Enhancer Of Zeste Homolog 2 Overexpression Research Articles

Enhancer of Zeste Homolog 2 Overexpression Has a Role in the Development of Anaplastic Thyroid Carcinomas

Enhancer of zeste homolog 2 (EZH2) is a histone lysine methyltransferase belonging to the polycomb group protein family. Overexpression of EZH2 has been found in several human malignancies including hematological and solid tumors. In this study we investigated the expression levels of EZH2 and its polycomb group protein partners in thyroid carcinoma tissues with different degrees of malignancy to identify potential new therapeutic targets for anaplastic thyroid carcinoma (ATC). We show that high EZH2 expression levels are characteristic of undifferentiated ATC, whereas no significant changes were observed in well-differentiated papillary and follicular thyroid carcinomas as compared with normal thyroid. Knockdown of EZH2 in ATC cell lines results in cell growth inhibition, loss of anchorage-independent growth, migration, and invasion properties. Moreover, we demonstrate that EZH2 directly controls differentiation of ATC cells by silencing the thyroid specific transcription factor paired-box gene 8 (PAX8). EZH2 is specifically overexpressed in ATC, and it directly contributes to transcriptional silencing of PAX8 gene and ATC differentiation.

Enhancer of zeste homolog 2: A potential target for tumor therapy

Enhancer of zeste homolog 2 (EZH2) is a subunit of the Polycomb-Repressive Complex 2 (PRC2), which catalyses the trimethylation of histone H3 on Lys 27 (H3K27) and involves in genes repression. EZH2 is amplified and overexpressed in a variety of cancers, including prostate and breast cancer. Overexpression of EZH2 has been associated with the invasion and progression of malignant cancers, especially with the progression of prostate cancer. Here, we review the structure and biological function of EZH2, especially focused on its activities in the tumorigenesis.

Enhanced Gene Expression of EZH2 In Patients with Primary Myelofibrosis

AbstractAbstract 4118 Introduction:The polycomb repressive complex (PRC) 2 contains 3 core proteins, EZH2 (enhancer of zeste homolog 2), SUZ12, and EED, in which the SET (suppressor of vaegation-enhancer of zeste-trithorax) domain of EZH2 mediates the histone methyltransferase activity. This induces trimethylation of lysine 27 on histone H3, regulates the expression of HOX genes, and promotes proliferation and aggressiveness of neoplastic cells. EZH2, a known repressor of gene transcription, has been reported to be overexpressed in many cancers and correlates with poor prognosis. EZH2 may also be involved in disease progression in patients with the classical Philadelphia-negative chronic myeloproliferative neoplasms (CMPNs) encompassing essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Since the potential oncogenic role of EZH2 in CMPNs has never been investigated, we have assessed gene expression of EZH2 in a cohort of patients with CMPNs. Patients and Methods:Using Affymetrix HG-U133 2.0 Plus microarrays, recognizing 54675 probe sets (38500 genes), gene expression profiling has been performed on control subjects (n=21) and patients with ET (n =19), PV (n=41), and PMF (n=9). All patients were diagnosed according to the WHO criteria of a CMPN. Total RNA was purified from whole blood and amplified to biotin-labeled RNA and hybridized to microarray chips. Results:We identified 20439, 25307, 17417, and 25421 probe sets which were differentially expressed between controls and patients with ET, PV, PMF, and CPMNs as a whole, respectively (false discovery rate (FDR) adjusted p values < 0.05). These genes included EZH2, which was highly significantly upregulated in patients with PMF as compared to controls (2.3 fold upregulated; uncorrected p-value=1.09×10-8 and FDR=1.75×10-6, and between PMF and non-PMF patients (fold change=2.0, FDR < 0.0005). No significant differences in EZH2 gene expression were recorded between controls and ET patients, controls and PV patients, or controls and the CMPN group as a whole. Within patients, the EZH2 gene was also differentially expressed with the highest levels being recorded in patients with PMF compared to PV patients (fold change=2.4, FDR < 7.5 ×10-6). Discussion and Conclusions:Using global gene expression profiling we have found the EZH2 gene to be significantly upregulated in CMPN patients, with the highest expression levels being found in PMF. We hypothesize that an altered expression of EZH2 may be involved in the transformation of ET and PV into myelofibrosis. It remains to be clarified if deregulation of EZH2 occurs consequent to mutations in the EZH2-gene. Enhanced EZH2 expression may also be associated with silencing of differentiation genes during myelofibrotic and leukemic transformation. An increased expression of EZH2 may provide a proliferative advantage of the malignant clone through interaction with the pathways of key elements controlling cell growth arrest and differentiation, (e.g. nuclear factor kappa beta and - the proteasome pathway). Studies are in progress to elucidate if genomic loss of distinct microRNAs (microRNA 101 leads to overexpression of EZH2 in cancer is associated with overexpression of EZH2 in CMPNs. Highly expressed EZH2 may be a new marker of an aggressive clinical phenotype which might imply EZH2 as a novel biomarker for predicting prognosis. If so, EZH2-blockade might be a novel approach to be incorporated in the strategies for developing epigenetic therapies in patients with CMPNs. Disclosures:No relevant conflicts of interest to declare.

