EZH2: Not EZHY (Easy) to Deal

Home , DNA methylation, Enhancer (genetics), EZH2, Histone code, Histone methylation, Inducer

Published OnlineFirst February 13, 2014; DOI: 10.1158/1541-7786.MCR-13-0546

Molecular Cancer Review Research

Gauri Deb1,4, Anup Kumar Singh5, and Sanjay Gupta1,2,3

Abstract Seminal discoveries have established that epigenetic modifications are important for driving tumor progression. Polycomb group (PcG) proteins are highly conserved epigenetic effectors that maintain, by posttranslational modification of histones, the silenced state of genes involved in critical biologic processes, including cellular development, stem cell plasticity, and tumor progression. PcG proteins are found in two multimeric protein complexes called Polycomb repressive complexes: PRC1 and PRC2. Enhancer of zeste homolog 2 (EZH2), catalytic core subunit of PRC2, epigenetically silences several tumor-suppressor genes by catalyzing the trimethylation of histone H3 at lysine 27, which serves as a docking site for DNA methyltransferases and histone deacetylases. Evidence suggests that overexpression of EZH2 is strongly associated with cancer progression and poor outcome in disparate cancers, including hematologic and epithelial malignancies. The regulatory circuit and molecular cues causing EZH2 deregulation vary in different cancer types. Therefore, this review provides a comprehensive overview on the oncogenic role of EZH2 during tumorigenesis and highlights the multifaceted role of EZH2, as either a transcriptional activator or repressor depending on the cellular context. Additional insight is provided on the recent understanding of the causes and consequences of EZH2 overexpression in specific cancer types. Finally, evidence is discussed on how EZH2 has emerged as a promising target in anticancer therapy and the prospects for targeting EZH2 without affecting global methylation status. Thus, a better understanding of the complex epigenetic regulatory network controlling EZH2 expression and target genes facilitates the design of novel therapeutic interventions. Mol Cancer Res; 12(5); 639–53. 2014 AACR.

Introduction alterations in DNA, which do not alter the nucleotide More than five decades ago, C.H. Waddington defined sequence, are inheritable and potentially reversible, unlike genetic changes. The transcription state of any gene may be "Epigenetics" as a discipline, which aims to describe changes fi in the development of organisms that could not be explained predicted by deciphering the histone modi cation pattern at by the changes in DNA sequence. Local chromatin config- the promoter, which is often referred to as "Histone Code" uration at the gene promoter determines the accessibility of (for a review see ref. 2). transcription machinery and associated proteins to bind to The Polycomb group (PcG) of proteins and their counter- the specific DNA sequences. They either directly interact parts, the Trithorax group (TrxG) of proteins, have long been with the nucleosome components or modulate the degree of found to be associated with the establishment of heritable compaction of nucleosome complexes into higher structures. gene transcription patterns. The antagonistic activities of the DNA methylation at CpG islands in the promoter region PcG proteins that act as epigenetic repressors and TrxG and covalent chemical modification of less structured, pro- families of proteins function as epigenetic activators, resulting truding N-terminal tails of core histones by methylation, in the maintenance of the spatial patterns of homeotic gene acetylation, ubiquitylation, and phosphorylation at certain expression in Drosophila, throughout development and adult- amino acid residues have emerged as two integral mechan- hood (3, 4). PcG proteins represent evolutionarily conserved isms of epigenetic regulation that principally modulate local multiprotein complexes that include the Polycomb repressive chromatin structure (for a review see ref. 1). These epigenetic complexes, PRC1 and PRC2. Histone H3 lysine 27 trimethylation (H3K27me3) is a distinct histone modification catalyzed by a histone methyltransferase (HMTase) enhancer Authors' Affiliations: 1Department of Urology, University Hospitals Case Medical Center; 2Department of Nutrition, Case Western Reserve Univer- of zeste homolog 2 (EZH2), a catalytic component of PRC2, sity; 3Division of General Medical Sciences, Case Comprehensive Cancer involved in the regulation of homeotic (Hox) gene expression Center, Cleveland, Ohio; 4Department of Biotechnology, Indian Institute of and in the early steps of X-chromosome inactivation in Technology, Guwahati, Assam; and 5Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India women (5, 6). Given the essential role of PcG proteins in establishing the repressed state of several genes during devel- Corresponding Author: Sanjay Gupta, University Hospitals Case Medical Center, Case Western Reserve University, 2109 Adelbert Road, Wood opment and maintenance of embryonic stem cell (ESC) Research Tower, 10900 Euclid Avenue, Cleveland, OH 44106. Phone: identity and pluripotency, recent studies imply that PcG 216-368-6162; Fax: 216-368-0213; E-mail: [email protected] proteins are often deregulated in various cancer types, and doi: 10.1158/1541-7786.MCR-13-0546 their overexpression is closely associated with carcinogenesis 2014 American Association for Cancer Research. (reviewed in refs. 7–9).

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In this review, we comprehensively discuss the role of one EZH2 and its role in the PRC2 complex of the most important component of the PRC2 complex, The composition of the PRC2 complex is dynamic, viz., EZH2, in the tumorigenesis of different cancer types. consisting of some core subunits responsible for catalyzing After evaluating the role of EZH2 in the mammalian PRC2 H3K27me3 and several accessory regulatory subunits con- complex, we systematically discuss its function and interac- trolling the enzymatic activity and function of the holoezyme tion with other epigenetic modiﬁers and elaborate in detail (10, 11). The core components of the mammalian PRC2 the causes and consequences of EZH2 overexpression, complex include EZH1/2, suppressor of zeste 12 (SUZ12), focusing on each cancer type. Recent literature focused on embryonic ectoderm development (EED), and retinoblas- studying the molecular circuit guiding EZH2 expression and toma (Rb)-associated protein 46/48 (RbAp46/48). Other function in normal cells, and its deregulation in tumor cells is accessory units that regulate PRC2 enzymatic activity and also highlighted. Finally, owing to the pivotal "oncogenic" function include AEBP2, PCLs, and JARID2 (Fig. 1). role of EZH2 in the development and progression of several EZH2 is the catalytic core of the PRC2 complex that cancer types, recent literature highlighting the potential of catalyzes the H3K27me3 repressive chromatin mark. Both EZH2 as a promising cancer target is discussed. genetic and biochemical evidence suggests that EED

Figure 1. Epigenetic cross-talk showing a functional link between PcG proteins (PRC1 and PRC2), HDACs, and DNMTs during transcriptional silencing of a PRC2 target gene. Initial deacetylation of histone H3 acetylated at lysine 27 (H3K27Ac) by HDACs allows the catalysis of trimethylation of lysine 27 (H3K27me3) by EZH2 containing the PRC2 complex. H3K27me3 enrichment facilitates the binding of the PRC1 complex that catalyzes the H2AK119ub and may also serve as a docking site for DNMTs, which methylate CpG islands in the promoter region. Methylated CpG islands are then bound by a group of proteins possessing methyl DNA-binding characteristics such as methyl CpG-binding protein 2 (MeCP2), methyl CpG-binding domain protein 1 (MBD1), which form part of other histone-modifying complexes such as NURD and Sin3A. These events assist in the recruitment of HDACs and HMTase-containing protein complexes, contributing further to target gene repression. H3K27me3 and occupancy of target promoter by other histone-modifying proteins lead to the formation of a highly compressed chromatin structure that is inaccessible to RNA Pol II, causing transcriptional silencing of the target gene.

