Review
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Epigenetic modification in gliomas: role of the histone methyltransferase EZH2 1.
Introduction
2.
EZH2
3.
The role of EZH2 in glioma
4.
Overview of DNA methylation
5.
The interaction of DNA methylation with EZH2 in gliomas
6.
The regulation of EZH2 by miRNAs in glioma
7.
Expert opinion
Er-Bao Bian, Jia Li, Xiao-Jun He, Gang Zong, Tao Jiang, Jun Li & Bing Zhao† †
The Second Affiliated Hospital of Anhui Medical University, Department of Neurosurgery, Hefei, China
Introduction: Gliomas are characterized by increased anaplasia, malignization, proliferation and invasion. They exhibit high resistance to standard treatment with combinations of radiotherapy and chemotherapy. They are currently the most common primary malignancy tumors in the brain that is related to a high mortality rate. Recently, increasing evidence suggests that EZH2 is involved in a number of glioma cell processes, including proliferation, apoptosis, invasion and angiogenesis. Areas covered: In this review, we emphasize the role of EZH2 in gliomas. We also address that EZH2 interacting with DNA methylation mediates transcriptional repression of specific genes in gliomas, and the regulation of EZH2 by microRNAs in gliomas. Expert opinion: Although the exact role of EZH2 in gliomas has not been fully elucidated, to understand the role of EZH2 proteins in epigenetic modification will provide valuable insights into the causes of gliomas, and pave the way to the potential future applications of EZH2 in the treatment of gliomas. Keywords: EZH2, glioma, methylation, microRNAs Expert Opin. Ther. Targets (2014) 18(10):1197-1206
1.
Introduction
Gliomas are the most common form of CNS primary tumors, accounting for > 50% of all primary CNS tumors [1]. Malignant gliomas possess a sequence of characteristics: transformation from a cell of origin, increased proliferative combined with abrogation of cell cycle control, clonal evolution, acquisition of invasive ability and activation of angiogenesis signal pathways [2]. Gliomas are tumors of neuroepithelial tissues, including several types that are astrocytic, oligodendroglial, oligoastrocytic, ependymal, choroid plexus-derived or others, according to the cell type with which they share [3]. Gliomas are clinicopathologically categorized into grades I -- IV according to the 2007 classification by the WHO [4]. Histological evaluation remains the gold standard for glioma diagnosis and serves as a criterion to estimate the prognosis of patients [5]. Among all gliomas cases diagnosed, most of them belong to grade IV, identified as high-grade astrocytoma or glioblastoma (GBM) with marked nuclear atypia and high mitotic rates [6,7]. Primary GBM arise de novo, however, secondary GBM derives from pre-existing low-grade astrocytomas [8]. Most patients with these glioma progress to GBM, with current treatment protocols, patients with GBM have a 5-year survival rate of no > 30% [9]. Despite aggressive therapeutic modalities are surgical resection combined with radiation and temozolomide chemotherapy, the prognostic statistics for GBM patients is discouraging [10-12]. GBM is highly infiltrative, however, complete elimination of the tumor by surgery is usually impossible. Further, radiation and chemotherapy can kill most of the remaining tumor cells, those that typically reoccur [13]. 10.1517/14728222.2014.941807 © 2014 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted
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Article highlights. . . .
The role of EZH2 in glioma. The interaction of DNA methylation with EZH2 in gliomas. The regulation of EZH2 by microRNAs in gliomas.
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This box summarizes key points contained in the article.
Accumulating evidences suggests that the presence of a subpopulation of cells with the potential to initiate and maintain growth of gliomas might be pivotal for their resistance to conventional therapies [14]. Very recently, GBM is thought to be derived from a small population of GBM stem cells (GSCs) [15-17]. GSCs and normal neural stem cells appear to share common features, including the expression of neural stem cell markers, the capacity for self-renewal and long-term proliferation, the formation of neurospheres and the capability of differentiating into multiple lineages. Intriguingly, recent studies revealed that in addition to GSCs differentiating into non-GSCs, non-GSCs can become GSCs or tumor-initiating cells and exhibit an enhanced ability to form neurospheres, thereby suggesting that the GSC state may be plastic [18,19]. This phenotypic plasticity between the GSC and non-GSC states may be regulated by the signaling pathways, genetic and epigenetic mechanisms. Recently, increasing evidences indicate that EZH2 have been implicated in gliomas initiation, progression, and importantly, in the formation, maintenance and plasticity of GSCs. A rapidly growing body of data describes the importance of EZH2 in gliomas, whereas most recent reports focus on the interplay between EZH2 and noncoding RNAs. Therefore, EZH2 may emerge as a potential therapeutic target in gliomas and in the development of novel treatment strategies that target both genetic and epigenetic mechanisms of tumorigenesis. With the knowledge that EZH2 is associated with aggressive gliomas, understanding how EZH2 promotes disease progression has gradually made possible the development of epigenetic drugs that directly or indirectly target EZH2. Herein, this review aims to provide a comprehensive overview of EZH2 involvement in gliomas pathogenesis and progression, with specific emphasis on the regulatory network of the interaction of EZH2 with microRNAs (miRNAs) and DNA methylation. 2.
EZH2
Human EZH2 gene is located on the long arm of chromosome 7 at position 7q35 and encodes a 746 amino acid protein that has histone methyltransferase (HMT) activity [20]. Besides its HMT activity, EZH2 protein contains several domains with potential functions (Figure 1). The major domains include CXC, cysteine-rich domain; noncoding RNA-binding domain; SANT (SWI3, ADA2, N-CoR) and TFIIIB DNA1198
binding domain; SET, Su(var)3-9, enhancer of zeste, trithorax domain [21]. The N-terminal SET domains provide binding sites for assembly with the required partner subunits. In addition, both the C-terminal SET domain and the adjacent cysteine-rich CXC domain are required for HMT activity [22-24]. The SET domain of EZH2 is identified as the signature of methyltransferases, which leads to trimethylation (me3) of histone 3 (H3) at lysine 27 (K27), as well as at lysine 9 (K9) albeit to a much lesser extent [25]. When the polycomb repressive complex 2 (PRC2) complex is recruited to chromatin, the HMT EZH2 catalyses the H3K27me3, which results in subsequent recruitment of the PRC1 complex that monoubiquitylates the lysine 119 of histone H2A (H2AK119ub1) to prevent RNA polymerase II-dependent transcriptional elongation and consolidate transcriptional repression [26,27]. More recently, a study reported that EZH2 mutations may either cause gain-of-function or complete loss of HMT activity in lymphoma and myeloid neoplasms [28]. In addition, somatic activating mutations in the catalytic (SET) domain of EZH2 have been identified in follicular lymphoma and germinal center B-cell diffuse large B-cell lymphoma, leading to increased H3K27me3 [29-33]. It is interesting to point out that the role of transcriptional regulation of EZH2 in epigenetics is complex. EZH2, identified as the core member of PRC2, combines with embryonic ectoderm development (EED) and SUZ12 and plays the role of HTMase to catalyze histone H3k27 and H3K9 methylation, and represses transcription at the chromatin level. Another role of EZH2 is direct interaction with DNA methyltransferases (DNMTs) to catalyze the methylation of promoter region in downstream genes. In addition, several reports have shown that EZH2 may function in target gene activation as a multifaceted molecule. 3.