MicroRNA-101 Is Down-Regulated In PV and ET Granulocytes and Its Decrease Is Associated with Over-Expression of Histone Methyltransferase In EZH2 In MPN Patients

AbstractAbstract 1989Polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF) are hematological malignancies due to clonal proliferation arising from a multilineage stem cell and have been classified as myeloproliferative neoplasms (MPNs). A somatic point JAK2 V617F mutation occurs in the majority (>95%) of PV patients and about 50–60% of ET and MF patients. The nonspecificity of this mutation, presence of JAK2V617F-negative PV patients, and evidence of both JAK2V617F-negative and positive PV clones demonstrate that the JAK2V617F mutation is not the initial or sole somatic event in the pathogenesis of PV. However, JAK2V617F plays an important role in the dysregulation of hematopoiesis in MPNs.MicroRNAs (miRs) have been shown to be important regulators of hematopoiesis. We searched for miRs that directly regulate JAK2 expression by in silico analysis. Two of these miRs, namely miR101-1 and miR101-2, had JAK2 as a putative target. Interestingly, miR101-2 is located on chromosome 9p adjacent to the JAK2 locus. miR101 also negatively regulates the enhancer of zeste homolog 2 (EZH2), a member of the polycomb group of proteins involved in controlling and maintaining stem cell pluripotency and cell proliferation. Loss of miR101 in the cancer genome leads to overexpression of EZH2, contributing to cancer progression.We analyzed the expression of miR101 in 35 PV, 15 ET and 3 MF patients and 20 normal controls by quantitative RT-PCR and found it was decreased in PV, ET and MF patients compared to normal controls (PV; P<0.03 and ET; P<0.01). We next investigated whether miR101 expression inversely correlates with EZH2 mRNA levels in MPN patients, as had been previously reported in human tumors. We examined the expression level of EZH2 for the same cohort of PV, ET and MF patients by quantitative RT-PCR and found a significant increase of EZH2 expression compared with normal controls (PV; P<0.02 and ET; P<0.05).Analysis of the molecular bases of these observations is in progress. We will test the hypothesis that the dysregulation of these miRs further increases the expression of EZH2 and contributes to the severity and pathophysiology of the MPNs. Disclosures:No relevant conflicts of interest to declare.