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physically associates with EZH2 and histone H3, and thus loss of di- and trimethylation on H3K27, indicating the functions as a scaffold protein. Molecular analysis of the presence of another HMTase catalyzing methylation on EZH2–EED interaction by X-ray crystallographic studies H3K27 (24). They reported EZH1 to be an HMTase that revealed that the EED-binding domain of EZH2 (EBD) is physically present in a noncanonical PRC2 complex, binds to the bottom of the Trp–Asp (WD) repeat domain of both in vivo and in vitro, and prevents derepression of PRC2 EED (12). SUZ12 is required for the nucleosome recogni- target genes. Margueron and colleagues investigated the tion, activity, and stability of the PRC2 complex in both cellular role of EZH1 in comparison with EZH2 and showed in vivo and in vitro conditions (13, 14). In addition, other that EZH1 is ubiquitously expressed, whereas the expres- PRC2 members, such as RbAp46/48, are involved in histone sion of EZH2 is found in proliferating tissues (23). Also, as binding, and AEBP2, a zinc-finger protein, enhances the part of similar PRC2 complexes, they control an overlapping enzymatic activity of the PRC2 complex (11, 15). set of PRC2 target genes. Surprisingly, the functional roles Human EZH2 is a 746 amino acid protein belonging to of EZH1 and EZH2 were distinctly different; PRC2– the histone–lysine methyltransferase family, all having a EZH1 exhibit low histone–lysine methyltransferase activity conserved SET (Su(var)3–9 enhancer of zeste trithorax) as compared with PRC2–EZH2, which may be attributed domain. In addition to the SET domain at the C-terminus, to their differential expression patterns and subfunc- EZH2 contains several other functional domains, such tionalization of Ez during evolution (23). This indicates as CXC (cysteine-rich domain) and ncRBD (noncoding that PRC2–EZH2 function in establishing the cellular RNA– and a DNA-binding domain), required for its inter- H3K27me3 repressive marks on PRC2 target genes, while action with other PRC2 members and regulatory proteins PRC2–EZH1 may help restore the repressive methylation (11, 15–17). Structural and biochemical analysis of SET pattern (H3K27me2/me3) of histones after demethylase domains in various HMTase has unraveled the molecular activity or histone exchange that might cause the loss of mechanism of histone methylation. Such studies highlight- their methylation mark. ed the presence of a conserved catalytic triad—the Asp–His– Ser (NHS) motif—responsible for recognition of the EZH2- and PRC2-dependent H3K27 trimethylation amino acid sequence of the histone peptide tail and for the The unstructured histone tails protruding from the nucle- binding of S-adenosyl-methionine (SAM; refs. 18, 19). osome assembly are subjected to various PTMs, including Mutation of any of these residues in the active site of lysine methylation, which alter the physical state of chro- EZH2 abolishes its HMTase activity. Focusing on highly matin and thus modulate gene expression. Lysine residues in evolutionarily conserved Tyr641 of EZH2, Yap and collea- the histone tail may be present in the unmethylated form gues demonstrated that EZH2 Y641–mutant protein-con- or covalently modified by HMTases into mono-, di-, and taining PRC2 complexes display enhanced H3K27me3 trimethylated lysine (H3K27me1, H3K27me2, and activity on dimethylated peptides (and not on unmethylated H3K27me3), with each form functionally different from histone peptides) as compared with wild-type containing the other. Methylation of histone H3 lysine 27 by the SET PRC2 complexes, which ultimately shifts the steady state domain of EZH2 (as part of the PRC2 complex) has been of H3K27 in favor of trimethylation in vivo (20). The reported to be a processive event in which H3K27me3 presence of the wild-type EZH2–PRC2 complex was found results from monomethylation of H3K27me2 (25). Di- and to be mandatory for the Y641 EZH2 mutant to act, so that trimethylated forms of histone H3 lysine 27 are associated previously methylated histone substrates remain available with a facultative heterochromatin region, whereas the for trimethylation. Several posttranslational modifications monomethylated form is associated with stably silent con- (PTM) of the EZH2 protein have also been reported to stitutive heterochromatin (26, 27). In ESCs, 50% of the H3 alter its H3K27me3 activity. For example, phosphorylation histone was reported to be dimethylated, 15% trimethy- of EZH2 by Akt1 at serine 21 reduces H3K27me3 activity, lated, and 15% monomethylated, which add up to a total of whereas phosphorylation of Thr 345 by CDK1 and CDK2 80% methylated histone H3 (28). H3K27me3 is a stable is required for the maintenance of H3K27me3 repressive repressive chromatin mark catalyzed by PRC2 complexes marks at target gene promoters (21, 22). containing either EZH1 or EZH2. H3K27me2, which is Notably, besides few exceptions, most invertebrates have also catalyzed by PRC2, represents an intermediary product only single copy of PcG genes; however, in vertebrates, that not only serves as a substrate for subsequent H3K27me3 multiple copies of PcG genes have been reported. Among but might also inhibit acetylation of H3K27, which is PRC2 components, mammalian homologs EZH1 and reported to be antagonistic to PRC2-mediated gene repres- EZH2 have been reported to be paralogs that remain an sion (29, 30). In plants, two monomethyltransferases, name- integral part of the PRC2 complex, but are generally asso- ly Arabidopsis trithorax–related protein 5 (ATXR5) and ciated with contrasting H3K27me2/3 repressive roles (23). ATXR6, which are not orthologous to Ez, are involved in Although EZH1 was the first Ez homolog to be cloned, it is generating H3K27me1 mark; but in mammals the appear- not as well characterized as its mammalian homolog EZH2 ance of H3K27me1 is still controversial (31). Some reports or its counterpart Drosophila Ez. Shen and colleagues dem- speculate that, in mammals, H3K27me1 might be catalyzed onstrated that EZH2 conditional knockout allele containing by an enzymatic activity distinct from PRC2, and its ESCs (EZH2 / ) sustains H3K27me3 at some develop- presence in actively transcribed genes may result from mental genes and displays H3K27me1, despite the global H3K27me2/3 demethylation by the demethylases UTX or