The role of EZH2 in glioma
EZH2 functions as a chromatin-modifying enzyme, which plays a critical role in epigenetic silencing of genes (especially TSGs) through complex mechanisms (Figure 2). Increasing evidences have appeared that the links between EZH2 and tumorigenesis have been found in diverse type of cancers: breast, prostate, bladder, colon, lung, pancreatic, sarcoma and lymphomas [34-41]. The common phenomenon is that the levels of EZH2 expression are abnormally upregulated in cancer tissues compared with normal tissues, and overexpression of EZH2 is related to advanced stages of disease and a poor clinical outcome. Recently, a number of evidences suggest that EZH2 plays a central role in gliomas. EZH2 was more expressed in GBM than in low-grade gliomas and highly expressed in U87 human glioma cells [42]. The knockdown of EZH2 expression by using RNA interference in U87 human glioma cells resulted in apoptosis and a cell cycle arrest in the G0/G1phase. Moreover, silencing of EZH2 altered the mitochondrial membrane potential and promoted the release of cytochrome c from the mitochondria. In addition, the reduced expression of EZH2 altered the Bax
Expert Opin. Ther. Targets (2014) 18(10)
Epigenetic modification in gliomas
EID EZH2
Domain I SANT
Domain II ncRBC
EED
CXC CXC
SANT
RNA
SET SET
SUZ12
Figure 1. Characterized domains with potential functions are displayed for EZH2. Both the C-terminal SET domain and the adjacent cysteine-rich CXC domain are required for histone methyltransferase activity. Robust methyltransferase requires EZH2 assembly with both EED and SUZ12 of domain II, and domains required for binding these noncatalytic subunits are indicated. Expert Opin. Ther. Targets Downloaded from informahealthcare.com by Universitat de Girona on 10/24/14 For personal use only.
EED: Embryonic ectoderm development; ncRBD: Noncoding RNA-binding domain; SANT: SWI3, ADA2, N-CoR.
GSC self-renewal
c-myc
STAT3
Apoptosis
Caspase9 Caspase3
Bax Bcl-2
EZH2
H3K27m
Puripotency
AXL
Proliferation
Invasion
Figure 2. EZH2 regulates the biology of gliomas by controlling of different pathways. Knockdown of EZH2 alters expression of apoptosis associated-genes and reduces transcription of AXL receptor kinase in gliomas. In addition, EZH2 regulates the biology of GSC by control of global levels of H3K27 trimethylation. AXL: AXL receptor kinase; GSC: Glioblastoma stem cell.
and Bcl-2 protein levels and led to the activation of caspase 9 and caspase 3 [43]. Smits et al. reported that inhibition of EZH2 in vivo by systemic 3-deazaneplanocin A (DZNep) administration in a U87-Fluc-mCherry GBM xenograft mouse imaging model resulted in reduced tumor growth [44]. Glioma progression is associated with EZH2 overexpression and PRC2 target gene silencing; EZH2 inhibits differentiation and activates cancer-, cell cycle- and cellular movement-related genes. In addition, EZH2 is both a prognostic factor and a promising therapy target in brain tumors [45]. Ott et al. demonstrated that EZH2 is expressed in human glioma and correlates with malignancy, whereas silencing of EZH2 reduced the proliferation and invasion of glioma cells. Microarray analyses of U87MG glioma cells after EZH2 knockdown demonstrated a strong transcriptional reduction of the AXL receptor kinase (AXL). Furthermore, histone modification appeared to be involved in the positive regulation of AXL by EZH2. Silencing AXL
mimicked the anti-invasive effects of EZH2 knockdown. Finally, AXL expression is found in human gliomas with high EZH2 expression [46]. Recently, both Bmi1 and EZH2 expressions in glioma tissues were significantly higher than those in corresponding nonneoplastic brain tissues. Additionally, the upregulations of Bmi1 and EZH2 proteins were both significantly related to advanced WHO grades and low Karnofsky. Moreover, the overall survival of patients with high Bmi1 protein expression or high EZH2 protein expression was obviously lower than those with low expressions [47]. Abdouh et al. reported that Bmi1 is expressed in human GBM tumors and highly enriched in CD133-positive cells. Stable Bmi1 knockdown using short hairpin RNA (shRNA)-expressing lentiviruses inhibited clonogenic potential in vitro and tumor formation in vivo. Bmi1 prevents CD133-positive cell apoptosis and/or differentiation into neurons and astrocytes, depending on the cellular
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context. Bmi1 represses alternate tumor suppressor pathways that attempt to compensate for INK4A/ARF/P53 deletion and PI(3)K/AKT hyperactivity [48]. More interestingly, glioma patients with combined overexpression of Bmi1 and EZH2 proteins had the shortest overall survival. Furthermore, expression of Bmi1 and EZH2 was independent prognostic factor for overall survival in glioma patients [47]. Natsume et al. reported that the biological interconversion between GSCs and differentiated non-GSCs is functionally plastic and accompanied by gain or loss of PRC2-mediated histone H3 K27 trimethylation on pluripotency [18]. H3K27M inhibits the enzymatic activity of the PRC2 through interaction with the EZH2 subunit. In addition, transgenes containing lysine-tomethionine substitutions at other known methylated lysines (H3K9 and H3K36) are sufficient to cause specific reduction in methylation through inhibition of SET-domain enzymes. Recent studies have identified a Lys 27-to-methionine (K27M) mutation at one allele of H3F3A, one of the two genes encoding histone H3 variant H3.3, in 60% of high-grade pediatric glioma cases. The levels of H3K27 di- and trimethylation (H3K27me2 and H3K27me3) are reduced globally in H3.3K27M patient samples due to the expression of the H3.3K27M mutant allele. Remarkably, H3K27me3 and EZH2 (the catalytic subunit of H3K27 methyltransferase) at chromatin are dramatically increased locally at hundreds of gene loci in H3.3K27M patient cells. Moreover, the gain of H3K27me3 and EZH2 at gene promoters alters the expression of genes. In addition, the development associated genes (e.g., Nanog, Wnt1, BMP5) together with alterations in the subcellular localization of EZH2 [18,49]. A recent study showed that EZH2 is highly expressed in murine and human GSC. Treatment with suberoylanilide hydroxamic acid caused significant upregulation of PRC2 predicted target genes, GSC disruption and decreased expression of EZH2 and of the stem cell marker CD133. Inhibition of EZH2 expression by shRNA was related to a significant decrease in the proliferation of glioma cells. Inhibition of EZH2, the main component of the PRC2, also impaired GBM tumor growth [42,48]. Targeted pharmacologic disruption of EZH2 by the S-adenosylhomocysteine hydrolase inhibitor DZNep, or its specific downregulation by shRNA, strongly impairs GBM cancer stem cell (CSC) self-renewal in vitro and tumor-initiating capacity in vivo. In DZNep-treated GBM CSCs, the expression of c-myc, known as a direct target of EZH2 in GBM CSCs, strongly repressed upon EZH2 depletion [50]. EZH2 binds to and methylates STAT3, resulting in enhanced STAT3 activity by increased tyrosine phosphorylation of STAT3. EZH2 knockdown greatly decreased global levels of H3K27 trimethylation in GSCs and reduced the levels of p-STAT3 in GSCs. P-STAT3 in GSCs was also rapidly decreased by treatment with either DZNep or GSK126 [51]. These results suggest that the inhibition of EZH2 reverses the silencing of Polycomb target genes and diminishes STAT3 activity. 1200
4.
