aberrations of ezh2 in cancer - clinical cancer research

Molecular Pathways

Aberrations of EZH2 in Cancer

Andrew Chase and Nicholas C.P. Cross

AbstractControl of gene expression is exerted at a number of different levels, one of which is the accessibility of

genes and their controlling elements to the transcriptional machinery. Accessibility is dictated broadly by

the degree of chromatin compaction, which is influenced in part by polycomb group proteins. EZH2,

together with SUZ12 and EED, forms the polycomb repressive complex 2 (PRC2), which catalyzes

trimethylation of histone H3 lysine 27 (H3K27me3). PRC2 may recruit other polycomb complexes,

DNA methyltransferases, and histone deacetylases, resulting in additional transcriptional repressive marks

and chromatin compaction at key developmental loci. Overexpression of EZH2 is a marker of advanced

andmetastatic disease inmany solid tumors, including prostate and breast cancer. Mutation of EZH2 Y641

is described in lymphoma and results in enhanced activity, whereas inactivatingmutations are seen in poor

prognosis myeloid neoplasms. No histone demethylating agents are currently available for treatment of

patients, but 3-deazaneplanocin (DZNep) reduces EZH2 levels and H3K27 trimethylation, resulting in

reduced cell proliferation in breast and prostate cancer cells in vitro. Furthermore, synergistic effects are seen

for combined treatment with DNA demethylating agents and histone deacetylation inhibitors, opening up

the possibility of refined epigenetic treatments in the future. Clin Cancer Res; 17(9); 2613–8.�2011 AACR.

Background

DNA is wrapped around histone complexes termednucleosomes, and gene accessibility is determined largelyby the local chromatin configuration. This configurationmay impact on the ability of cofactors and enhancers tobind specific DNA sequences, either via direct interactionswith nucleosome components or, more generally, by influ-encing the degree to which DNA and/or nucleosome com-plexes are compacted into higher structures and, thus, thephysical accessibility of specific recognition sequences.Local chromatin structure is influenced, in part, by histoneusage but principally by covalent modifications to histonetails and DNAmethylation. Both PcG and Trx proteins playimportant roles in this process bymodifying lysine residueswithin histone tails, resulting in repression or enhance-ment of transcription.Two histone modifications, in particular, play crucial

opposing roles in the epigenetic control of proliferationand differentiation. Trimethylation of histone H3 lysine 27(H3K27me3), catalyzed by the PcG enhancer of zestehomolog 2 (EZH2), is associated with transcriptionalrepression, whereas H3K4 methylation, catalyzed by the

trithorax homolog myeloid-lymphoid leukemia (MLL), isassociated with transcriptional activation (Fig. 1A). Impor-tantly, many genes involved in development, stem cellmaintenance, and differentiation are targets of H3K27and H3K4 methylation. In embryonic (1, 2) and hemo-poietic (3) stem cells (HSC), promoters may carry bothmarks, termed a "bivalent" domain, and genes thusmarkedare thought to be poised for transcriptional activation orrepression with loss of H3K27 or H3K4 methylation,respectively.

H3K27 methylation is catalyzed by the SET domain ofEZH2 and requires the presence of 2 additional proteins,embryonic ectoderm development (EED) and suppressorof zeste 12 (SUZ12). These proteins, together with thehistone binding proteins retinoblastoma binding protein4 (RBBP4) and RBBP7, comprise the core components ofthe polycomb repressive complex 2 (PRC2; Fig. 1B). Otherproteins such as PHD finger protein 1 (PHF1), whichspecifically stimulates H3K27 trimethylation rather thandimethylation (4), sirtuin 1 (SIRT1; ref. 5), and jumonji, ATrich interactive domain 2 (Jarid2; refs. 6, 7) have also beendescribed in PRC2 complexes and probably serve modulat-ing functions. A second polycomb complex, PRC1, cata-lyzes ubiquitination of histone H2A K119, which is alsoassociated with a repressive chromatin structure. PRC1consists of several proteins including chromobox homolog2 (CBX2) or related homologs, which bind H3K27me3,ring finger protein 1A (RING1A), or RING1B, which cat-alyze ubiquitination and BMI1 polycomb ring finger onco-gene (BMI1) or polycomb group ring finger 6/Mel18(PCGF2), which modulate ubiquitination activity.