Overexpression of EZH2 contributes to acquired cisplatin resistance in ovarian cancer cells in vitro and in vivo

Enhancer of Zeste Homologue 2 (EZH2), a specific histone 3 lysine 27 (H3K27) methyltransferase, plays a critical role in tumorigenesis and cancer progression through epigenetic gene silencing and chromatin remodeling. However, the role of EZH2 in chemotherapy resistance is unknown. In this study, we found that EZH2 was overexpressed in cisplatin-resistant ovarian cancer cells compared with cisplatin-sensitive cells. Knockdown of EZH2 by RNA interference (RNAi) resensitized drug-resistant ovarian cancer A2780/DDP cells to cisplatin and decreased the level of H3K27 trimethylation (H3K27me3). Moreover, EZH2 downregulation suppressed cell proliferation and caused G2/M cell cycle arrest in A2780/DDP cells. Loss of EZH2 also enhanced sensibility of tumor xenografts to cisplatin and inhibited tumor growth in vivo. Our results indicate that EZH2 is essential for chemotherapy resistance in cisplatin-resistant cancer cells in vitro and in vivo, which is probably through H3K27 methylation as well as regulation of cell proliferation. EZH2 could be a potential novel epigenetic target to overcome drug resistance.

EZH2 supports ovarian carcinoma cell invasion and/or metastasis via regulation of TGF-β1 and is a predictor of outcome in ovarian carcinoma patients

It was suggested that the enhancer of zeste homolog 2 (EZH2) gene is a putative candidate oncogene in several types of human cancer. The potential oncogenic role of EZH2 and its clinical/prognostic significance, however, in ovarian carcinoma are unclear. In this study, EZH2 expression was examined by immunohistochemistry (IHC) in cohorts of normal and tumorous ovarian tissues. High expression of EZH2 was examined in none of the normal ovaries, in 3% of the cystadenomas, in 23% of the borderline tumors and in 50% of the ovarian carcinomas, respectively. In the ovarian carcinomas, high expression of EZH2 was positively correlated with an ascending histological grade and/or advanced stage of the disease (P < 0.05). Moreover, high expression of EZH2 in ovarian carcinoma was determined to be a strong and an independent predictor of short overall survival (P < 0.05). In ovarian carcinoma HO-8910 and UACC-326 cell lines, EZH2 knockdown by RNA interference led to a G(1) phase cell cycle arrest, reduced cell growth/proliferation and inhibited cell migration and/or invasion in vitro. In addition, EZH2 knockdown was found to reduce transforming growth factor-beta1 (TGF-beta1) expression and increase E-cadherin expression either in the transcript or in the protein levels. Furthermore, a significant positive correlation between overexpression of EZH2 and TGF-beta1 in ovarian carcinoma tissues was observed (P < 0.001). These findings suggest a potential important role of EZH2 in the control of cell migration and/or invasion via the regulation of TGF-beta1 expression, and the high expression of EZH2, as detected by IHC, is an independent molecular marker for shortened survival time of patients with ovarian carcinoma.

Abstract 4881: Polycomb protein EZH2 activates Wnt/β-catenin signaling to promote hepatocellular carcinoma development

Abstract Aberrant activation of the canonical Wnt pathway due to accumulation of β-catenin occurs in approximately 70% of hepatocellular carcinomas (HCCs) and contributes to their initiation, development and progression. Genetic mutations in the components of this pathway account for only a subset of HCCs with β-catenin accumulation, suggesting that an alternative mechanism for Wnt/β-catenin activation is prevailing. Enhancer of zeste homolog 2 (EZH2) is known to promote tumorigenesis by down-regulating tumor-suppressor genes; however, whether EZH2 regulates oncogenic pathways is unclear. Using chromatin immunoprecipitation microarray, we uncovered 12 Wnt/β-catenin signal antagonists whose promoters were concordantly occupied by EZH2 and repressive histone modifications in hepatocellular HCC cells. EZH2 over-expressed in 42% (75/179) of human HCCs and significantly associated with β-catenin accumulation. Concomitant inhibition of EZH2 and histone deacetylase transcriptionally activated Wnt/β-catenin signal antagonists, suppressed T-cell factor-dependent transcriptional activity and down-regulated β-catenin transcriptional targets in HCC cells. Conversely, ectopic EZH2 over-expression in nontumorigenic hepatocytes increased cellular proliferation in a β-catenin-dependent manner and promoted tumorigenicity. This study uncovers an EZH2-mediated epigenetic mechanism that leads to Wnt/β-catenin signaling dyregulation in human HCC and suggests that therapeutic interventions targeting EZH2 may confer a clinical benefit in β-catenin-driven malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4881.