Overview of DNA methylation
DNA methylation is an enzyme-mediated chemical modification of DNA by the addition of a methyl group from S-adenosyl-L-methionine substrates to the 5-position of cytosine (5-methylcytosine, 5mC) [52]. In humans and other mammals, DNA methylation is catalyzed by two general classes of DNMTs: maintenance DNMT1 and de novo DNMT3A and DNMT3B [53,54]. DNMT1, which maintains the existing methylation patterns following DNA replication, and DNMT3A and DNMT3B, de novo enzymes that prefer unmethylated and hemimethylated DNA and establish new DNA methylation patterns early in development [55,56]. DNA methylation can regulate gene expression directly, by repressing the binding of key transcription factors, and indirectly, by recruiting methyl-CpG-binding domain (MBD) proteins to the promoter of gene. So far, six members of the methyl-CpGbinding proteins family have been identified: MECP2, MBD1, MBD2, MBD3, MBD4 and Kaiso [57]. MECP2 recognizes methylated CpG sites and, when cooperation with DNMT1, forms a complex to copy the parental DNA methylation to the daughter DNA strands during cell division [58]. In addition, DNMTs interact with polycomb group (PcG) protein EZH2 to methylate EZH2-binding promoters, suggesting that epigenetic transcriptional repression complex is closely connected in regulating the expression of gene [58]. DNA demethylation is regulated by the ten--eleven translocation (TET) family. To date, three members of the TETs family, including TET1 TET2 TET3, have been identified in mammals. TETs convert 5mC to 5-hydroxymethylcytosine (5hmC), provide a way for dysregulation of DNA methylation [59]. TETs can promote to transcriptional repression, and to a minor extent also transcriptional activation, and that the majority of TETs-mediated transcriptional effects are independent of conversion of 5mC to 5hmC [60,61]. TET1 and TET3, but not TET2, is frequently found in chromatinbinding proteins such as DNMT1 and 5mC-binding proteins (MBDs), which is involved in regulating the expression of genes [60]. TET1 contributes to silencing of a subgroup of genes by facilitating recruitment of PRC2 to CpG-rich target gene promoters [62,63]. Another mechanism by which transcriptional repression of target genes are mediated is by the interaction of TET1 with the Sin3A co-repressor complex [62]. TETs regulate gene expression by preventing the gene promoter from hypermethylation mediated by DNMTs [64].
The interaction of DNA methylation with EZH2 in gliomas
5.
DNA hypermethylation is frequently recruited by PcGmediated repressive mark to a number of tumor suppressor genes that are epigenetic silencing in cancer cells, suggesting that links EZH2 with DNMTs has established a role for this protein during the induction and targeting of DNA
Expert Opin. Ther. Targets (2014) 18(10)
Epigenetic modification in gliomas
A.
Transcrptional repression CpG H3K27m Me EZH2
DNMTs
B.
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EZH2
5hmc
5mc TETs
C.
Transcrptional activation
EZH2
MeCP2 Transcrptional repression
Methylated CpGs
Figure 3. miRNAs mediate the signal pathways of gliomas by targeting EZH2. EZH2 interacts with miRNAs and other upstream and downstream molecules. EZH2 is under the negative control of several tumor-suppressive miRNAs and inhibits cell cycle regulators and molecules involved in gliomas. 5hmC: 5-hydroxymethylcytosine; 5mC: 5-methylcytosine; DNMTs: DNA methyltransferases; miRNA: MicroRNAs; TET: Ten--eleven translocation.
methylation [65-67]. The expression levels of DNMT1 and DNMT3B were strongly upregulated in malignant glioma tissue compared with normal brain samples, whereas DNMT1 and DNMT3B knockdown resulted in the restored expression of 308 genes that also contained promoter region hypermethylation [68]. EZH2 interacts with DNMTs and associates with DNMT activity. Binding of DNMTs to several EZH2-repressed genes depends on the presence of EZH2. Furthermore, EZH2 is required for DNA methylation of EZH2-target promoters, suggesting that EZH2 serves as a recruitment platform for DNMTs, thus highlighting a previously unrecognized direct connection between two key epigenetic repression systems [66]. Both MeCP2 and EZH2 have recently been shown to be recruited and appear to be involved in the maintenance of PPARg repression in mouse stellate cells undergoing liver fibrogenesis [69]. Consistently, MeCP2 and EZH2 levels inversely correlate with PPARg expression in the colorectal cancer cells. MeCP2 and EZH2 silencing re-activate PPARg, confirming their crucial role in PPARg epigenetic repression [70]. Cartron et al. reported that U251 cells presenting a low level of EZH2 induced by the knockdown of EZH2. EZH2 downregulation affected the TET1/EZH2 interactions. The downexpression of EZH2 decreased TET1 recruitment on the HOXD12 genes. The decrease of TET1/EZH2 recruitment on the HOXD12 gene in cells treated with siRNA-EZH2 produced an increase in methylation of the HOX12D gene. These results
suggest that EZH2 plays a role as anchors for TET1 recruitment on the HOXD12 genes [71]. These findings suggest that the interaction of EHZ2, known as an epigenetic driver, with several specific genes associated with regulation of DNA methylation mediated transcriptional repression of genes (Figure 3).
The regulation of EZH2 by miRNAs in glioma
6.
miRNAs are now recognized as single-stranded noncoding RNA molecules, about 19 -- 25 nucleotides in length [72]. miRNA regulates gene expression by the sequence-selective targeting of mRNAs, promoting degradation and inhibiting translation of cellular mRNAs, depending on the degree of complementarity with target mRNA sequences [72-76]. As much knowledge about the biological characteristics of the miRNA has been accumulated, miRNA has been found to be closely associated with cellular events, including cell proliferation, apoptosis, differentiation, development and tumorigenesis [77-79]. Increasing evidences suggest that miRNAs are pivotal players in glioma biology exhibiting oncogenic or tumorsuppressive functions. The gliomas-specific expression pattern of miRNAs highlights their potential use as diagnostic biomarkers and target for the treatment of gliomas. The interactions of the epigenetic machinery modulate important cellular pathways leading to oncogenesis.