According to the hierarchical model of PRC recruitmentfirst developed through genetic studies in Drosophila (8),

Authors' Affiliation: Wessex Regional Genetics Laboratory, Salisbury,and Human Genetics Division, University of Southampton Faculty ofMedicine, Southampton, United Kingdom

Corresponding Author: Andrew Chase, Wessex Regional GeneticsLaboratory, Salisbury District Hospital, Salisbury SP2 8BJ, United King-dom. Phone: 44-1722 336262; Fax: 44-1722 338095; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-10-2156

�2011 American Association for Cancer Research.

ClinicalCancer

Research

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Published OnlineFirst March 2, 2011; DOI: 10.1158/1078-0432.CCR-10-2156

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Figure 1. A, PRC2 catalyzes H3K27me associated with transcriptional repression, whereas myeloid-lymphoid leukemia catalyzes methylation of H3K4associated with transcriptional activation. In bivalent domains, both marks are present and associated with a low level of transcription; genes thus marked arethought to be poised for later transcriptional repression or activation. B, the PRC2 core components are EZH2, SUZ12, EED, and RBBP4 or RBBP7. C,PRC2 is recruited to DNA, resulting in H3K27me3. This mark facilitates recruitment of PRC1, which ubiquitinates H2AK119, DNMT, and HDACs, whichcontribute to chromatin compaction and transcriptional repression. D, Levels of EZH2 and H3K27me3 can be indirectly reduced by treatment with DZNep,resulting in transcriptional derepression.

Chase and Cross

Clin Cancer Res; 17(9) May 1, 2011 Clinical Cancer Research2614




PRC2 is first recruited to target genes and catalyzes methy-lation of H3K27. This step is followed by recruitment ofPRC1 and ubiquitination of H2AK119 (Fig. 1C). Thismodel would predict that targets of PRC1 and PRC2 largelyoverlap, as has indeed been shown by RNA interference(RNAi) depletion studies in human embryonic fibroblasts(9). In HSCs, however, there is evidence for opposing rolesfor PRC1 and PRC2 (10–13). BMI1 is required for HSCrepopulation (10, 12), whereas reduced levels of Suz12,Ezh2, or Eed result in increased HSC-progenitor prolifera-tion (13). In addition, genes known to be repressed byCCAAT/enhancer binding protein alpha (CEBPA) or acti-vated by homeobox A9 (HoxA9) have been found to bedifferentially regulated by PRC1 and PRC2 (13). It iscurrently not known whether the differential effects ofPRC1 and PRC2 seen in HSC also occur in other tissues.Although PRC2 in Drosophila is recruited to defined

Polycomb Response Elements upstream of target genes,similar motifs have not been identified in vertebrates. It hasbeen suggested that recruitment may occur via intermedi-ary molecules, with evidence for involvement of the longnoncoding RNA hox transcript antisense RNA (HOTAIR;refs. 14, 15), nuclear inhibitor of protein Ser/Thr phospha-tase-1 (NIPP1; ref. 16), Pho repressive complex (PhoRC;refs. 8, 17), and Jarid2 (18).It is clear that activating and repressive histone methyla-

tion marks do not act in isolation but are intimatelycoordinated with the action of other epigenetic modifica-tions (Fig. 1C). EZH2 can bind the DNAmethyltransferasesDNMT1, DNMT3A, andDNMT3B (19), which can result inDNA methylation, at least in certain circumstances (20).Knockdown of EZH2 can result in reexpression of geneswith H3K27 methylation and minimal DNA methylationbut not genes that are hypermethylated (21). Recruitmentof histone deacetylase (HDAC) proteins by PRC2 has beensuggested as a further mechanism for transcriptionalrepression. PRC2 can bind HDAC via EED (22), andtreatment of nonerythroid cells with the HDAC inhibitortrichostatin A results in depletion of PRC2 and induction ofa-globin expression, a locus that is normally tightlyrepressed andmarked by hypoacetylation and the presenceof PRC2 and H3K27me3 (23).

