catenin in transfected HKCI-10 cells. b-Catenin expression or control plasmids were transiently transfected into HKCI-10 cells for 48 hours before protein extraction. Arrow indicates the positions of the exogenous b-catenin. The altered electrophoretic mobility of the exogenous b-catenin is likely caused by the amino-terminal epitope tags, as previously described (35). D, effect of ectopic b-catenin overexpression on HKCI-10 cell growth following RNA interference. Relative cell number represents the cell count divided by that of control (siControl-pcDNA3.1). Two independent experiments were conducted and each error bar represents SD for a representative experiment done in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

expression levels of EZH2 and b-catenin by using a TMA EZH2-mediated epigenetic mechanism and support the consisting of 179 human HCCs and matched nontumorous notion that their concordant overexpression contributes to livers. EZH2 was predominantly localized in the nucleus of the the HCC development. HCC cells, whereas the b-catenin accumulation was mainly found in the cytoplasm and, to a lesser extent, both in the Discussion nucleus and cytoplasm (5:1). Overall, EZH2 and b-catenin was expressed in 42% (75/179) and 69% (123/179) of the HCC The canonical Wnt signaling pathway, which regulates the cases, respectively; and coexpression of both proteins was ability of the b-catenin protein to drive the activation of specific found in 34% (61/179) of cases. The majority of EZH2-expres- target genes, is aberrantly activated in the development of sing HCCs (81%) showed an accumulation of b-catenin various human cancers, particularly colorectal and liver can- (Fig. 7A). Notably, EZH2 expression was significantly asso- cers (8–10). In comparison with colorectal cancers, the loss of ciated both with the nuclear and cytoplasmic b-catenin adenomatous polyposis coli rarely occurs in HCC of which expression (both P < 0.005; Supplementary Table S5). In a CTNNB1, AXIN1, and AXIN2 mutations have been suggested to subset of HCCs showing concordant overexpression of EZH2 be the preferred routes to chronic Wnt signaling dysfunction (7, and b-catenin (n ¼ 25), the examination of CTNNB1, AXIN1, 10). However, these genetic defects can be detected in only 3% and AXIN2 hot-spot mutation regions revealed 2 CTNNB1 to 40% of HCCs (37–39), which are far less frequent than those missense mutations (Supplementary Table S6), thus suggest- showing abnormal accumulation of b-catenin, that is, approxi- ing that the b-catenin accumulation in the majority (>90%) of mately 70% (40; data presented herein). Here, we provide these HCCs might not be caused by genetic mutations. evidence for the Wnt/b-catenin signaling activation through Furthermore, the poorly differentiated HCC cases showed an EZH2-mediated epigenetic mechanism. We found a strong stronger EZH2 and b-catenin staining than the moderately positive correlation between the EZH2 and b-catenin expres- and well-differentiated HCC cases (P < 0.05 and 0.005, respec- sion in primary HCCs and showed concordant overexpression tively; Fig. 7B). In contrast, neither EZH2 nor b-catenin nuclear of both proteins in one-third (61/179) of cases. The finding that staining could be detected in the surrounding liver tissues or the majority of the EZH2- and b-catenin–coexpressing HCCs in the normal liver specimens (Supplementary Fig. S9). These do not harbor CTNNB1/AXIN1/AXIN2 mutations further data highlight the significance of Wnt/b-catenin activation by implies that aberrant EZH2 overexpression plays a significant

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EZH2 Activates Wnt/b-Catenin Signaling in HCC

AB a ehicle nt3 V W EGFP-MIHAEGH2-MIHA EGFP-MIHA β-Catenin* EZH2 EZH2-MIHA

β-Catenin β-Catenin* 4 ** 3 β-Actin β-Catenin 2 * CCND1 1

C l 0

o mRNA level Relative tr EGFR CCND1 EGFR -catenin siCon siβ β-Actin β-Catenin D β-Actin siCtl-EGFP-MIHA 9 * * siCtl-EZH2-MIHA siControl 8 siβ-cat-EZH2-MIHA 1.6 siβ-catenin 1.4 7 1.2 6 1.0 5 0.8 4 0.6 3 0.4 2

0.2 cell number Relative 1 Relative mRNA level Relative 0.0 0 CCND1 EGFR 0 2 4 Day (posttransfection)