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Downregulation of miR-101 was observed in primary glioma samples of different WHO grades, whereas upregulation of miR-101 by pre-miR-101 resulted in a significant decrease in the migration of U87 glioma cells. Pre-miR-101 notably repressed EZH2 protein expression and reduced the levels of trimethylation of H3K27, suggesting inhibition of EZH2 function. Inhibition of EZH2 by pre-miR-101 attenuated GBM cell growth, migration/invasion and GBM-induced endothelial tubule formation [44]. It is known that EZH2 and EED, known as the member of PRC2 complexes, are direct targets of miR-101. MiR-101 downregulated the expression of cytoplasmic polyadenylation element-binding protein 1 (CPEB1) through reversing the methylation status of the CPEB1 promoter by regulating the presence on the promoter of the methylation-related histones H3K4me2, H3K27me3, H3K9me3 and H4K20me3. CPEB1 is hypomethylated and overexpressed in glioma cells and tissues. The knockdown of CPEB1 reduced cell senescence by regulating the expression or distribution of p53 in glioma cells. The epigenetic regulation of H3K27me3 on CPEB1 promoter is mediated by EZH2 and EED. CPEB1 is also regulated directly by miR-101 [80]. Guo et al. reported that the expression of miR-708 was downregulated in GBM cells. The overexpression of miR-708 inhibited invasion in the human GBM cell lines. Furthermore, EZH2 was reduced when miR-708 was overexpressed in GBM cells [81]. EZH2 plays an important role in promoting invasive of several types of tumor cells [82,83]. These findings suggest that the regulation of EZH2 provides support for miR-708 as a tumor suppressor that targets cell invasion. Qiu et al. have demonstrated that miR-138 is reduced in both GBM clinical specimens and cell lines, and is effective to inhibit EZH2 expression. Moreover, high levels of miR-138 are associated with long overall and progression-free survival of GBM patients. Ectopic expression of miR-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. In addition miR-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Furthermore, an EZH2-mediated signal loop, EZH2CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miR-138 [84]. These findings suggest that the 3¢-untranslated region (3¢-UTR) of EZH2 targeted directly by miRNAs is involved in regulating the growth, migration and invasion of glioma cells. Lee et al. reported that transfection of let-7 reduced expression of pan-RAS, N-RAS and K-RAS in the GBM cells. Let-7 also reduced the proliferation and migration of the cells, and reduced the sizes of the tumors produced after transplantation into nude mice [85]. Overexpression of let-7 by transfection of let-7 precursors decreased EZH2 expression and repressed clonogenic ability and sphere-forming capacity of prostate cancer cells, which was consistent with inhibition of EZH2 3¢-UTR luciferase activity [86]. The expression of miR-98 in glioma tissues was significantly lower than that in normal brain tissues. Moreover, forced expression of miR-98 accelerated the 1202
inhibition of glioma cell invasion [87]. In Eca109 cells, overexpression of miR-98 significantly inhibited the migration and invasion of esophageal squamous cell carcinoma cells, which was reversed by transfection of EZH2. MiR-98 was validated as bona fide regulators of EZH2 expression. In particular, miR-98 was underexpressed in relapsed patient samples, strongly suggesting an important role for the miR-98 and EZH2 axis in nasopharyngeal carcinoma (NPC) biology [88]. MiR-124 is dramatically downregulated in human GBM tumor cell lines and clinical specimen of glioma, and is negatively correlated with the tumor pathological grading [89,90]. MiR-124 could directly target the 3¢-UTR of EZH2 mRNA, and suppress mRNA and protein expressions of EZH2 in hepatocellular carcinoma [91]. MiR-137 expression is significantly downregulated in a cohort of 35 oligodendroglial tumors and 9 glioma cell lines compared with normal brains. Lower miR-137 expression is associated with shorter progressive-free and overall survival. Restoration of miR-137 expression significantly suppressed cell growth, anchorage-independent growth, as well as invasion of GBM cells [92]. The decreased invasion caused by miR-137 overexpression could be phenocopied by small-interfering RNA knockdown of EZH2 [93]. The patients displayed a significantly decreased expression of miR-137 levels, as well as significantly increased expression of the validated downstream target EZH2 [94]. Forced expression of miR-26a in glioma cells significantly increased both growth rate and colony formation in vitro and tumor growth and angiogenesis in vivo, while reduced expression of miR-26a played opposite roles [95]. MiR-26a strongly reduced the expression of EZH2 oncogene in NPC cells. In addition, miR-26a suppressed the expression of c-myc, the cyclin D3 and E2, and the cyclindependent kinase CDK4 and CDK6 while enhancing the expression of CDK inhibitors p14(ARF) and p21(CIP1) in an EZH2-dependent manner [96]. These results suggest that miRNAs may be involved in controlling the biology of gliomas by targeting EZH2 (Figure 4). 7.
Expert opinion
It is speculated that epigenetic control of EZH2 provides a potential target for the treatment of gliomas. In this review, we summarize our current view of the role EZH2 in the epigenetic modifications and that EZH2 is involved in cellular homeostasis affecting cell proliferation, cell growth, invasion and apoptosis, while their disruption results in plasticity of GSCs. Gene repression mediated by EZH2 acts in a highly interconnected and coordinated manner, epigenetically regulating a diverse cohort of genes involved in gliomas. Together, understanding the regulatory mechanisms of EZH2 and the function of EZH2 target genes will shed light on the development of novel glioma treatments targeting EZH2-mediated gliomas to suppress oncogenic and stemness-associated pathways, which will pave the way to prevention and remission of gliomas. Several miRNAs bind directly to the 3¢-UTR of EZH2 through which they exert their function in gliomas.
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miR-708 miR-101
C-myc CyclinD3 E2 CDK4 CDK6
miR-138
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miR-26a
Let-7 miR-98 miR-124 miR-137
EZH2 p14 p21
CDK4/6-PRb-E2F1 CPEB
P53
Figure 4. The interaction of EZH2 with several specific genes mediated by DNA methylation regulates transcriptional repression or activation in gliomas. A. EZH2 mediates H3K27 methylation and recruits DNMT1, resulting in further modifications such as maintenance of DNA methylation that is responsible for stable repression of specific genes. B. The oxidation of 5mC to 5hmC by ten--eleven translocation (TET) proteins may therefore ease the repressive effects of 5mC. In addition, TETs binding may prevent access to EZH2, further contributing to the maintenance of an unmethylated promoter. C. DNA methylation at promoter regions is normally associated with silenced genes, where binding of MeCP2, EZH2 promotes to 5mC, mediating gene repression. 5mC: 5-methylcytosine; CPEB: Cytoplasmic polyadenylation element-binding protein; DNMT1: DNA methyltransferase-1.
MiRNAs are capable of regulating EZH2 expression at both transcriptional and posttranscriptional levels. Through the interaction with EZH2 they affect H3K27 methylation and in consequence they are involved in the biology of gliomas. In addition, EZH2 interacted with DNA methylation related to genes such as DNMTs, TETs, MeCP2, mediates transcriptional repression of glioma-mediated genes. EZH2 has a mediator role in transcriptional repression of genes, and if an EZH2 role exists and is experimentally verified, epigenetic modifications will also affect gliomas biology by forming important epigenetic regulatory network in which EZH2 plays the role of the pathway-stabilizing factor. Although there has been progress in generating EZH2 inhibitors for treatment of cancer, a more deeply understanding of the multifaceted biological roles of EZH2 is yet to be clarified, in order to promote the development of pharmacological agents. Inhibitors of EZH2, including GSK-A, GSK126, GSK-343 and DZNep, represent the beginning of a promising era that on-target epigenetic drugs can be applied to clinics and be effectively treated in EZH2-high tumors. In addition, Hypericin is a potent inhibitor of glioma growth by inducing unique epigenetic downregulations of EZH2, remodeling chromatin structure. UNC1999, the first orally bioavailable inhibitor that was highly selective for EZH2, may be more efficient and effective for treatment of gliomas. Although increasing evidences support the epigenetic biomarkers and therapeutic potential of EZH2 for gliomas,
there are largely unknown about its regulatory mechanism. For example, it is needed to clarify why the expression of EZH2 is upregulated in gliomas, and what the underlying mechanisms are? What is the relationship between the DNA methylation and miRNA about EZH2? Is histone modification such as histone acetylation involved in regulation of the expression of EZH2 in gliomas? It is also not clear whether targeted EZH2 protein has a beneficial effect for treatment of gliomas. More experiments are needed to unravel how DNA methylation together with other repression mechanisms such as miRNA or histone acetylation, generates distinct patterns of EZH2 silencing in gliomas. Better understanding of the epigenetics involved in gliomas by using in vitro in cells, in vivo in rodents and gliomas specimen with advanced techniques, such as microarray and next-generation sequencing, may provide a big step toward epigenetic drug translating into clinical applications treatment of gliomas.
Declaration of interest The authors were supported by the National Science Foundation of China (No.81072066). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
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Affiliation Er-Bao Bian1,2, Jia Li1,2, Xiao-Jun He1,2, Gang Zong1,2, Tao Jiang1,2, Jun Li3 & Bing Zhao†1,2 † Author for correspondence 1 The Second Affiliated Hospital of Anhui Medical University, Department of Neurosurgery, Hefei 230601, China E-mail: [email protected] 2 Anhui Medical University, Cerebral Vascular Disease Research Center, Hefei 230601, China 3 Anhui Medical University, School of Pharmacy, Hefei 230032, China
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Epigenetic modification in gliomas: role of the histone methyltransferase EZH2 1.