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JMJD3 (32). In contrast, few reports suggest that in Dro- Akt1 at serine 21, which allows its association with AR. sophila, the generation of the H3K27me1 mark is dependent Furthermore, it was demonstrated that depletion of EZH2 on Ez, while in mammals the PRC2 complex containing causes a decrease in AR-associated lysine methylation at either EZH1 or EZH2 function redundantly controls the lysine 630 and 632, but the AR levels remain unchanged. global H3K27me1 levels. EED was also shown to be The noncanonical role of EZH2 independent of its required for the H3 lysine monomethylation (24, 33). HMTase activity was also observed in a highly aggressive Further experimental studies in mammals are needed to lymphoid malignancy called natural killer/T-cell lymphoma characterize PRC2-type complexes capable of HMTase (NKTL). Interestingly, EZH2 overexpression in NKTL was activity, so that the origin and function of H3K27me1 mark found not to be associated with H3K27 trimethylation (41). could be comprehensively understood. Also, in NKTL cell lines, ectopic expression of EZH2 Supported by various experimental studies in the past mutant lacking HMTase activity was found to confer growth several years, it is a well-established fact that H3K27me3 advantage and prevent growth inhibition upon endogenous enrichment is associated with gene silencing (34). Probing EZH2 depletion (41). Jacob and colleagues showed that in into the molecular mechanism reveals that this chromatin differentiated Th cells, binding of polycomb proteins YY1, mark may serve as a docking site and facilitate the binding of Mel-18, Ring1A, Ezh2, and Eed to the Il4 and Ifng gene loci the PRC1 complex that catalyze the monoubiquitylation exhibited a differential pattern and induced transcription of histone 2A at lysine 119 (H2AK119ub), and thus main- (42). In summary, recent findings support the hypothesis tain repressed state of the target gene (Fig. 1). Also, the that EZH2 functions as a dual-faced molecule, which may H3K27me3 mark might indirectly regulate transcription by function both as a transcriptional repressor or activator posing a steric hindrance to the binding of transcription depending on its association with other members of the machinery to the promoter region of the target gene. The PRC2 complex and cellular context. presence of RNA polymerase II (RNA Pol II) phosphorylated at serine 5 of its C-terminal tail in a wide fraction of EZH2 and its interaction with other epigenetic modifiers H3K27me3-enriched promoters and low transcript levels Early evidence in Drosophila and Caenorhabditis elegans suggests that RNA Pol II might be paused at PcG-targeted showed that polycomb-mediated H3K27me3 was deployed genes causing transcriptional repression (35). Studies reveal in transcriptional silencing, but DNA methylation was that the RNA Pol II transcription complex that is recruited to absent in their chromatin, emphasizing the notion that the PcG target genes gets involved in early transcription but polycomb-based silencing was independent from DNA fails to proceed to the elongation stage. Kanhere and col- methylation (43). However, recent studies have completely leagues demonstrated that short RNAs that are transcribed changed this outlook, and it has now become apparent that from the 50 end of polycomb target genes marked by the there exists a cross-talk between various epigenetic modifiers, transcription initiation marker H3K4me3 form a stem-loop such as DNA methylation and histone modification path- structure resembling the PRC2-binding site that could ways, which are mediated at the molecular level by bio- recruit and interact with the PRC2 complex (36). chemical interactions between DNA methyltransferases (DNMT) and SET domain containing HMTase, respec- EZH2- and PRC2-independent function tively (Fig. 1). A key study by Vire and colleagues first Apart from the canonical role of EZH2 as a transcriptional described that PRC2-dependent EZH2-mediated silencing repressor, recent literature reports highlight its less-known and DNA methylation systems are mechanistically linked function as a transcriptional activator. In our previous (44). Using glutathione S-transferase (GST) pull-down review, we have comprehensively discussed the mechanism assays, they demonstrated that all three DNMTs (in vitro of action of EZH2 as a transcriptional inducer, where it translated 35S-labeled DNMT1, DNMT3A, and functions independently (not as part of the PRC2 complex), DNMT3B) can interact and bind to full-length GST– in endocrine-related cancers of the breast and prostate EZH2. Furthermore, they showed that PRC2 members (reviewed in ref. 37). Depending on the estrogen receptor EZH2 and EED coimmunoprecipitated with all three (ER) status of the breast cancer cells, the EZH2-mediated DNMTs in HeLa nuclear extracts. The physical interaction target gene activation mechanism varies. In ER-positive, between EZH2 and DNMTs facilitated the binding of the luminal-like MCF-7 cells, it was demonstrated that EZH2 DNMTs to EZH2 target genes, such as MYT1 and WNT1, physically links b-catenin and T-cell factor (TCF; ER-a and and was essential for CpG methylation of the target pro- Wnt signaling components) on the target gene promoters of moters. Interestingly, the EZH2–DNMT interaction was cyclin B1 and c-Myc. The domain II of EZH2 interacts with reported to be dispensable for EZH2 binding to the target the mediator complex and induces transcription (38). How- gene promoter, suggesting that EZH2 acts upstream in the ever, in ER-negative MDA-MB-231 cells, EZH2 activates silencing pathway and serves as a recruiting platform for the transcription of NF-kB target genes TNF and interleu- DNMTs. On the contrary, McGarvey and colleagues pro- kin (IL)-6 by forming a ternary complex with RelA and RelB posed a dominant role for CpG DNA methylation in gene (39). In castration-resistant prostate cancer (CRPC), meth- silencing and maintenance of heritable repressive states of ylation of androgen receptor (AR) or AR-associated proteins target gene promoters by demonstrating that EZH2 deple- has emerged as a potential mechanism for EZH2-mediated tion neither affected the methylation status nor induced the transcriptional induction (40). EZH2 is phosphorylated by hypermethylated genes such as p16INK4a in U2OS cells

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(45). This study also suggests that EZH2 may not be pathways, some of them are universal for all cancer types, essential for the maintenance of repressed state and silencing while others are more specific and limited to particular of a target gene promoter, once it becomes CpG hyper- malignancies. The causes and consequences of EZH2 dereg- methylated. Subsequent findings provided more inputs to ulation in different cancer types are discussed. the EZH2–DNA methylation link by comparing gene profiles of EZH2 target genes in normal versus cancer cells, EZH2 and hematologic malignancies and revealed that EZH2 targets that display H3K27Me3 in Overexpression of PcG proteins, particularly EZH2, has normal cells are correlated to the aberrantly hypermethylated been linked to the pathogenesis of complex and heteroge- corresponding genes in cancer cells (46, 47). This suggests neous hematologic malignancies such as myelodysplastic that genes that acquire EZH2-catalyzed H3K27me3 marks syndromes and acute myeloid leukemia (AML; ref. 53). It are susceptible to subsequent DNA methylation and silenc- is well established that deregulated epigenetic factors coupled ing during cellular transformation. On the basis of the to genetic mutations contribute to their pathogenesis by methylated CpG island microarray (MCAM) analysis of causing abnormal silencing of critical tumor-suppressor H3K27me3-modified CpG islands in PC-3 and MCF-7 cell genes (TSG). A study by Xu and colleagues demonstrated lines, Kondo and colleagues proposed that DNA methyla- that in comparison with the patients with nonclonal cyto- tion and H3K27me3-based silencing may be independent penia diseases, patients with myelodysplastic syndromes/ mechanisms, and overlapping genes targeted by both path- AML have overexpression of EZH2, RING1, and BMI1 ways are relatively rare (48). In conclusion, the current genes, leading to poor prognostic scoring (53). Elevated opinion supported by various experimental studies empha- levels of DNMTs and hypermethylation of TSGs, such as sizes that DNA methylation and polycomb-mediated silenc- p15, DAPK, and SOCS-1, are frequently observed in pati- ing may act together in the epigenetic repression of some ents with myelodysplastic syndromes. In patients with genes depending on the cellular context. However, there are p15INK4b methylation, EZH2 levels were found to be discrepancies among studies suggesting a direct mechanistic high as compared with patients without methylation. In a link between these silencing pathways. study using high-resolution chromatin immunoprecipita- In human cells, the PRC2 complex containing EZH2 tion (ChIP-on-chip), Paul and colleagues demonstrated that has been shown to physically interact with histone deace- as compared with unmethylated p15INK4b samples, there tylases (HDAC) such as HDAC1 and HDAC2 (49, 50). On was lower enrichment of the p15INK4b locus with active the basis of the available biochemical data, HDACs are chromatin mark H3K4me3 in AML cells with p15INK4b not the core subunits of the PRC2 complex, but they DNA methylation (54). However, the repressive chromatin function in synergy during transient interactions in gene mark H3K27me3 catalyzed by EZH2 was found at silencing. H3K27 acetylation has been shown to be func- p15INK4b as well as in neighboring TSGs p14ARF and tionally antagonistic to H3K27me3-mediated silencing and p16INK4a, in both samples with and without p15INK4b both are mutually exclusive (29, 30). Possibly, HDACs may DNA methylation. Therefore, it was concluded that the function in deacetylation of the K27 side chains and allow p15INK4b locus was enriched by bivalent histone marks PRC2-mediated methylation of e-amino groups. Also, H3K27me3/H3K4me3 in AML cells without DNA meth- HDACs may alter the chromatin configuration by modu- ylation. They further reported that epigenetic therapies lating local histone code favorable to polycomb-mediated could restore the loss of H3K4me3 and reactivate silencing by deacetylation of other histone lysines such as p15INK4b expression, but the H3K27me3 mark is retained, H3-K9, H3-K14, or H4-K8. In addition, H3K4me3 and which ultimately causes the return of the p15INK4b locus H3K36me2, which mark actively transcribing genes, have to a bivalent state. been shown to antagonize EZH2-mediated H3K27me3 The genomic organization and revised mapping of the silencing (51, 52). Although current literature reports advo- EZH2 gene by Cardoso and colleagues revealed that it is cate the fact that EZH2-mediated gene silencing has evolved located on 7q35, a critical region associated with malignant to sense the surrounding epigenetic landscape and is linked myeloid disorders (55). Recent findings suggest that there are to other epigenetic silencing mechanisms, further experi- multiple mechanisms causing EZH2 deregulation in myeloid mental studies are needed to address the direct mechanistic malignancies. Mutations in the EZH2 gene have been and functional link among them in driving oncogenesis. described in myelodysplasia–myeloproliferative neoplasms (MPNs) (10%–13%), myelofibrosis (13%), and various subtypes of myelodysplastic syndromes (9, 56–58). In mye- Role of EZH2 in Cancer Progression loid neoplasms, EZH2 mutations were found to be inactivat- Accumulated evidence reveals that EZH2 deregulation ing/hypomorphic and distributed throughout the gene, which and overexpression are frequently observed in a variety of includes missense, nonsense, and premature stop codons (9). cancer types, including solid tumors and hematologic malig- HMTase activity was predicted to be lost in all EZH2 nancies. EZH2 levels have been reported to increase steadily missense and nonsense mutants, as the SET domain respon- as the tumor progresses, and elevated EZH2 levels often are sible for catalyzing H3K27 trimethylation was located in the directly correlated with advanced metastatic stages of cancer C-terminal domain of the EZH2 protein (56, 57). Assessment progression and poor prognosis. Studies suggest that EZH2 of mutational status in 469 cases of myeloid malignancy overexpression may be caused by a variety of signals or revealed that EZH2 mutations were present in 8% cases,