Abstract 1111: Enhancer of Zeste homologue 2 (EZH2) regulates BRCA1 transiently nuclear export during the cell cycle, and induces tetraploidy of breast cells through AKT1

Abstract EZH2 is overexpressed in breast carcinomas with high propensity to metastasize. EZH2 is an oncogene essential to promote proliferation and capable to trigger neoplastic conversion in the breast. We have reported that EZH2 promotes proliferation of breast cancer through downregulation of BRCA1 protein, but the mechanism is unknown. BRCA1 shuttles between the cytoplasm and nucleus to maintain genomic stability. AKT1 up regulation disrupts BRCA1 function by altering its nuclear localization and inducing genomic instability. We hypothesize that EZH2 regulates nuclear-cytoplasmic shuttling of BRCA1 in breast cells through AKT1. Methods: We developed two cell culture models: an inducible EZH2 up regulation system in MCF10A benign mammary cells and a lentivirus-mediated shRNA interference targeting EZH2 model in CAL51 breast cancer cells (tetraploid, hormone receptor negative, and with intact p53 and BRCA1 genes). Our recently developed EZH2 transgenic mouse model was analyzed to determine the in vivo relevance of our in vitro data obtained by Western blots, immunofluorescence, proliferation, and FACS. Results: EZH2 shRNA knock-down in CAL51 cells increased BRCA1 nuclear localization in early S phase, decreased cell growth by delaying cell cycle progression, and tetraploidy. EZH2 overexpression in MCF10A cells decreased BRCA1 nuclear localization, increased proliferation and caused mitotic spindle defects and tetraploidy. EZH2 overexpression increased total and phoshporylated AKT1 at serine 473. Specific pharmacologic inhibition of AKT reverted the effect of EZH2 on BRCA1 nuclear localization, proliferation, mitotic defects and tetraploidy. We also found that EZH2 directly interacts with AKT1 protein. Mammary glands of EZH2 transgenic mice show up-regulation of AKT1 and a decreased in nuclear BRCA1 protein compared to wild type mice. Conclusion: EZH2 regulates the nuclear-cytoplasmic shuttling of BRCA1 protein and influences BRCA1 functions on proliferation, mitotic spindle control, and maintenance of genomic stability in benign breast cells and in breast cancer cells. The regulation of BRCA1 localization and function by EZH2 requires AKT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1111.

195 Correlation between hormonal receptors and HERr2 status of primary and metastatic breast cancer according to the metastatic organ involved

EZH2 (Enhancer of zeste homolog 2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2), which is involved in repressing gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation. EZH2 overexpression is implicated in tumorigenesis and has been a candidate oncogene in several tumor types. Recently, point mutations of EZH2 at Tyr641 and Ala677 were identified in diffuse large B cell lymphoma and follicular lymphoma, where they drive H3K27 hypertrimethylation and cancer progression. Here, we reported a novel, highly potent and selective small molecule inhibitor of EZH2, ZLD10A, which inhibited wild-type and mutant versions of EZH2 with nanomolar potency and had greater than 1000-fold selectivity against 10 other histone methyltransferases. Our results have shown that the compound suppressed global H3K27 methylation and cause the anti-proliferation effects in a concentration- and time-dependent manner in DLBCL cell lines. These results demonstrated that ZLD10A, as a novel EZH2 inhibitor, could be a potential promising agent for the treatment of EZH2 mutant lymphoma.

Tyrosine 641 of the EZH2 Oncogene Is Frequently Mutated in Follicular and Diffuse Large B-Cell Lymphomas of Germinal Center Origin.