Introduction
2.
EZH2
3.
The role of EZH2 in glioma
4.
Overview of DNA methylation
5.
The interaction of DNA methylation with EZH2 in gliomas
6.
The regulation of EZH2 by miRNAs in glioma
7.
Expert opinion
Er-Bao Bian, Jia Li, Xiao-Jun He, Gang Zong, Tao Jiang, Jun Li & Bing Zhao† †
The Second Affiliated Hospital of Anhui Medical University, Department of Neurosurgery, Hefei, China
Introduction: Gliomas are characterized by increased anaplasia, malignization, proliferation and invasion. They exhibit high resistance to standard treatment with combinations of radiotherapy and chemotherapy. They are currently the most common primary malignancy tumors in the brain that is related to a high mortality rate. Recently, increasing evidence suggests that EZH2 is involved in a number of glioma cell processes, including proliferation, apoptosis, invasion and angiogenesis. Areas covered: In this review, we emphasize the role of EZH2 in gliomas. We also address that EZH2 interacting with DNA methylation mediates transcriptional repression of specific genes in gliomas, and the regulation of EZH2 by microRNAs in gliomas. Expert opinion: Although the exact role of EZH2 in gliomas has not been fully elucidated, to understand the role of EZH2 proteins in epigenetic modification will provide valuable insights into the causes of gliomas, and pave the way to the potential future applications of EZH2 in the treatment of gliomas. Keywords: EZH2, glioma, methylation, microRNAs Expert Opin. Ther. Targets (2014) 18(10):1197-1206
1.
Introduction
Gliomas are the most common form of CNS primary tumors, accounting for > 50% of all primary CNS tumors [1]. Malignant gliomas possess a sequence of characteristics: transformation from a cell of origin, increased proliferative combined with abrogation of cell cycle control, clonal evolution, acquisition of invasive ability and activation of angiogenesis signal pathways [2]. Gliomas are tumors of neuroepithelial tissues, including several types that are astrocytic, oligodendroglial, oligoastrocytic, ependymal, choroid plexus-derived or others, according to the cell type with which they share [3]. Gliomas are clinicopathologically categorized into grades I -- IV according to the 2007 classification by the WHO [4]. Histological evaluation remains the gold standard for glioma diagnosis and serves as a criterion to estimate the prognosis of patients [5]. Among all gliomas cases diagnosed, most of them belong to grade IV, identified as high-grade astrocytoma or glioblastoma (GBM) with marked nuclear atypia and high mitotic rates [6,7]. Primary GBM arise de novo, however, secondary GBM derives from pre-existing low-grade astrocytomas [8]. Most patients with these glioma progress to GBM, with current treatment protocols, patients with GBM have a 5-year survival rate of no > 30% [9]. Despite aggressive therapeutic modalities are surgical resection combined with radiation and temozolomide chemotherapy, the prognostic statistics for GBM patients is discouraging [10-12]. GBM is highly infiltrative, however, complete elimination of the tumor by surgery is usually impossible. Further, radiation and chemotherapy can kill most of the remaining tumor cells, those that typically reoccur [13]. 10.1517/14728222.2014.941807 © 2014 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted
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Article highlights. . . .
The role of EZH2 in glioma. The interaction of DNA methylation with EZH2 in gliomas. The regulation of EZH2 by microRNAs in gliomas.
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This box summarizes key points contained in the article.
Accumulating evidences suggests that the presence of a subpopulation of cells with the potential to initiate and maintain growth of gliomas might be pivotal for their resistance to conventional therapies [14]. Very recently, GBM is thought to be derived from a small population of GBM stem cells (GSCs) [15-17]. GSCs and normal neural stem cells appear to share common features, including the expression of neural stem cell markers, the capacity for self-renewal and long-term proliferation, the formation of neurospheres and the capability of differentiating into multiple lineages. Intriguingly, recent studies revealed that in addition to GSCs differentiating into non-GSCs, non-GSCs can become GSCs or tumor-initiating cells and exhibit an enhanced ability to form neurospheres, thereby suggesting that the GSC state may be plastic [18,19]. This phenotypic plasticity between the GSC and non-GSC states may be regulated by the signaling pathways, genetic and epigenetic mechanisms. Recently, increasing evidences indicate that EZH2 have been implicated in gliomas initiation, progression, and importantly, in the formation, maintenance and plasticity of GSCs. A rapidly growing body of data describes the importance of EZH2 in gliomas, whereas most recent reports focus on the interplay between EZH2 and noncoding RNAs. Therefore, EZH2 may emerge as a potential therapeutic target in gliomas and in the development of novel treatment strategies that target both genetic and epigenetic mechanisms of tumorigenesis. With the knowledge that EZH2 is associated with aggressive gliomas, understanding how EZH2 promotes disease progression has gradually made possible the development of epigenetic drugs that directly or indirectly target EZH2. Herein, this review aims to provide a comprehensive overview of EZH2 involvement in gliomas pathogenesis and progression, with specific emphasis on the regulatory network of the interaction of EZH2 with microRNAs (miRNAs) and DNA methylation. 2.
EZH2
Human EZH2 gene is located on the long arm of chromosome 7 at position 7q35 and encodes a 746 amino acid protein that has histone methyltransferase (HMT) activity [20]. Besides its HMT activity, EZH2 protein contains several domains with potential functions (Figure 1). The major domains include CXC, cysteine-rich domain; noncoding RNA-binding domain; SANT (SWI3, ADA2, N-CoR) and TFIIIB DNA1198
binding domain; SET, Su(var)3-9, enhancer of zeste, trithorax domain [21]. The N-terminal SET domains provide binding sites for assembly with the required partner subunits. In addition, both the C-terminal SET domain and the adjacent cysteine-rich CXC domain are required for HMT activity [22-24]. The SET domain of EZH2 is identified as the signature of methyltransferases, which leads to trimethylation (me3) of histone 3 (H3) at lysine 27 (K27), as well as at lysine 9 (K9) albeit to a much lesser extent [25]. When the polycomb repressive complex 2 (PRC2) complex is recruited to chromatin, the HMT EZH2 catalyses the H3K27me3, which results in subsequent recruitment of the PRC1 complex that monoubiquitylates the lysine 119 of histone H2A (H2AK119ub1) to prevent RNA polymerase II-dependent transcriptional elongation and consolidate transcriptional repression [26,27]. More recently, a study reported that EZH2 mutations may either cause gain-of-function or complete loss of HMT activity in lymphoma and myeloid neoplasms [28]. In addition, somatic activating mutations in the catalytic (SET) domain of EZH2 have been identified in follicular lymphoma and germinal center B-cell diffuse large B-cell lymphoma, leading to increased H3K27me3 [29-33]. It is interesting to point out that the role of transcriptional regulation of EZH2 in epigenetics is complex. EZH2, identified as the core member of PRC2, combines with embryonic ectoderm development (EED) and SUZ12 and plays the role of HTMase to catalyze histone H3k27 and H3K9 methylation, and represses transcription at the chromatin level. Another role of EZH2 is direct interaction with DNA methyltransferases (DNMTs) to catalyze the methylation of promoter region in downstream genes. In addition, several reports have shown that EZH2 may function in target gene activation as a multifaceted molecule. 3.