Figure 6. Ectopic overexpression of EZH2 activates Wnt/b-catenin signaling to promote cellular proliferation. A, Western blot analysis of active (b-catenin*) and total b-catenin expression in MIHA cells. MIHA cells were treated with Wnt3a ligand (250 ng/mL) or vehicle (PBS) for 24 hours before protein extraction. B, Western blot (left) and real-time PCR (right) analysis of CCND1, EGFR, Myc (for EZH2), and active and total b-catenin expression in EGFP- or EZH2-MIHA cells. Each error bar represents SD calculated from triplicates. C, Western blot analysis of b-catenin (above) and real-time RT-PCR analysis of CCND1 and EGFR expression (below) in EZH2-MIHA cells following RNA interference. D, effect of silencing b-catenin on EZH2-stimulated cellular proliferation. Relative cell number represents the cell count divided by that obtained from the day of transfection (Day 0). Two independent experiments were conducted and each error bar represents SD for a representative experiment done in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

role in the oncogenic dysregulation of the Wnt/b-catenin nin level in immortalized human hepatocytes. Our data pathway in human HCC. further show that EZH2 positively regulated the expression Our findings illustrate that the epigenetic repression of the of the b-catenin downstream targets CCND1 and EGFR, both natural Wnt pathway inhibitors by EZH2 allows constitutive by ectopic expression and knockdown experiments. Consis- Wnt/b-catenin signaling. First, ChIP-chip analysis in HCC tent with the role of CCND1 and EGFR in promoting hepato- cells showed that EZH2 bound to the promoters of a panel cellular proliferation, the inactivation of EZH2 significantly of Wnt signal antagonists where the H3K27me3-repressive reduced HCC cell growth which could be partially rescued by mark was also enriched. These natural inhibitors antagonize increasing the b-catenin level. Conversely, EZH2 induced Wnt/b-catenin signaling in different subcellular compart- cellular proliferation in a b-catenin–dependent manner. These ments through the following processes: (i) inhibition of recep- results strongly suggest that EZH2 stimulates HCC cell pro- tor–ligand interactions (CER1, DKK1, and SFRP5); (ii), liferation via activation of Wnt/b-catenin signaling. Further formation of the b-catenin "destruction box" complex (AXIN2, evidence supporting this notion comes from a recent trans- CK1a, PPP2CB, and PPP2R2B); (iii), interference with the genic study which showed that targeted overexpression of intracellular mediator Dishevelled (NKD1, PRICKLE1); and EZH2 in the mammary gland induces b-catenin nuclear (iv) disruption of the b-catenin/TCF complex formation accumulation and causes epithelial hyperplasia (41). (EP300, NLK, and TP53; Supplementary Table S3). Second, Emerging data suggest that histone modifications might EZH2, in cooperation with other PRC2 components and control key epigenetic regulators of the Wnt/b-catenin HDAC, directly altered the histone modifications and tran- signaling pathway in cancers. Jiang and colleagues have scription of Wnt antagonists as shown by RNA interference, recently found that the transcriptional repression of DACT3, ChIP, and gene expression analyses. Third, the simultaneous a Wnt antagonist interacting with Dishevelled, leads to the blockage of EZH2 and HDAC resulted in the transcriptional constitutive activation of Wnt/b-catenin signaling in color- derepression of the Wnt antagonists and the inhibition of the ectal cancer (33). Unlike some Wnt antagonists that are b-catenin/TCF-dependent transcription in the HCC cells. silenced by DNA methylation (42), DACT3 repression in Fourth, ectopic EZH2 expression increased the active b-cate- cancer cells is associated with a bivalent chromatin state

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A Case 1 Case 2

EZH2 β-Catenin EZH2 β-Catenin

CER1 EZH2 3 SFRP5 DKK1 β-Catenin Wnt

2 LRP Frizzled NKD1 AXIN PPP2R2B DVL CK1 GSK3 AXIN2 PPP2CB PRICKLE1 1 CK1 β-Catenin APC

Staining intensity GSK3 β-Catenin β -Catenin β-Catenin β-Catenin 0 β-Catenin β-Catenin NT Well Moderately Poorly degradation

HCC β-Catenin TCF/LEF Wnt target genes Cases (n) 41 122 16 NLK e.g. CCND1, EGFR

Figure 7. EZH2 overexpression significantly associates with b-catenin accumulation and tumor progression in human HCCs. A, immunohistochemical staining of EZH2 and b-catenin in consecutive sections of HCC TMA. Low-power (top, original magnification 50) and high-power (bottom, original magnification 400) pictures of 2 HCC cases. Examples of nuclear and cytoplasmic staining are indicated by pink arrowheads and pink arrows, respectively. B, analysis of EZH2 and b-catenin protein expression in HCCs of different tumor differentiation and their matched nontumorous liver tissues. Immunohistochemical staining was scored semiquantitatively as shown in Supplementary Table S5. Data are represented as mean SEM. *, P < 0.05; ***, P < 0.005. C, a model of EZH2-mediated activation of Wnt/b-catenin signaling in HCC. EZH2 hypersilences Wnt pathway antagonists (grey ovals) operating at several levels, which together releases tumor-suppressive brakes on the oncogenic potency of highly active Wnt/b-catenin signaling.