EZH2 and cancerOverexpression of EZH2 was first linked to cancer by

microarray studies of prostate cancer (24) and breast cancer(25). In prostate cancer, EZH2 overexpression is associatedwith aggressive and metastatic disease. Similarly in breastcancer, EZH2 expression is elevated in invasive carcinomaand metastases, and increased expression is strongly asso-ciated with a poor clinical outcome (25). Overexpression ofEZH2 has also been described in other cancers includingbladder (26–28), gastric (29), lung (30), and hepatocel-lular carcinoma (31). Deletions of microRNA-101, a nega-tive regulator of EZH2 expression, have been described inprostate cancer, thus providing a mechanism for EZH2overexpression (32). Experimental support for the onco-genic action of EZH2 has been provided by induction of

anchorage-independent colony growth and promotion ofinvasion in vitro by overexpression of EZH2 in the breastepithelial cell line H16N2 (25). In addition, ectopic EZH2increased the proliferation of mouse embryonic fibroblasts(33).

A remarkable connection has been described betweenPRC2 occupancy and the aberrant methylation of CpGislands at promoters seen in some cancers (34–36). Pro-moters found to have aberrant CpG island hypermethyla-tion were also found to be enriched for PRC2 and H3K27methylation. These sites were also found to be preciselythose occupied by PRC2 in stem cells. Because PRC2 canrecruit DNMTs (19), it seems that PRC2 may act as aplatform for aberrant de novo promoter methylation,although the precise contexts in which this occurs is stillunclear.

EZH2 mutationsRecent work has identified acquired EZH2 mutations in

lymphoma and myeloid neoplasms. In lymphoma, a het-erozygous missense mutation at amino acid Y641, withinthe SET domain, was initially identified by high throughputtranscriptome sequencing. Various heterozygous muta-tions at Y641 were subsequently found in 7% of follicularlymphomas and 22%of diffuse large cell B-cell lymphomasof germinal center origin (37). Mutations elsewhere inEZH2 were not seen. Although initially thought to resultin a loss of catalytic activity, more detailed analysis showedthat Y641 mutants actually result in a gain of function.Specifically, normal EZH2 displays greatest catalytic activityfor monomethylation of H3K27 and relatively weak effi-ciency for the subsequent (mono- to di- and di- to tri-methylation) reactions. Remarkably, Y641 mutants displayvery limited ability to do the first methylation reaction buthave enhanced catalytic efficiency for the subsequent reac-tions (38). Heterozygous Y641 mutants, thus, work inconjunction with wild-type EZH2 to increase levels ofH3K27me3 and may be functionally equivalent to EZH2overexpression.

In myeloid neoplasms, mutations have been describedlargely in poor prognosis myelodysplasia-myeloprolifera-tive neoplasms (10 to 13%), myelofibrosis (13%), andvarious subtypes of myelodysplastic syndromes (6%; refs.39, 40). In contrast to the mutation of a single residue andgain of function seen in lymphoma, mutations were spreadthroughout the gene and comprised missense, nonsense,and premature stop codons, some of which were homo-zygous. Because the catalytic SET domain lies at the far Cterminus, all nonsense and stop codonmutations would bepredicted to result in loss of histone methyltransferaseactivity and, thus, are clearly different functionally fromY641 mutants. The fact that both activating and inactivat-ing mutations of EZH2 are associated with malignancy isremarkable and reflects the complex role that PcG genesplay in cell fate decisions (41).

Mutations of other PRC2 components have notbeen reported, although SUZ12 (also known as JJAZ1)fuses to juxtaposed with another zinc finger 1 (JAZF1) in


www.aacrjournals.org Clin Cancer Res; 17(9) May 1, 2011 2615




endometrial stromal tumors (42). In addition, mutationshave been reported in a growing number of genes encodingfunctionally related epigenetic components. For example,inactivating mutations of ubiquitously transcribed tetratri-copeptide repeat gene on X chromosome (UTX), an H3K27demethylase, are seen in a wide range of malignancies andmay also be functionally equivalent to EZH2 overexpression(43). Inactivating mutations of the PRC2 downstream sub-strate DNMT3A are seen in acute myeloid leukemia (AML;ref. 44), and other PcG members such as ASXL1 (additionalsex combs-like 1) aremutated inmyeloidmalignancies (45).