The role of EZH2 in glioma
EZH2 functions as a chromatin-modifying enzyme, which plays a critical role in epigenetic silencing of genes (especially TSGs) through complex mechanisms (Figure 2). Increasing evidences have appeared that the links between EZH2 and tumorigenesis have been found in diverse type of cancers: breast, prostate, bladder, colon, lung, pancreatic, sarcoma and lymphomas [34-41]. The common phenomenon is that the levels of EZH2 expression are abnormally upregulated in cancer tissues compared with normal tissues, and overexpression of EZH2 is related to advanced stages of disease and a poor clinical outcome. Recently, a number of evidences suggest that EZH2 plays a central role in gliomas. EZH2 was more expressed in GBM than in low-grade gliomas and highly expressed in U87 human glioma cells [42]. The knockdown of EZH2 expression by using RNA interference in U87 human glioma cells resulted in apoptosis and a cell cycle arrest in the G0/G1phase. Moreover, silencing of EZH2 altered the mitochondrial membrane potential and promoted the release of cytochrome c from the mitochondria. In addition, the reduced expression of EZH2 altered the Bax
Expert Opin. Ther. Targets (2014) 18(10)
Epigenetic modification in gliomas
EID EZH2
Domain I SANT
Domain II ncRBC
EED
CXC CXC
SANT
RNA
SET SET
SUZ12
Figure 1. Characterized domains with potential functions are displayed for EZH2. Both the C-terminal SET domain and the adjacent cysteine-rich CXC domain are required for histone methyltransferase activity. Robust methyltransferase requires EZH2 assembly with both EED and SUZ12 of domain II, and domains required for binding these noncatalytic subunits are indicated. Expert Opin. Ther. Targets Downloaded from informahealthcare.com by Universitat de Girona on 10/24/14 For personal use only.
EED: Embryonic ectoderm development; ncRBD: Noncoding RNA-binding domain; SANT: SWI3, ADA2, N-CoR.
GSC self-renewal
c-myc
STAT3
Apoptosis
Caspase9 Caspase3
Bax Bcl-2
EZH2
H3K27m
Puripotency
AXL
Proliferation
Invasion
Figure 2. EZH2 regulates the biology of gliomas by controlling of different pathways. Knockdown of EZH2 alters expression of apoptosis associated-genes and reduces transcription of AXL receptor kinase in gliomas. In addition, EZH2 regulates the biology of GSC by control of global levels of H3K27 trimethylation. AXL: AXL receptor kinase; GSC: Glioblastoma stem cell.
and Bcl-2 protein levels and led to the activation of caspase 9 and caspase 3 [43]. Smits et al. reported that inhibition of EZH2 in vivo by systemic 3-deazaneplanocin A (DZNep) administration in a U87-Fluc-mCherry GBM xenograft mouse imaging model resulted in reduced tumor growth [44]. Glioma progression is associated with EZH2 overexpression and PRC2 target gene silencing; EZH2 inhibits differentiation and activates cancer-, cell cycle- and cellular movement-related genes. In addition, EZH2 is both a prognostic factor and a promising therapy target in brain tumors [45]. Ott et al. demonstrated that EZH2 is expressed in human glioma and correlates with malignancy, whereas silencing of EZH2 reduced the proliferation and invasion of glioma cells. Microarray analyses of U87MG glioma cells after EZH2 knockdown demonstrated a strong transcriptional reduction of the AXL receptor kinase (AXL). Furthermore, histone modification appeared to be involved in the positive regulation of AXL by EZH2. Silencing AXL
mimicked the anti-invasive effects of EZH2 knockdown. Finally, AXL expression is found in human gliomas with high EZH2 expression [46]. Recently, both Bmi1 and EZH2 expressions in glioma tissues were significantly higher than those in corresponding nonneoplastic brain tissues. Additionally, the upregulations of Bmi1 and EZH2 proteins were both significantly related to advanced WHO grades and low Karnofsky. Moreover, the overall survival of patients with high Bmi1 protein expression or high EZH2 protein expression was obviously lower than those with low expressions [47]. Abdouh et al. reported that Bmi1 is expressed in human GBM tumors and highly enriched in CD133-positive cells. Stable Bmi1 knockdown using short hairpin RNA (shRNA)-expressing lentiviruses inhibited clonogenic potential in vitro and tumor formation in vivo. Bmi1 prevents CD133-positive cell apoptosis and/or differentiation into neurons and astrocytes, depending on the cellular
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context. Bmi1 represses alternate tumor suppressor pathways that attempt to compensate for INK4A/ARF/P53 deletion and PI(3)K/AKT hyperactivity [48]. More interestingly, glioma patients with combined overexpression of Bmi1 and EZH2 proteins had the shortest overall survival. Furthermore, expression of Bmi1 and EZH2 was independent prognostic factor for overall survival in glioma patients [47]. Natsume et al. reported that the biological interconversion between GSCs and differentiated non-GSCs is functionally plastic and accompanied by gain or loss of PRC2-mediated histone H3 K27 trimethylation on pluripotency [18]. H3K27M inhibits the enzymatic activity of the PRC2 through interaction with the EZH2 subunit. In addition, transgenes containing lysine-tomethionine substitutions at other known methylated lysines (H3K9 and H3K36) are sufficient to cause specific reduction in methylation through inhibition of SET-domain enzymes. Recent studies have identified a Lys 27-to-methionine (K27M) mutation at one allele of H3F3A, one of the two genes encoding histone H3 variant H3.3, in 60% of high-grade pediatric glioma cases. The levels of H3K27 di- and trimethylation (H3K27me2 and H3K27me3) are reduced globally in H3.3K27M patient samples due to the expression of the H3.3K27M mutant allele. Remarkably, H3K27me3 and EZH2 (the catalytic subunit of H3K27 methyltransferase) at chromatin are dramatically increased locally at hundreds of gene loci in H3.3K27M patient cells. Moreover, the gain of H3K27me3 and EZH2 at gene promoters alters the expression of genes. In addition, the development associated genes (e.g., Nanog, Wnt1, BMP5) together with alterations in the subcellular localization of EZH2 [18,49]. A recent study showed that EZH2 is highly expressed in murine and human GSC. Treatment with suberoylanilide hydroxamic acid caused significant upregulation of PRC2 predicted target genes, GSC disruption and decreased expression of EZH2 and of the stem cell marker CD133. Inhibition of EZH2 expression by shRNA was related to a significant decrease in the proliferation of glioma cells. Inhibition of EZH2, the main component of the PRC2, also impaired GBM tumor growth [42,48]. Targeted pharmacologic disruption of EZH2 by the S-adenosylhomocysteine hydrolase inhibitor DZNep, or its specific downregulation by shRNA, strongly impairs GBM cancer stem cell (CSC) self-renewal in vitro and tumor-initiating capacity in vivo. In DZNep-treated GBM CSCs, the expression of c-myc, known as a direct target of EZH2 in GBM CSCs, strongly repressed upon EZH2 depletion [50]. EZH2 binds to and methylates STAT3, resulting in enhanced STAT3 activity by increased tyrosine phosphorylation of STAT3. EZH2 knockdown greatly decreased global levels of H3K27 trimethylation in GSCs and reduced the levels of p-STAT3 in GSCs. P-STAT3 in GSCs was also rapidly decreased by treatment with either DZNep or GSK126 [51]. These results suggest that the inhibition of EZH2 reverses the silencing of Polycomb target genes and diminishes STAT3 activity. 1200
4.