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while EED/SUZ12 mutations were reported in 3.3% cases. in vivo (mouse xenograft CRPC model using CWR22Rv1 In addition to EZH2 mutation, decreased EZH2 expression cells). The study further investigated the factors causing was found be associated with hemizygous deletion (7/ EZH2 to switch to a gene activation function in CRPC. del7q) involving EZH2 locus (78% of cases), diploid chro- EZH2 phosphorylation at serine 21 by the phosphoinositide mosome 7 (41% of cases), and spliceosomal U2AF1/SRSF2 3-kinase (PI3K)–Akt pathway was demonstrated to be mutations (63% of cases). EZH2 mutation was associated crucial for EZH2-mediated androgen-independent growth, with reduced H3K27me3ethylation and derepression of gene activation, and association with AR-containing EZH2 target genes, which may contribute to leukemogen- complexes. esis. HOXA9, one of the major downstream genes involved Recent reports indicate that androgens and the AR sig- in stem cell renewal, was found to be elevated in cases with naling pathway play a critical role in regulating EZH2 levels, reduced EZH2 expression. Unlike myeloid neoplasm, in suggesting a potential reason for increased EZH2 levels after follicular lymphomas and diffused B-cell lymphomas, a androgen deprivation therapy (ADT) in metastatic and heterozygous missense somatic mutation at tyrosine 641 hormone-refractory prostate cancer. Androgens have been (Y641) in the EZH2–SET domain causes a gain-of-function shown to repress EZH2 expression (62). Rb- and p130- leading to increased H3K27me3 (59). The complex role of dependent pathways mediate EZH2 repression in the pres- EZH2 in tumorigenesis is reflected by the fact that both ence of functional AR. Using coimmunoprecipitation, Xu activating and inactivating mutations in the EZH2 gene and colleagues reported that EZH2 and AR physically contribute to oncogenesis and malignancy. interact in androgen-independent abl cells, and the interaction was lost in EZH2 deletion mutants either in domain I EZH2 and prostate cancer (N-terminal protein–protein interaction domain) or in the Prostate cancer is one of most common noncutaneous C-terminal SET domain, emphasizing the importance of malignancies responsible for the majority of cancer-related these two domains for the interaction (40). They have also deaths among men in the United States. Like other cancer shown that the AR mRNA or protein levels did not change types, the interplay between genetic and epigenetic factors upon EZH2 depletion, but there was decrease in AR- has been demonstrated to play a major role in the patho- associated lysine methylation. It was further demonstrated genesis and progression of prostate cancer. Epigenetic that through their cooperative recruitment or binding, alterations, such as aberrant DNA methylation (hypo- or EZH2 and AR activate a set of target genes. Another study, hypermethylation), changes in chromatin remodeling pat- by Zhao and colleagues, has shown that EZH2 and AR terns due to dysfunction in histone-modifying enzymes, collaborate with each other in repressing developmental and microRNA (miRNA, miR) deregulation have been regulators involved in nonprostatic pathways (63). The reported to be the major players in prostate carcinogenesis AR-dependent transcriptional silencing is mediated by (reviewed in ref. 60). Focusing on aberrant histone PTMs, EZH2 in an androgen-deprived environment. evidence indicates that critical histone-modifying enzymes, In prostate cancer, various molecular mechanisms have particularly HDACs and HMTase, are deregulated in pros- been proposed to be responsible for EZH2 overexpression, tate tumors. which includes EZH2 gene amplification, miR-101 dele- In recent years, the lack of success in defining useful tion, and transcriptional regulation by MYC and ETS gene molecular markers to evaluate disease progression and clin- family members (reviewed in ref. 37). Using the FISH ical outcome had led to the application of high-throughput technique, Saramaki and colleagues determined the copy genomic approaches, such as microarray expression analysis, number of the EZH2 gene in prostate cancer cell lines to predict differences in the gene expression profiles of LNCaP, DU145, PC-3, 22Rv1, xenografts, and clinical normal versus tumor samples at the molecular level. Using tumors (64). The data showed increased EZH2 copy num- cDNA microarray analysis, Varambally and colleagues ber in all the cell lines. Unlike early prostate cancer, in late- reported that increased expression of the PcG protein EZH2 stage tumor samples, EZH2 gene amplification was associ- in metastatic prostate tumor samples distinguished them ated with its overexpression. Also, quantitative real-time from organ-confined localized tumors (61). EZH2 has been reverse transcriptase (RT)-PCR analysis indicated that found to promote prostate carcinogenesis by PRC2-medi- EZH2 expression was elevated in hormone-refractory pro- ated silencing of critical TSGs such as DAB2IP, SLIT2, state cancer as compared with benign prostate hyperplasia TIMP-3, and MSMB (reviewed in ref. 37). However, a or untreated prostate cancer. In another study, Varambally recent study by Xu and colleagues highlighted that the and colleagues proposed that miR-101 negatively regulates oncogenic role of EZH2 in CRPC cells is independent of EZH2 expression, and somatic loss of one or both miR-101 its transcriptional repressor function (40). Using an andro- genomic loci elevates EZH2 levels, causing deregulation gen-dependent LNCaP cell line and its androgen-indepen- of various epigenetic pathways in prostate cancer (65). dent derivative LNCaP-abl as models to study prostate Their study revealed that there was a negative correlation cancer progression, they have shown that EZH2 levels were between miR-101 and EZH2 levels during prostate cancer higher in abl cells than in LNCaP cells. Also, unlike andro- progression. An increase in EZH2 levels paralleled a con- gen-dependent growth of LNCaP cells, EZH2 silencing has comitant decrease in miR-101 levels in human prostate been demonstrated to have a more profound effect in cancer samples. In addition, some studies reported that androgen-independent growth, both in vitro (abl cells) and the ETS transcriptional network consisting of ERG and