Abstract 139 Background:Diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) constitute 70% of all non-Hodgkin lymphomas (NHL). Both malignancies derive from germinal center (GC) B-cells and are characterized by clinical and genomic heterogeneity. Chromosomal alterations that deregulate oncogenes as well as mutations in genes involved in cell proliferation and apoptosis have been described in DLBCL. This disease can also be divided into distinct molecular subtypes by gene expression profiling. Differences observed between the activated B-cell (ABC) and germinal center B cell (GCB) subtypes result in part from distinct genomic alterations (Lenz, PNAS 2008) . For example, recent targeted re-sequencing efforts have revealed mutations in various genes in the NFkB signaling pathway, which likely contribute mainly to the ABC subtype (Compagno, Nature 2009). Thus far, few GCB-specific mutations other than t(14;18) have been identified. Methods:Targeted re-sequencing studies are only able to reveal mutations in pre-selected candidate genes and so much of the genome has not yet been investigated in lymphomas. To obtain a more global view of the mutational landscape of NHL, we applied Illumina massively parallel second-generation sequencing to sequence the genomic DNA of a FL tumor sample. In parallel, we sequenced the transcriptomes (i.e. mRNA) of 31 DLBCL tumor samples using the same technology. Results:Genomic sequencing revealed a mutation altering tyrosine 641 (Y641) in the polycomb group oncogene Enhancer of Zeste Homolog 2 (EZH2), a gene responsible for trimethylating lysine 27 on histone H3 (H3K27). Mutations affecting the same codon were also observed in 4 DLBCL samples. These mutations were confirmed to be somatic in nature by Sanger sequencing exon 15 in tumor DNA and germline DNA derived from peripheral blood in these 5 patients. Sanger sequencing all coding exons in EZH2 from an additional 25 FL samples revealed that mutations were restricted to codon 641. These mutations could change the tyrosine residue to Asparagine, Serine, Phenylalanine or Histidine. The frequency of EZH2 Y641 mutations in GCB-derived lymphomas was determined by Sanger sequencing exon 15 in a total of 479 lymphoma samples. EZH2 (Y641) mutations occurred predominantly in lymphomas of the GCB type: 18/80 (22%) GCB-type de novo DLBCL, 6/52 (11.5)% of “transformed” DLBCL derived from FL and 16/225 (7.1%) of pre-treatment FL samples. No EZH2 mutations were found in 42 DLBCL of the ABC subype, 25 mantle cell lymphomas, 30 small lymphocytic lymphomas or 25 T cell lymphomas or 23 reactive tonsils. Introducing each of the four Y641 mutations into wild-type EZH2 resulted in 15-fold reduction in the ability to trimethylate lysine 27 on H3 peptides in-vitro. Discussion:Over-expression of EZH2 is thought to drive malignancy in a variety of solid tumors but mutations in this gene have never been described. The trimethylated H3K27 epigenetic mark is used to silence the transcription of genes involved in differentiation and hematopoiesis. In Drosophila, the phenotypic consequence of mutating the orthologous tyrosine residue in the E(z)(“Enhancer of Zeste”) gene indicates an apparent gain-of-function (Jones, Genetics 1990). Taken together, our mutation frequency and biochemical data are compatible with the notion that alteration of the ability of germinal center B-cells to trimethylate H3K27 may promote the development of FL and the GCB subtype of DLBCL. Disclosures:Zhu:BPS Biosciences: Employment. Kimbara:BPS Biosciences: Employment. Shashkin:BPS Biosciences: Employment. Charlot:BPS Biosciences: Employment. Tcherpakov:BPS Biosciences: Employment.

Targeted Overexpression of EZH2 in the Mammary Gland Disrupts Ductal Morphogenesis and Causes Epithelial Hyperplasia