Overview of DNA methylation
DNA methylation is an enzyme-mediated chemical modification of DNA by the addition of a methyl group from S-adenosyl-L-methionine substrates to the 5-position of cytosine (5-methylcytosine, 5mC) [52]. In humans and other mammals, DNA methylation is catalyzed by two general classes of DNMTs: maintenance DNMT1 and de novo DNMT3A and DNMT3B [53,54]. DNMT1, which maintains the existing methylation patterns following DNA replication, and DNMT3A and DNMT3B, de novo enzymes that prefer unmethylated and hemimethylated DNA and establish new DNA methylation patterns early in development [55,56]. DNA methylation can regulate gene expression directly, by repressing the binding of key transcription factors, and indirectly, by recruiting methyl-CpG-binding domain (MBD) proteins to the promoter of gene. So far, six members of the methyl-CpGbinding proteins family have been identified: MECP2, MBD1, MBD2, MBD3, MBD4 and Kaiso [57]. MECP2 recognizes methylated CpG sites and, when cooperation with DNMT1, forms a complex to copy the parental DNA methylation to the daughter DNA strands during cell division [58]. In addition, DNMTs interact with polycomb group (PcG) protein EZH2 to methylate EZH2-binding promoters, suggesting that epigenetic transcriptional repression complex is closely connected in regulating the expression of gene [58]. DNA demethylation is regulated by the ten--eleven translocation (TET) family. To date, three members of the TETs family, including TET1 TET2 TET3, have been identified in mammals. TETs convert 5mC to 5-hydroxymethylcytosine (5hmC), provide a way for dysregulation of DNA methylation [59]. TETs can promote to transcriptional repression, and to a minor extent also transcriptional activation, and that the majority of TETs-mediated transcriptional effects are independent of conversion of 5mC to 5hmC [60,61]. TET1 and TET3, but not TET2, is frequently found in chromatinbinding proteins such as DNMT1 and 5mC-binding proteins (MBDs), which is involved in regulating the expression of genes [60]. TET1 contributes to silencing of a subgroup of genes by facilitating recruitment of PRC2 to CpG-rich target gene promoters [62,63]. Another mechanism by which transcriptional repression of target genes are mediated is by the interaction of TET1 with the Sin3A co-repressor complex [62]. TETs regulate gene expression by preventing the gene promoter from hypermethylation mediated by DNMTs [64].
The interaction of DNA methylation with EZH2 in gliomas
5.
DNA hypermethylation is frequently recruited by PcGmediated repressive mark to a number of tumor suppressor genes that are epigenetic silencing in cancer cells, suggesting that links EZH2 with DNMTs has established a role for this protein during the induction and targeting of DNA
Expert Opin. Ther. Targets (2014) 18(10)
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Transcrptional repression CpG H3K27m Me EZH2
DNMTs
B.
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EZH2
5hmc
5mc TETs
C.
Transcrptional activation
EZH2
MeCP2 Transcrptional repression
Methylated CpGs
Figure 3. miRNAs mediate the signal pathways of gliomas by targeting EZH2. EZH2 interacts with miRNAs and other upstream and downstream molecules. EZH2 is under the negative control of several tumor-suppressive miRNAs and inhibits cell cycle regulators and molecules involved in gliomas. 5hmC: 5-hydroxymethylcytosine; 5mC: 5-methylcytosine; DNMTs: DNA methyltransferases; miRNA: MicroRNAs; TET: Ten--eleven translocation.
methylation [65-67]. The expression levels of DNMT1 and DNMT3B were strongly upregulated in malignant glioma tissue compared with normal brain samples, whereas DNMT1 and DNMT3B knockdown resulted in the restored expression of 308 genes that also contained promoter region hypermethylation [68]. EZH2 interacts with DNMTs and associates with DNMT activity. Binding of DNMTs to several EZH2-repressed genes depends on the presence of EZH2. Furthermore, EZH2 is required for DNA methylation of EZH2-target promoters, suggesting that EZH2 serves as a recruitment platform for DNMTs, thus highlighting a previously unrecognized direct connection between two key epigenetic repression systems [66]. Both MeCP2 and EZH2 have recently been shown to be recruited and appear to be involved in the maintenance of PPARg repression in mouse stellate cells undergoing liver fibrogenesis [69]. Consistently, MeCP2 and EZH2 levels inversely correlate with PPARg expression in the colorectal cancer cells. MeCP2 and EZH2 silencing re-activate PPARg, confirming their crucial role in PPARg epigenetic repression [70]. Cartron et al. reported that U251 cells presenting a low level of EZH2 induced by the knockdown of EZH2. EZH2 downregulation affected the TET1/EZH2 interactions. The downexpression of EZH2 decreased TET1 recruitment on the HOXD12 genes. The decrease of TET1/EZH2 recruitment on the HOXD12 gene in cells treated with siRNA-EZH2 produced an increase in methylation of the HOX12D gene. These results
suggest that EZH2 plays a role as anchors for TET1 recruitment on the HOXD12 genes [71]. These findings suggest that the interaction of EHZ2, known as an epigenetic driver, with several specific genes associated with regulation of DNA methylation mediated transcriptional repression of genes (Figure 3).
The regulation of EZH2 by miRNAs in glioma
6.
miRNAs are now recognized as single-stranded noncoding RNA molecules, about 19 -- 25 nucleotides in length [72]. miRNA regulates gene expression by the sequence-selective targeting of mRNAs, promoting degradation and inhibiting translation of cellular mRNAs, depending on the degree of complementarity with target mRNA sequences [72-76]. As much knowledge about the biological characteristics of the miRNA has been accumulated, miRNA has been found to be closely associated with cellular events, including cell proliferation, apoptosis, differentiation, development and tumorigenesis [77-79]. Increasing evidences suggest that miRNAs are pivotal players in glioma biology exhibiting oncogenic or tumorsuppressive functions. The gliomas-specific expression pattern of miRNAs highlights their potential use as diagnostic biomarkers and target for the treatment of gliomas. The interactions of the epigenetic machinery modulate important cellular pathways leading to oncogenesis.