that can be strongly reactivated by simultaneous pharma- tained by promoter DNA methylation (24). On the other cologic inhibition of histone methylation and deacetylation hand, the observation that AXIN2 repression in HCC (33). Our present data also suggest that both histone methy- wasassociatedbothwithH3K27andDNAmethylation, lation and deacetylation might cooperate for the concordant as reported in other cancers (43), supports the notion transcriptional repression of several Wnt antagonists in that reversible polycomb-mediated repression can result HCC cells. On the one hand, these findings are consistent in stable silencing (44–46). Collectively, the differential with our previous observation that not all genes suppressed patterns of epigenetic marks might reflect the independence by polycomb-mediated methylation are necessarily main- and interplay of gene silencing mechanisms in the

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repression of Wnt antagonists, leading to constitutive Wnt/ the experimental and the bioinformatics methods to decipher b-catenin signaling activation. the cross talk between a master transcriptional regulator and Our integrative genomics analysis indicated 2 other fre- a signaling pathway that are both frequently deregulated in quently deregulated pathways in human HCC namely, the human cancers. mitogen-activated protein kinase (MAPK) and the TGF-b pathways (2, 4–6; Fig. 1A). It is conceivable that the dysregula- Disclosure of Potential Conflicts of Interest tion of multiple signaling pathways cooperatively yields the maximal oncogenic effects of EZH2 in HCCs. Recent studies No potential conflicts of interest were disclosed. have revealed recurrent somatic mutations of EZH2 in hema- Acknowledgments topoietic malignancies (47, 48). Together with the inactivating UTX somatic mutations of the H3K27 demethylase, , in multi- We thank Drs. Anthony Chan, Wei Kang, Martin Chan, Ranxu Zhu, Ms. Daisy ple cancers (49), these findings suggest that a precise balance Tsang, and Wandy Liu for their technical supports; Drs. Henry Chan and Vincent Wong (CUHK) and Dr. Alice Wong from the University of Hong Kong for their of H3K27 methylation is critical for normal cell growth and input and helpful discussion; Drs. B. Vogelstein and K. Kinzler from the Johns differentiation. Further studies should be directed to dissect Hopkins University for the pTOPflash and pFOPflash reporter plasmids; and the oncogenic pathways that are activated by the dysregula- Dr. R. Grosschedl from the University of California, San Francisco for Wnt-1, wild-type-, and DP-b-catenin expression plasmids. tion of H3K27 methylation. In conclusion, this study uncovers an EZH2-mediated epigenetic mechanism that leads to Wnt/b-catenin signaling Grant Support dysregulation in human HCC (Fig. 7C). The complexity of b This work was supported by the General Research Fund (Ref No. CUHK4623/ the Wnt/ -catenin pathway makes it amenable to therapeutic 09M) from the Research Grants Council, the Research Fund for the Control of intervention at many levels (10). Given the molecular diversity, Infectious Diseases (Ref Nos. 09080042 and 08070332) from the Food and Health Bureau, and the Direct Grant (Ref No. 2007.1.033) from the Chinese University of cancer specificity and reversibility of the Wnt antagonist Hong Kong. repression by EZH2, it might be prudent to combine drugs The costs of publication of this article were defrayed in part by the payment that block PRC2-mediated gene silencing (50) or viral vectors of page charges. This article must therefore be hereby marked advertisement in targeting EZH2 (21) with conventional chemotherapy for accordance with 18 U.S.C. Section 1734 solely to indicate this fact. effective treatment of Wnt-addicted cancers such as HCC. Received September 10, 2010; revised March 29, 2011; accepted March 30, These findings also exemplify the power of integrating both 2011; published OnlineFirst April 21, 2011.

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OF12 Cancer Res; 71(11) June 1, 2011 Cancer Research

EZH2-Mediated Concordant Repression of Wnt Antagonists Promotes β-Catenin−Dependent Hepatocarcinogenesis

Alfred S.L. Cheng, Suki S. Lau, Yangchao Chen, et al.

Cancer Res Published OnlineFirst April 21, 2011.

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