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epithelial-specific ETS factor, ESE3, are important molec- state in otherwise morphologically normal breast epithelial ular players with opposing effects regulating EZH2 ex- cells (72). Both in vitro and in vivo genetic knockdown pression in prostate cancer cells (66). ESE3 have been studies targeting EZH2 demonstrated that a decrease in demonstrated to suppress EZH2 expression, whereas ERG EZH2 levels results in decreased breast cancer cell prolifer- – competes with ESE3 for promoter occupancy and opposes ation, delayed G2 M cell-cycle transition, reduced breast its effects. Although prior studies have proposed various xenograft growth, and improved survival. Upregulation of molecular mechanisms for EZH2 upregulation in prostate EZH2 levels caused H3K27me3-mediated aberrant silenc- cancer cells, they were insufficient in explaining the reason ing of various essential TSGs such as FOXC1, CDKN1C for elevated EZH2 levels in primary prostate cancer and (p57KIP2), RUNX3, RKIP, CIITA, IL-6, and IL-8 (for a high-grade prostatic intraepithelial neoplasia (PIN). Inter- review see ref. 37). estingly, a study by Koh and colleagues proposed two Over the years, several molecular mechanisms have been additional, yet complementary, Myc-regulated molecular proposed to address the causes and consequences of EZH2 mechanisms responsible for EZH2 upregulation in prostate overexpression in breast cancer cells. Bracken and colleagues carcinogenesis (67). Because both Myc and EZH2 are reported that the expression of two PRC2 complex compo- frequently overexpressed in human PIN and primary carci- nents, viz., EZH2 and EED, is regulated by E2F transcrip- noma lesions, the study demonstrates that there exists a tion factors (73). It is well established that defects in the molecular link between them. In prostate cancer, elevated pRb–E2F pathway are a mandatory event in the pathogen- Myc induces EZH2 expression by directly binding to the esis of almost all human malignancies. E2F transcription E-box containing promoter region of EZH2 and activa- factors are known for regulating genes, such as CyclinE1, ting transcription. Similar findings were also reported by CyclinA2, CDC6, DHFR, and TK1, that control entry into Salvatori and colleagues in AML (68). Interestingly, Myc S-phase and DNA replication, which are well-studied targets is also known to regulate miRNAs. In LNCaP and PC-3 of the pRb pathway. Upon pRb phosphorylation, E2F cell lines, promoter regions of CTDSPL, CTDSP2, and dissociates from the pRb–E2F complex, and the activated CTDSP1 genes (miR-26a and miR-26b primary transcripts E2F directly binds to the promoters of EZH2 or EED are embedded in the intron region) were found to be containing putative E2F-binding sites and transactivates enriched by Myc, which results in transcriptional repres- their transcription. The study further confirmed that, sion (67). When Myc levels are low, miR-26a/miR-26b although the abrogation of EZH2 or EED expression could are actively transcribed and incorporated into the RISC significantly decrease positive regulators of cell proliferation, complex. It is then targeted to the 30-untranslated region such as cyclin D1 (CCND1), cyclin E1 (CCNE1), cyclin (30-UTR) of EZH2, which destabilizes EZH2 mRNA, A2 (CCNA2), and cyclin B1 (CCNB1), there was no ultimately repressing its translation. Repression of miR- increase in the expression of negative regulators of the cell 26a/miR-26b by elevated Myc levels contributes to EZH2 cycle, viz., p14ARF. This study provides a direct link overexpression by increasing the stability of EZH2 mRNA between the pRb–E2F pathway–mediated growth control due to the less availability of the miR-26a/miR-26b–RISC and PcG-regulated essential chromatin modifications. complex binding to EZH2–30-UTR. In conclusion, elevated In another study, Fujii and colleagues reported that the Myc levels enforce EZH2 overexpression by altering both MEK–ERK–Elk-1 pathway, which is commonly upregu- transcriptional and posttranscriptional regulatory mechan- lated in various cancer types, is linked to EZH2 over- isms controlling EZH2 expression in prostate cancer cells. expression in triple-negative and ERBB2-overexpressing breast cancer cells (74). Computational analysis revealed EZH2 and breast cancer that the EZH2 promoter harbor three Elk-1–binding Recent experimental advances in breast cancer research motifs and other sequence elements for NF-kB, c-Myb, have established EZH2 as a promising novel biomarker STAT1, and SRF (serum response factor) recruitment. indicating cancer progression and disease aggressiveness. Treatment with MAP–ERK kinase (MEK) inhibitor, EZH2 levels have been found to increase steadily as the U0126, decreased EZH2 levels in triple-negative breast disease progresses from a benign tumor to clinically evident cancer type MDA-MB-231 cells and ERBB2-overexpres- metastasis (69, 70). Highest EZH2 levels are associated with sing SKBr3 cells. Also, siRNA-mediated knockdown of the ER-negative basal-like phenotype of breast cancer cells Elk-1 caused a significant decrease in EZH2 mRNA that represents the most aggressive form of breast carcinoma expression, which was similar to the decrease caused by characterized by nuclear polymorphism, lack of ER, and U0126 treatment. Furthermore, they demonstrated that BRCA1 protein (71). Using a high-density tissue micro- MEK inhibitor treatment significantly decreased the asso- array, Kleer and colleagues showed that as compared with ciation of phospho-Elk-1 with the EZH2 promoter in normal or patients with atypical hyperplasia, EZH2 levels breast cancer cells. In conclusion, the study suggests that were significantly higher in patients with invasive breast the MEK–ERK pathway activated via KRAS mutation, carcinoma (69). Interestingly, Ding and Kleer reported that EGF receptor (EGFR) amplification, and ERBB2 ampli- in breast tissue samples that seemed to be histologically fication in triple-negative and ERBB2-overexpressing cells normal, but at a high risk of developing cancer, showed causes EZH2 overexpression. increased EZH2 expression, and hence identified EZH2 as a Under cancer-predisposed hypoxic conditions in breast potential in vivo biomarker that may detect a precancerous tumor–initiating cells (BTIC), EZH2 expression was