The Polycomb group protein enhancer of zeste homolog 2 (EZH2), which has roles during development of numerous tissues, is a critical regulator of cell type identity. Overexpression of EZH2 has been detected in invasive breast carcinoma tissue samples and is observed in human breast tissue samples of morphologically normal lobules up to 12 years before the development of breast cancer. The function of EZH2 during preneoplastic progression in the mammary gland is unknown. To investigate the role of EZH2 in the mammary gland, we targeted the expression of EZH2 to mammary epithelial cells using the mouse mammary tumor virus long terminal repeat. EZH2 overexpression resulted in aberrant terminal end bud architecture. By the age of 4 months, 100% of female mouse mammary tumor virus-EZH2 virgin mice developed intraductal epithelial hyperplasia resembling the human counterpart accompanied by premature differentiation of ductal epithelial cells and up-regulation of the luminal marker GATA-3. In addition, remodeling of the mammary gland after parturition was impaired and EZH2 overexpression caused delayed involution. Mechanistically, we found that EZH2 physically interacts with beta-catenin, inducing beta-catenin nuclear accumulation in mammary epithelial cells and activating Wnt/beta-catenin signaling. The biological significance of these data to human hyperplasias is demonstrated by EZH2 up-regulation and colocalization with beta-catenin in human intraductal epithelial hyperplasia, the earliest histologically identifiable precursor of breast carcinoma.

6640 Enhancer of zeste homolog 2 expression is associated with tumour cell proliferation and metastasis in gastric cancer

EZH2 (Enhancer of zeste homolog 2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2), which is involved in repressing gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation. EZH2 overexpression is implicated in tumorigenesis and has been a candidate oncogene in several tumor types. Recently, point mutations of EZH2 at Tyr641 and Ala677 were identified in diffuse large B cell lymphoma and follicular lymphoma, where they drive H3K27 hypertrimethylation and cancer progression. Here, we reported a novel, highly potent and selective small molecule inhibitor of EZH2, ZLD10A, which inhibited wild-type and mutant versions of EZH2 with nanomolar potency and had greater than 1000-fold selectivity against 10 other histone methyltransferases. Our results have shown that the compound suppressed global H3K27 methylation and cause the anti-proliferation effects in a concentration- and time-dependent manner in DLBCL cell lines. These results demonstrated that ZLD10A, as a novel EZH2 inhibitor, could be a potential promising agent for the treatment of EZH2 mutant lymphoma.

Downregulation of EZH2 decreases growth of estrogen receptor-negative invasive breast carcinoma and requires BRCA1

Increased levels of EZH2, a critical regulator of cellular memory, are associated with negative estrogen receptor (ER) expression and disease progression in breast cancer. High levels of EZH2 signal the presence of metastasis and poor outcome in breast cancer patients. To test the hypothesis that deregulation of EZH2 contributes to ER negative breast cancer progression, EZH2 expression was inhibited in ER negative breast cancer cells MDA-MB-231 and CAL51 using a lentivirus system. EZH2 knockdown decreased proliferation and delayed the G2/M cell cycle transition, while not affecting apoptosis. In vivo, EZH2 down-regulation significantly decreased breast xenograft growth and improved survival. EZH2 knockdown up regulated BRCA1 protein. Of note, BRCA1 knockdown was sufficient to rescue the effects of EZH2 down-regulation in proliferation, G2/M arrest, and on the levels of hyperphosphorlated mitotic Cdc25C and Cyclin B1 proteins, crucial for entry into mitosis. Invasive ER negative breast carcinomas show significant overexpression of EZH2 and down-regulation of BRCA1 proteins. Taken together, we show that EZH2 plays a role in ER negative breast cancer progression in vivo and in vitro, and that BRCA1 is required for the proliferative effects of EZH2. Blockade of EZH2 may provide a prime target to prevent and/or halt ER negative breast cancer progression.

Genomic loss of microRNA-101 leads to overexpression of histone methyltransferase EZH2 in cancer.

Enhancer of zeste homolog 2 (EZH2) is a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes and regulates the survival and metastasis of cancer cells. EZH2 is overexpressed in aggressive solid tumors by mechanisms that remain unclear. Here we show that the expression and function of EZH2 in cancer cell lines are inhibited by microRNA-101 (miR-101). Analysis of human prostate tumors revealed that miR-101 expression decreases during cancer progression, paralleling an increase in EZH2 expression. One or both of the two genomic loci encoding miR-101 were somatically lost in 37.5% of clinically localized prostate cancer cells (6 of 16) and 66.7% of metastatic disease cells (22 of 33). We propose that the genomic loss of miR-101 in cancer leads to overexpression of EZH2 and concomitant dysregulation of epigenetic pathways, resulting in cancer progression.