Clinical-Translational Advances

The therapeutic modulation of epigenetic marks has ledto improvements in treatment of some cancers, for exam-ple, the use of DNA demethylating agents azacytidine anddecitabine for myelodysplastic syndrome (46, 47) and theHDAC inhibitor suberoylanilide hydroxamic acid (vorino-stat) for cutaneous T-cell lymphoma (48). In contrast toDNA demethylating agents andHDAC inhibitors, no thera-pies that directly target histone methylation are clinicallyavailable, despite the fact that there is experimental evi-dence for a potential therapeutic benefit for this approach.Most experimental work on the inhibition of EZH2 activityhas used the carbocyclic adenosine analog 3-deazanepla-nocin (DZNep), a derivative of the naturally occurringantibiotic neplanocin-A, in which ribose is replaced by acyclopentyl ring (49). DZNep inhibits S-adenosylhomo-cysteine hydrolase, a component of the methionine cycle,resulting in accumulation of the inhibitory S-adenosylho-mocysteine with a knock-on disruption of methylation ofsubstrates by EZH2 (Fig. 1D). The effect of DZNep uponhistone methylation is, therefore, global rather than EZH2specific (50). A comparison of DZNep action and specificknockdown of EZH2 by targeted RNAi has shown inter-esting differences; DZNep reduces levels of EZH2 proteinbut not the mRNA, whereas RNAi reduces both protein andmRNA levels. Both strategies result in loss of H3K27methylation but differ somewhat in the pattern of genesthat are reexpressed (51). Inhibition of EZH2 by eitherDZNep or RNAi leads to reduced proliferation in a subsetof breast cancer cell lines (51) and reduced proliferationand invasiveness in prostate cancer cell lines (24, 51, 52).

After initial work on DZNep as a single agent histonemethylation inhibitor, in vitro experimental work pro-gressed to the use of combinatorial approaches to modifyhistonemethylation, acetylation, andDNAmethylation. Inbreast cancer cell lines, DZNep treatment and HDACinhibition result in synergistic effects upon proliferation,apoptosis, differentiation, and cell cycle arrest (53). Chro-matin immunoprecipitation experiments have shown dif-

ferential effects on gene reexpression patterns with differentcombinatorial approaches with DZNep, DNA demethylat-ing agents, and HDAC inhibitors (54). In AML, combinedtreatment with DZNep and the HDAC inhibitor panobino-stat induced a synergistic apoptotic effect upon primaryleukemia cells compared with normal cells (55). The com-plexity of the interactions between different epigeneticmarks is highlighted by recent work showing downregula-tion of BMI1 and EZH2, by treatment with the HDACinhibitors valproic acid or sodium butyrate by mechanismsthat are currently unclear (56).

The concept of "synthetic lethality" refers to the depen-dency of a tumor on the presence of either 1 of 2 genes, butwith resulting cell death if both are lost. Recently, a possible"synthetic lethal" relationship between EZH2 and BRCA1was described. BRCA1 plays a role in differentiation ofbreast stem cells in addition to a well-documented role inDNA repair. In amousemodel of basal-type tumors (whichare generally poorly differentiated and do not expressERBB2, estrogen, or progesterone receptors and are, there-fore, without rational therapeutic targets), EZH2 expressionwas found to be higher in BRCA1-deficient tumors com-pared with those that were wild-type. Remarkably, knock-down of EZH2 by RNAi or by treatment with DZNep was20 times more efficient at killing BRCA1-deficient cells(57). Further work has shown that EZH2 knockdowncan result in increased BRCA1 expression with a concomi-tant contribution to reduced proliferation (58).

Although the development of histone demethylatingagents is an area of great interest, currently no compoundsare approved for treatment or in clinical trials. However, itis clear that modulation of EZH2 levels and H3K27 methy-lation can be achieved by therapies primarily directed atDNA methylation or histone acetylation. Although there isan obvious rationale for inhibiting EZH2 function inmalignancies in which this gene is overactive as a conse-quence of overexpression or Y641 mutation, the finding ofinactivating mutations in myeloid neoplasms suggests thatthis approach will need to be employed with caution.Understanding how to effectively combine current treat-ments with future histone demethylating agents will be amajor challenge in the coming years.

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.

Received November 24, 2010; revised January 10, 2011; accepted January11, 2011; published OnlineFirst March 2, 2011.

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2011;17:2613-2618. Published OnlineFirst March 2, 2011.Clin Cancer Res Andrew Chase and Nicholas C.P. Cross

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