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reported to be regulated by hypoxia-inducible factor-1a poor prognosis (78–80). In a recent study, Coe and collea- HIF-1a. Chang and colleagues identified a consensus gues found a strong correlation between deregulation of sequence for HIF response element (HRE) by promoter the E2F–Rb pathway in 96% SCLC samples and EZH2 analysis of the EZH2 promoter (75). They further demon- overexpression (81). Their study revealed that genomic loss strated that a hypoxic microenvironment induces HIF-1a of the E2F–Rb pathway by loss of either Rb1 or E2F binding to the HRE and transactivates EZH2 expression. amplification leads to increased EZH2 expression. Gener- Increased EZH2 expression downregulates double-strand ation of a "stem cell–like" hypermethylator profile in SCLC break repair protein RAD51 expression, which ultimately tumors due to aberrant methylation of PRC2 target genes results in impaired DNA repair and accumulation of geno- was found to be associated with elevated EZH2 levels. mic abnormalities. Furthermore, they demonstrated that In addition, the finding that lentivirus-mediated knockdown EZH2-mediated downregulation of RAD51 causes expan- of EZH2 in two SCLC cell lines caused a significant decrease sion of self-renewing BTIC population and RAF1 ampli- in growth as compared with empty-vector controls empha- fication, which further activates downstream p-ERK–b-cate- sized the proproliferative and oncogenic role of EZH2 nin signaling. EZH2-mediated RAF1–ERK signaling in SCLC. Using microarray analysis on a genome-wide scale, promotes BTIC expansion and aggravates breast cancer Sato and colleagues revealed that the expression levels of condition. EZH2 and other PRC2 members, such as SUZ12 and EED, Recent evidence indicates that PTMs also play a crucial were significantly higher in SCLC samples than in normal role in regulating EZH2 levels in breast cancer cells. Studies tissues, including the lung (78). They also demonstrated that by Cha and colleagues have shown that Akt-mediated H3K27me3-mediated repression of PRC2 target genes, such phosphorylation of EZH2 at a highly conserved serine 21 as JUB, in both SCLC cell lines and clinical samples residue inhibited its H3K27 methyltransferase activity (21). correlated with poor prognosis. Findings by Hubaux and Although EZH2 phosphorylation had no impact on the colleagues highlighted the implication of EZH2 in cell integrity of the PRC2 complex, there was a marked decline in death and cell-cycle regulation by demonstrating that short the affinity of EZH2 for histone H3, which ultimately results hairpin RNA (shRNA)–mediated knockdown of EZH2 in decreased H3K27me3. Interestingly, in another study, in SCLC cells induced apoptosis by elevating proapoptotic Gonzalez and colleagues reported that elevated EZH2 in factors Puma and Bad, increased p21 protein levels, and – – breast cancer cells induces the PI3K Akt pathway by spe- decreased the fraction of cells in S or G2 M cell-cycle phases. cifically activating Akt isoform1 (76). Increased EZH2 levels NSCLCs, including squamous cell carcinoma (SCC), positively correlated with elevated phospho-Akt1 (Ser473) adenocarcinoma, large cell carcinoma, and several other and decreased nuclear localization of phospho-BRCA1 types that occur less frequently, account for more than (Ser1423). The role of EZH2 in nuclear shutting of BRCA1 85% of all lung cancer cases and are associated with poor may be regarded as one of the non–PRC2-dependent EZH2 prognosis mainly due to late diagnosis, with an overall 5-year functions, as discussed in the previous sections. The accu- survival rate of approximately 11%. Immunohistochemical mulation of the BRCA1 protein in the nucleus promotes analysis of 157 surgically resected NSCL samples by Kikuchi tumorigenesis by causing aberrant mitosis, aneuploidy, and and colleagues revealed a strong correlation between elevated genomic instability. The multifaceted role of EZH2 in EZH2 protein levels with advanced pathologic tumor stage, promoting breast tumorigenesis, where it may act as a moderate or poor differentiation, nonadenocarcinoma his- transcriptional activator or repressor depending on the tology, and high Ki-67 and cyclin E levels (79). Corrobo- cellular context, has been highlighted in the previous section rating with the previous findings, Huqun and colleagues on non–PRC2-dependent EZH2 function. reported that there was a significant association of increased EZH2 expression with larger tumor size and shorter overall EZH2 and lung cancer survival in NSCLC (82). These studies highlighted EZH2 Lung cancer is a leading cause of cancer-related mortality protein levels as a negative prognostic indicator in NSCLC. among both men and women worldwide, accounting for In recent years, studies focusing on the identification of almost 1.4 million deaths each year (77). Small cell lung molecular mechanisms causing lung carcinogenesis have cancer (SCLC) is a highly aggressive neuroendocrine carci- revealed that deregulation of several miRNAs, such as noma and represents a histologic subtype that is distinct from miR-126, miR-21, miR-200c, miR-145, and miR-107, other lung cancer types called non–small cell lung cancers miR-185, and miR-101, contributes to tumor cell growth, (NSCLC). SCLC has been found to be strongly correlated invasion, and apoptosis. Interestingly, Zhang and colleagues to cigarette smoking and accounts for approximately 15% demonstrated EZH2 to be a direct functional target of miR- of all lung cancer cases. Like other cancer types, the accu- 138 in NSCLC and highlighted its therapeutic importance mulation of several genetic and epigenetic aberrations has in NSCLC treatment strategy (83). They showed that miR- been implicated in the initiation and progression of lung 138 expression was downregulated in NSCLC tissues and cancer. Focusing on the role of HMTase EZH2 in the A549, SPC-A1, SK-MES-1, and H460 cell lines as com- pathogenesis of lung cancer, recent evidence suggests that pared with the matched normal tissue samples from the same EZH2 overexpression and polycomb-mediated H3K27me3 patients and normal bronchial epithelial cell line 16HBE, promote malignancy in various lung cancer types, including respectively. Lentivirus-mediated overexpression of miR- – SCLC and NSCLCs, which is commonly associated with 138 caused decreased viability, G0 G1 arrest, and apoptosis

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in A549 and H460 cell lines. Furthermore, using bioinfor- findings suggest that aberration in the pRb–E2F pathway matics analysis, EZH2 30-UTR was found to harbor a highly may cause elevated EZH2 levels at successive stages of conserved miR-138–binding site. Transfection of miR-138 bladder carcinogenesis. In another study, Tang and collea- overexpressing A549 and H460 cells with the wild-type gues revealed a novel link between p53 TSG and EZH2 by 30-UTR EZH2 luciferase reporter construct resulted in demonstrating that activated p53 suppresses EZH2 gene decreased luciferase activity as compared with cells trans- expression by binding to its promoter (93). This raises an fected with the 30-UTR EZH2-mutant plasmid construct. interesting question of whether alterations in p53 may lead Also, miR-138 overexpression caused significant decrease to increased EZH2 expression in bladder cancer. In fact, in EZH2 mRNA and protein levels in both cell lines. In Yamada and colleagues showed that in SCC of the esophagus addition, ectopic expression of EZH2 was shown to rescue (SCCE), elevated EZH2 expression was associated with p53 miR-138–induced growth inhibition, cell-cycle arrest, and alteration and activated p53 reduced EZH2 mRNA levels in apoptosis in the lung cancer cells. In another study, Cho and SCCE cell lines (94). Furthermore, EZH2 was found to be a colleagues demonstrated an inverse correlation between miR-144 target gene, and miR-144 downregulation in miR-101 and EZH2 expression levels in lung cancer tissue bladder cancer cells was demonstrated to restore EZH2 samples (84). miR-101 was shown to inhibit lung cancer expression, which further resulted in the activation of the cell invasion by regulating EZH2 and H3K27me3 levels. In Wnt/b-catenin pathway and subsequent cell proliferation conclusion, miRNA deregulation has emerged as a major (95). cause of EZH2 overexpression in lung carcinomas, and novel therapeutic strategies should consider targeting specific EZH2 and ovarian cancer miRNAs, such as miR-138, miR-101, and miR-26a, in Ovarian cancer develops in tissues of the ovary and can be lung cancer treatment. categorized into two major types based on the tissue of origin—ovarian epithelial carcinomas (cancer forms in the EZH2 and bladder cancer cells on the surface of the ovary) or malignant germ cell Bladder cancer is one of the most common malignancies in tumors (cancer develops in egg cells). Epithelial ovarian the United States, and based on National Cancer Institute's cancer (EOC) accounts for the majority of gynecologic (Bethesda, Maryland, USA) statistics, it accounted for cancer–related deaths in the United States. Accumulated approximately 72,570 new cases and 15,210 deaths in evidence indicates that EZH2 overexpression plays a major 2013. On the basis of the site of origin, bladder cancer may role in the etiology and pathogenesis of EOC. Li and be categorized into various types, which include transitional colleagues demonstrated that, although elevated EZH2 cell carcinomas (TCC; cancer develops in the inner cells levels were positively correlated with cell proliferation mar- lining the bladder), SCC (cancer originates in thin, flat cells), kers such as Ki-67 and tumor grade, there was no association and adenocarcinoma (cancer begins in cells that produce and of EZH2 expression with tumor stage and overall or disease- secrete mucus and other fluids). TCCs, which account for free survival in patients with high-grade serous histotype >90% of bladder cancer, are associated with superficial, EOC (96). Interestingly, knockdown of EZH2 expression non–muscle-invasive tumors, which may be treated by local in both in vitro and in vivo xenograft models resulted in transurethral resection. However, the risk of recurrence is decreased H3K27me3 levels, induced apoptosis, suppressed high, and the noninvasive lesions may progress to more lethal growth, and invasion of human EOC cells. Rao and collea- and metastatic carcinoma. Although epigenetic molecular gues reported that EZH2 overexpression was absent in alterations involved in bladder cancer initiation and pro- normal ovaries, while high EZH2 expression in the ovarian gression are not as comprehensively studied as genetic carcinomas (50% cases) was positively associated with abnormalities of TSGs or oncogenes, recent studies correlate increasing histologic tumor grade and advanced stage of the elevated EZH2 levels to high-grade lesions and invasive disease (97). Furthermore, this study underscores the poten- bladder carcinomas (85–87). Raman and colleagues dem- tial role of EZH2 in regulating cell proliferation, migration, onstrated increased EZH2 mRNA and protein expression in or invasion via modulation of TGF-b1 expression and TCC compared with adjacent nontumorous tissue (88). demonstrated a positive correlation between high EZH2 Takawa and colleagues analyzed EZH2 mRNA and protein and TGF-b1 levels in ovarian carcinoma tissues. EZH2 levels in clinical bladder cancer samples by quantitative real- knockdown was shown to reduce expression of TGF-b1 time PCR and immunohistochemistry (89). Their findings and increase E-cadherin expression either at the transcript were in line with previous studies showing significant upre- or protein levels. Together, recent findings suggest that gulation of EZH2 levels in tumor cells as compared with elevated EZH2 levels in EOC promote carcinogenesis by normal cells. Zhang and colleagues showed that inhibition of H3K27me3-mediated silencing of target genes, which ulti- EZH2 expression by RNA interference resulted in decreased mately induce cell proliferation, invasiveness, and suppress cell proliferation and G1 arrest in bladder cancer EJ cells (90). apoptosis. Interestingly, some studies have highlighted overexpression Notably, Hu and colleagues found that EZH2 was over- of E2F transcription factors, including E2F3, and deregu- expressed in cisplatin-resistant ovarian cancer cell line lation of the pRb–E2F pathway, which directly regulate A2780/DDP as compared with cisplatin-sensitive A2780 EZH2 expression, as one of the fundamental factors driving cell line (98). siRNA-mediated knockdown of EZH2 human bladder tumorigenesis (91, 92). Collectively, these resulted in resensitization of A2780 cells to cisplatin and