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Downregulation of miR-101 was observed in primary glioma samples of different WHO grades, whereas upregulation of miR-101 by pre-miR-101 resulted in a significant decrease in the migration of U87 glioma cells. Pre-miR-101 notably repressed EZH2 protein expression and reduced the levels of trimethylation of H3K27, suggesting inhibition of EZH2 function. Inhibition of EZH2 by pre-miR-101 attenuated GBM cell growth, migration/invasion and GBM-induced endothelial tubule formation [44]. It is known that EZH2 and EED, known as the member of PRC2 complexes, are direct targets of miR-101. MiR-101 downregulated the expression of cytoplasmic polyadenylation element-binding protein 1 (CPEB1) through reversing the methylation status of the CPEB1 promoter by regulating the presence on the promoter of the methylation-related histones H3K4me2, H3K27me3, H3K9me3 and H4K20me3. CPEB1 is hypomethylated and overexpressed in glioma cells and tissues. The knockdown of CPEB1 reduced cell senescence by regulating the expression or distribution of p53 in glioma cells. The epigenetic regulation of H3K27me3 on CPEB1 promoter is mediated by EZH2 and EED. CPEB1 is also regulated directly by miR-101 [80]. Guo et al. reported that the expression of miR-708 was downregulated in GBM cells. The overexpression of miR-708 inhibited invasion in the human GBM cell lines. Furthermore, EZH2 was reduced when miR-708 was overexpressed in GBM cells [81]. EZH2 plays an important role in promoting invasive of several types of tumor cells [82,83]. These findings suggest that the regulation of EZH2 provides support for miR-708 as a tumor suppressor that targets cell invasion. Qiu et al. have demonstrated that miR-138 is reduced in both GBM clinical specimens and cell lines, and is effective to inhibit EZH2 expression. Moreover, high levels of miR-138 are associated with long overall and progression-free survival of GBM patients. Ectopic expression of miR-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. In addition miR-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Furthermore, an EZH2-mediated signal loop, EZH2CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miR-138 [84]. These findings suggest that the 3¢-untranslated region (3¢-UTR) of EZH2 targeted directly by miRNAs is involved in regulating the growth, migration and invasion of glioma cells. Lee et al. reported that transfection of let-7 reduced expression of pan-RAS, N-RAS and K-RAS in the GBM cells. Let-7 also reduced the proliferation and migration of the cells, and reduced the sizes of the tumors produced after transplantation into nude mice [85]. Overexpression of let-7 by transfection of let-7 precursors decreased EZH2 expression and repressed clonogenic ability and sphere-forming capacity of prostate cancer cells, which was consistent with inhibition of EZH2 3¢-UTR luciferase activity [86]. The expression of miR-98 in glioma tissues was significantly lower than that in normal brain tissues. Moreover, forced expression of miR-98 accelerated the 1202
inhibition of glioma cell invasion [87]. In Eca109 cells, overexpression of miR-98 significantly inhibited the migration and invasion of esophageal squamous cell carcinoma cells, which was reversed by transfection of EZH2. MiR-98 was validated as bona fide regulators of EZH2 expression. In particular, miR-98 was underexpressed in relapsed patient samples, strongly suggesting an important role for the miR-98 and EZH2 axis in nasopharyngeal carcinoma (NPC) biology [88]. MiR-124 is dramatically downregulated in human GBM tumor cell lines and clinical specimen of glioma, and is negatively correlated with the tumor pathological grading [89,90]. MiR-124 could directly target the 3¢-UTR of EZH2 mRNA, and suppress mRNA and protein expressions of EZH2 in hepatocellular carcinoma [91]. MiR-137 expression is significantly downregulated in a cohort of 35 oligodendroglial tumors and 9 glioma cell lines compared with normal brains. Lower miR-137 expression is associated with shorter progressive-free and overall survival. Restoration of miR-137 expression significantly suppressed cell growth, anchorage-independent growth, as well as invasion of GBM cells [92]. The decreased invasion caused by miR-137 overexpression could be phenocopied by small-interfering RNA knockdown of EZH2 [93]. The patients displayed a significantly decreased expression of miR-137 levels, as well as significantly increased expression of the validated downstream target EZH2 [94]. Forced expression of miR-26a in glioma cells significantly increased both growth rate and colony formation in vitro and tumor growth and angiogenesis in vivo, while reduced expression of miR-26a played opposite roles [95]. MiR-26a strongly reduced the expression of EZH2 oncogene in NPC cells. In addition, miR-26a suppressed the expression of c-myc, the cyclin D3 and E2, and the cyclindependent kinase CDK4 and CDK6 while enhancing the expression of CDK inhibitors p14(ARF) and p21(CIP1) in an EZH2-dependent manner [96]. These results suggest that miRNAs may be involved in controlling the biology of gliomas by targeting EZH2 (Figure 4). 7.
Expert opinion
It is speculated that epigenetic control of EZH2 provides a potential target for the treatment of gliomas. In this review, we summarize our current view of the role EZH2 in the epigenetic modifications and that EZH2 is involved in cellular homeostasis affecting cell proliferation, cell growth, invasion and apoptosis, while their disruption results in plasticity of GSCs. Gene repression mediated by EZH2 acts in a highly interconnected and coordinated manner, epigenetically regulating a diverse cohort of genes involved in gliomas. Together, understanding the regulatory mechanisms of EZH2 and the function of EZH2 target genes will shed light on the development of novel glioma treatments targeting EZH2-mediated gliomas to suppress oncogenic and stemness-associated pathways, which will pave the way to prevention and remission of gliomas. Several miRNAs bind directly to the 3¢-UTR of EZH2 through which they exert their function in gliomas.
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miR-708 miR-101
C-myc CyclinD3 E2 CDK4 CDK6
miR-138
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miR-26a
Let-7 miR-98 miR-124 miR-137
EZH2 p14 p21
CDK4/6-PRb-E2F1 CPEB
P53
Figure 4. The interaction of EZH2 with several specific genes mediated by DNA methylation regulates transcriptional repression or activation in gliomas. A. EZH2 mediates H3K27 methylation and recruits DNMT1, resulting in further modifications such as maintenance of DNA methylation that is responsible for stable repression of specific genes. B. The oxidation of 5mC to 5hmC by ten--eleven translocation (TET) proteins may therefore ease the repressive effects of 5mC. In addition, TETs binding may prevent access to EZH2, further contributing to the maintenance of an unmethylated promoter. C. DNA methylation at promoter regions is normally associated with silenced genes, where binding of MeCP2, EZH2 promotes to 5mC, mediating gene repression. 5mC: 5-methylcytosine; CPEB: Cytoplasmic polyadenylation element-binding protein; DNMT1: DNA methyltransferase-1.
MiRNAs are capable of regulating EZH2 expression at both transcriptional and posttranscriptional levels. Through the interaction with EZH2 they affect H3K27 methylation and in consequence they are involved in the biology of gliomas. In addition, EZH2 interacted with DNA methylation related to genes such as DNMTs, TETs, MeCP2, mediates transcriptional repression of glioma-mediated genes. EZH2 has a mediator role in transcriptional repression of genes, and if an EZH2 role exists and is experimentally verified, epigenetic modifications will also affect gliomas biology by forming important epigenetic regulatory network in which EZH2 plays the role of the pathway-stabilizing factor. Although there has been progress in generating EZH2 inhibitors for treatment of cancer, a more deeply understanding of the multifaceted biological roles of EZH2 is yet to be clarified, in order to promote the development of pharmacological agents. Inhibitors of EZH2, including GSK-A, GSK126, GSK-343 and DZNep, represent the beginning of a promising era that on-target epigenetic drugs can be applied to clinics and be effectively treated in EZH2-high tumors. In addition, Hypericin is a potent inhibitor of glioma growth by inducing unique epigenetic downregulations of EZH2, remodeling chromatin structure. UNC1999, the first orally bioavailable inhibitor that was highly selective for EZH2, may be more efficient and effective for treatment of gliomas. Although increasing evidences support the epigenetic biomarkers and therapeutic potential of EZH2 for gliomas,
there are largely unknown about its regulatory mechanism. For example, it is needed to clarify why the expression of EZH2 is upregulated in gliomas, and what the underlying mechanisms are? What is the relationship between the DNA methylation and miRNA about EZH2? Is histone modification such as histone acetylation involved in regulation of the expression of EZH2 in gliomas? It is also not clear whether targeted EZH2 protein has a beneficial effect for treatment of gliomas. More experiments are needed to unravel how DNA methylation together with other repression mechanisms such as miRNA or histone acetylation, generates distinct patterns of EZH2 silencing in gliomas. Better understanding of the epigenetics involved in gliomas by using in vitro in cells, in vivo in rodents and gliomas specimen with advanced techniques, such as microarray and next-generation sequencing, may provide a big step toward epigenetic drug translating into clinical applications treatment of gliomas.
Declaration of interest The authors were supported by the National Science Foundation of China (No.81072066). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
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Affiliation Er-Bao Bian1,2, Jia Li1,2, Xiao-Jun He1,2, Gang Zong1,2, Tao Jiang1,2, Jun Li3 & Bing Zhao†1,2 † Author for correspondence 1 The Second Affiliated Hospital of Anhui Medical University, Department of Neurosurgery, Hefei 230601, China E-mail: [email protected] 2 Anhui Medical University, Cerebral Vascular Disease Research Center, Hefei 230601, China 3 Anhui Medical University, School of Pharmacy, Hefei 230032, China