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also decreased H3K27me3 levels. Similarly, Rizzo and A key oncogenic event reported in the majority of mel- colleagues reported EZH2 overexpression in ovarian anoma cases is the BRAFT1799A mutation that results in the tumor–derived side population (SP) cells, which are stem constitutively activated BRAFV600E kinase. The epigenetic cell–like cells enriched by chemotherapy, and demonstrated mechanism of action of BRAFV600E, which is well known to that siRNA knockdown of EZH2 caused loss of SP and play an important role in melanoma progression, was reduced anchorage-independent growth in ovarian tumor recently studied as one of the potential mechanisms for models (99). This evidence suggests that EZH2 regulates BRAFV600E-mediated gene hypermethylation via upregula- stem cell–like attributes of ovarian cancer cells, which may tion of DNMT1 and EZH2 in melanoma cells (108). contribute to chemoresistance in EOCs. During tumor initiation, oncogenic stimuli in melanocytes In a recent study, Garipov and colleagues examined the exacerbate an oncogene-induced senescence, known as mel- molecular mechanism underlying EZH2 overexpression in anocytic nevus, which is a benign precursor of melanoma. EOCs (100). Their study highlighted the role of NF-YA, Cells overexpressing EZH2 escape this senescence through the regulatory subunit of CCAAT-binding transcription the inhibition of p21. In contrast, EZH2 depletion results in factor NF-Y, in regulating EZH2 transcription by asso- p21 activation and senescence induction in human mela- ciating with two CCAAT boxes in the proximal EZH2 noma cells (109). The PRC2 complex inhibits the expression promoter in EOC cells. They also showed that there is a of several tumor-suppressor miRNAs in various cancer types. positive correlation between EZH2 and NF-YA expression Luo and colleagues demonstrated that overexpression of levels in EOC, and elevated NF-YA levels predict shorter miR-101 inhibited melanoma cell invasion and proliferation overall survival in patients with EOC. Furthermore, NY-YA by downregulation of microphthalmia-associated transcrip- knockdown downregulated EZH2 expression as well as tion factor (MITF) and EZH2 protein levels (110). Simi- H3K27me3 levels, induced apoptosis, and reduced both larly, miR-137 was also demonstrated to have a tumor- in vitro and in vivo growth of human EOC cells. Conse- suppressive role, and c-Met, YB1, MITF, and EZH2 were quently, ectopic EZH2 expression rescued NF-YA–induced identified as its direct targets in malignant melanoma (111). apoptosis in EOC cells. In another study, Guo and collea- Downregulation of miR-31 is a common event in melano- gues emphasized the oncogenic role of EZH2 in ovarian ma, which is associated with genomic loss in a subset of cancer by possible downregulation of anti-oncogene p57 samples. Decreased miR-31 gene expression was found to be and showed that there is an inverse correlation between a result of epigenetic silencing by DNA methylation and via EZH2 and p57 mRNA expression levels in ovarian tissue EZH2-mediated histone methylation (112). Importantly, samples (101). Lu and colleagues showed that EZH2 is an miR-31 has emerged as a complex player in a number of important regulator of tumor angiogenesis (102). EZH2 cancers, and evidence suggests that miR-31 can act either as overexpression in EOC-associated endothelial cells was an oncomiR or a tumor-suppressive miR in a tumor type– demonstrated to be a direct result of paracrine circuit specific manner (113). Another critical TSG Rap1GAP, involving VEGF stimulation. EZH2 was shown to promote which is downregulated in multiple aggressive tumors, angiogenesis by silencing vasohibin1. Furthermore, siRNA- including melanoma, pancreatic, and thyroid cancer, was mediated EZH2 silencing caused significant growth inhibi- demonstrated to be epigenetically silenced by promoter tion in tumor-associated endothelial cells and reduced angio- hypermethylation in melanoma cells (114), whereas genesis. Together, accumulated evidence proposes EZH2 to EZH2-mediated Rap1GAP transcriptional repression by be a promising novel target in antiangiogenesis therapy. H3K27 trimethylation was reported in SCC of head and neck (115). EZH2 and skin cancer As per the latest National Cancer Institute's statistics, skin Role of EZH2 in cancer stem cells cancer remains as one of the most common cancers in the Cancer stem cells (CSC) are responsible for the initiation United States, with nearly more than 2 million people and proliferation of a tumor mass, and their recent in vivo treated for nonmelanoma (basal cell or SCC) and 76,690 validation in certain solid tumors has attracted considerable new melanoma cases each year. Melanoma is a type of cancer scientific attention mainly because of their potential clinical that develops in the melanocytes in the skin and represents a significance (116). CSCs were first identified in AML and genetically and epigenetically complex group of malignancy later on found to be present in various solid tumors (117). A characterized by deregulation of multiple tumor-suppressor modern CSC model postulates that there exists a subset of and oncogenic pathways, including BRAF, NRAS, PTEN, tumor cells with stem cell–like properties, including self- and CDKN2A (103–105). Like several other human cancers, renewal as well as multi-lineage differentiation capability. EZH2 overexpression has been implicated in the progression CSCs are characterized by their ability to seed tumor in vivo, of benign nevi to invasive or metastatic melanoma (70, 106). self-renewal property and to spawn differentiated non-CSC Athanassiadou and colleagues investigated EZH2 expression progeny lacking tumor-initiating capabilities (118). in SCC and actinic keratosis (107). In this study, weak With the emergence of the CSC model, it was obvious to EZH2 immunostaining was reported in actinic keratosis analyze the shared features of normal stem cells with CSCs. cases (62.8%), whereas moderate expression was found in PcG proteins play their important role in stem cell main- SCCs (42.1%) and 77.8% EZH2 expression levels in tenance by reversibly repressing the genes encoding tran- "mixed" SCC/actinic keratosis cases. scription factors required for differentiation (119). This

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feature of normal stem cells was also generalized for CSCs are highly selective over EZH1 and other histone methyl and it hypothesizes that the acquisition of promoter DNA transferases and directly inhibit EZH2 by binding to the methylation at these repressed genes could lock in a stem cell active site through competitive inhibition with methyl phenotype to initiate abnormal clonal expansion to develop group donor SAM. EPZ005687 displayed considerable cancer (120). In various cancers, including breast, prostate, specificity and selectivity when tested on tyrosine 641 or ovary, pancreas, lung, and liver, the isolated CSC popula- alanine 677 mutation harboring lymphoma cells (125). tions have been reported to overexpress EZH2, which is Another EZH2 inhibitor El1, which also acts in a SAM- proposed to be essential for the maintenance of an intact competitive manner, has been developed by Qi and collea- CSC population (121). In a limited number of studies, the gues (126). El1 has demonstrated remarkable selectivity epigenetic mechanism of gene silencing by EZH2 has been across an HMT panel and decreased H3K27me3 levels linked to CSC-associated features such as invasion and without altering other histone H3 methylation marks. They chemoresistance. The role of EZH2 in cancer could be also showed that El1-mediated selective inhibition of EZH2 related to the interesting mechanism of silencing through caused reduced proliferation, cell cycle arrest and apoptosis the repression of differentiating and antimetastatic genes. in DLBCL cells harbouring Y641 mutation and other EZH2 CSCs maintain their pool by suppressing cell-differentiating overexpressing cancer cell lines. Interestingly, Kim and genes, such as p16 and p19, and show other characteristics colleagues reported an inhibition strategy of targeting EZH2 such as decrease of E-cadherin expression to make them that is distinct compared with other small-molecule inhibi- metastatic (121). EZH2 mediates p16, p19, and E-cadherin tors that target the EZH2 catalytic site (127). They reported silencing through histone H3K27 trimethylation. Further- the dose-dependent disruption of the EZH2–EED complex more, EZH2 has been shown to repress the Forkhead box by stabilized a-helix of EZH2 (SAH–EZH2) peptides, transcription factor C1, thereby enhancing the invasive which inhibited H3K27me3 and reduced EZH2 protein potential of breast cancer cells (122). EZH2 is also a key levels. Furthermore, upon treatment with SAH–EZH2, mediator of DNA damage response in tumor cells and PRC2-dependent MLL-AF9 leukemic cells were demon- contributes to CSC chemoresistance, suggesting its addi- strated to undergo growth arrest, monocyte-macrophage tional role in therapeutic resistance, apart from tumorigen- differentiation, and changes in PRC2-regulated lineage- esis (123). Altogether, EZH2 has emerged as an important specific marker genes. molecular component required for the onset of the CSC Recent studies highlighted some natural chemopreventive phenotype in tumor cells as well as maintaining its associated phytochemicals, such as green tea polyphenols and curcu- features. min, as potential EZH2 inhibitors that can reactivate PRC2- silenced target genes in various cancer types. Choudhury and colleagues showed that epigallocatechin-3-gallate (EGCG), EZH2 as a Therapeutic Target a major constituent of green tea and DZNep, alone or in Emerging experimental evidence clearly demonstrates that combination, reduced PcG protein levels, including EZH2, EZH2 plays critical roles in driving carcinogenesis, from by increased ubiquitylation and proteasomal-associated tumor initiation to clinically evident metastasis in various degradation (128). Consistently, there were decreased cancer types. Like other epigenetic enzymes, such as H3K27me3 levels and upregulation of TSG expression. DNMTs and HDACs, EZH2 has emerged as a potential Hua and colleagues demonstrated that curcumin, a natural anticancer target in present epigenetic strategies. In recent phytochemical present in turmeric, downregulates EZH2 years, the search for novel small-molecule EZH2 inhibitors levels by modulating the mitogen-activated protein kinase has yielded some promising results. One of the best known (MAPK) pathway in MDA-MB-435 breast cancer cells and widely used EZH2 inhibitors, 3-dezaneplanocin-A (129). Curcumin treatment showed antiproliferative effect fi (DZNep), was identi ed by Tan and colleagues through and induced G1 arrest in MDA-MB-435 cells. Dietary library-based drug screening. DZNep has demonstrated a omega-3(v-3) polyunsaturated fatty acids (PUFA) were also significant antitumor activity in various cancer types, includ- shown to downregulate EZH2 expression by enhancing its ing breast, prostate, lung, liver, and brain cancer cells (124). proteasomal degradation, which resulted in the re-activation Notably, treatment with DZNep depletes EZH2 levels of EZH2-regulated TSGs such as E-cadherin and insulin- and reactivates PRC2 target genes. DZNep targets S- like growth factor–binding protein (130). adenosyl-L-homocysteine (SAH) hydrolase and causes SAH Because EZH2 plays key roles in the normal functioning levels to increase, which further inhibits SAH cofactor of the cells by establishing the repressive state of genes that dependent methyltransferases such as EZH2 by a feed back function as developmental regulators and maintain stem cell inhibitory mechanism. Although DZNep has exhibited pluripotency, alternative inhibition strategies are needed some promising results in in vitro and in vivo studies, there that focus on the regulatory network, causing EZH2 dereg- are concerns about its specificity as a potential therapeutic ulation in a specific cancer type. Direct EZH2 inhibition compound, as it may also affect other SAM-dependent by small molecules targeting the enzyme active site may have processes. Also, questions on its short plasma half-life, effect detrimental effects on global methylation patterns in normal on global methylation status, and toxicity profile in animal cells. For example, a recent study proposed that inhibition models remain open. More recently, the quest for more of CDK1/2-mediated Thr 350 phosphorylation of EZH2 specific EZH2 inhibitors has identified some molecules that may emerge as an alternative therapeutic option to diminish

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EZH2 activity in cancer cells without affecting the global and colleagues HIF inhibitors, which showed marked EZH2-mediated gene silencing (22). Further experimental decrease in tumor growth and have been used in clinical studies may also focus on the development of "combined trials, may prove effective in EZH2 inhibition because treatment strategies" targeting the epigenetic regulatory HIF-1a regulates EZH2 expression in BTICs (75). Also, network involving EZH2 in tumor cells. the development of CDK1/2, which controls EZH2 phosphorylation, may emerge as an alternative treatment strat- Conclusion and Future Perspectives egy. Notably, administration of EZH2 inhibitors by a fi On the basis of a large body of in vivo and in vitro cancer cell or tumor-speci c delivery mechanism may also experimental evidence, it is now well established that reduce the unwanted side effects and toxicity to the normal EZH2 functions as a key mediator of tumorigenesis, and or stem cells. In conclusion, understanding the complex its overexpression is associated with invasive growth, tumor epigenetic regulatory network controlling EZH2 expres- aggressiveness, and poor clinical outcomes in hematologic sion and target genes in tumor cells may help design better as well as epithelial cancers. Traditionally, the oncogenic therapeutic intervention strategies. role of EZH2 depends on its ability to act as a "transcrip- fl tional repressor," which causes H3K27me3-based silencing Disclosure of Potential Con icts of Interest No potential conflicts of interest were disclosed. of critical TSGs and contributes to tumorigenesis. How- ever, recent studies in hormone-refractory breast cancer Acknowledgments and CRPC demonstrated that EZH2 is a multifaceted We acknowledge Qiagen Pathway Central (http://www.qiagen.com/products/ genes%20and%20pathways/pathway%20central/) for the PowerPoint template used molecule that could switch to "transcriptional activator" fi function and act independent of the PRC2 complex on in gure preparation in this manuscript. nonhistone substrates. As discussed in previously, EZH2 Grant Support can be regulated through multiple mechanisms, depending This work was partially supported by funds from the United States Public on cellular context and cancer type. Together, this evidence Health Service Grants (RO1CA115491, RO1CA108512, and R21109424 to fi S. Gupta). The authors acknowledge Shyama Prasad Mukherjee (SPM) fellowship warrants the need for the identi cation of EZH2 target provided to G. Deb by the Council of Scientific and Industrial Research (CSIR), genes, which are either activated or repressed, in specific India, and the Fulbright–Nehru Doctoral and Professional Research fellowship cancer type, so that effective therapeutic strategies could provided by the United States–India Educational Foundation (USIEF) for her be developed. There is a scope for the development of more work in the United States. fi speci c EZH2 inhibitors that are highly selective and Received October 11, 2013; revised February 4, 2014; accepted February 4, 2014; exhibit less toxicity in normal cells. As suggested by Chang published OnlineFirst February 13, 2014.

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www.aacrjournals.org Mol Cancer Res; 12(5) May 2014 653

EZH2: Not EZHY (Easy) to Deal

Gauri Deb, Anup Kumar Singh and Sanjay Gupta

Mol Cancer Res 2014;12:639-653. Published OnlineFirst February 13, 2014.

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