Regulation of Pancreatic Tumor Cell Proliferation and Chemoresistance by the Histone Methyltransferase Enhancer of Zeste Homologue 2

Enhancer of zeste homologue 2 (EZH2), a histone methyltransferase, plays a key role in transcriptional repression through chromatin remodeling. Our objectives were to determine the expression pattern of EZH2 and to assess the anticancer effect of EZH2 depletion in pancreatic cancer cells. Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the expression pattern of EZH2 in normal pancreas and human pancreatic tumors. We used RNA interference, Western blotting, reverse transcription-PCR, and chromatin immunoprecipitation to study the effect of EZH2 depletion on pancreatic cancer cell proliferation and survival. We detected nuclear overexpression of EZH2 in pancreatic cancer cell lines and in 71 of 104 (68%) cases of human pancreatic adenocarcinomas. EZH2 nuclear accumulation was more frequent in poorly differentiated pancreatic adenocarcinomas (31 of 34 cases; P<0.001). We found that genetic depletion of EZH2 results in reexpression of p27(Kip1) and decreased pancreatic cancer cell proliferation. Moreover, we showed that EZH2 depletion sensitized pancreatic cancer cells to doxorubicin and gemcitabine, which leads to a significant induction of apoptosis, suggesting that the combination of EZH2 inhibitors and standard chemotherapy could be a superior potential treatment for pancreatic cancer. Our results show nuclear accumulation of EZH2 as a hallmark of poorly differentiated pancreatic adenocarcinoma; identify the tumor suppressor p27(Kip1) as a new target gene of EZH2; show that EZH2 nuclear overexpression contributes to pancreatic cancer cell proliferation; and suggest EZH2 as a potential therapeutic target for the treatment of pancreatic cancer.

High expression of EZH2 is associated with tumor proliferation and prognosis in human oral squamous cell carcinomas

Enhancer of zeste homolog 2 (EZH2) is a member of the polycomb group of genes and is important in cell cycle regulation. Overexpression of EZH2 protein has been associated with biological malignancy of prostate cancer and several other cancers. The aim of the present study was to evaluate the expression of EZH2 protein in human oral normal mucosa, dysplasia and oral squamous cell carcinoma (OSCC) with clinicopathological profiles. EZH2 expression was assessed by Western blotting and immunohistochemistry in 3 OSCC cell lines, 10 normal mucosae, 50 dysplasias and 102 OSCCs. The labeling indices (LIs) of EZH2, Ki-67, P53, and the apoptotic index (AI) were evaluated by immunohistochemistry and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) method. Western blot analysis detected EZH2 protein as a single band at 91kDa in the 3 OSCC cell lines, but it was almost absent in non-tumoral oral mucosae. The LI of EZH2 was highest in the OSCCs, followed by the dysplasias (p<0.05) and normal mucosae (p<0.05) with significant difference. The LI of EZH2 correlated with the clinical stage, tumor size, lymph node metastasis and LIs of Ki-67 and P53, but not with the AI in OSCCs, and inversely correlated with the histological differentiation of OSCCs. The survival rate calculated by the Kaplan-Meier method revealed that OSCC patients with higher EZH2 expression showed poorer prognosis than those with a lower EZH2 expression (p<0.01). These results suggest that overexpression of EZH2 is correlated with malignant potential and poor prognosis in OSCCs. EZH2 might serve as a novel biomarker for predicting prognosis in patients with OSCCs.

Enhancer of Zeste Homologue 2 (EZH2) Down-regulates RUNX3 by Increasing Histone H3 Methylation

Overexpression of enhancer of zeste homologue 2 (EZH2) occurs in various malignancies and is associated with a poor prognosis, especially because of increased cancer cell proliferation. In this study we found an inverse correlation between EZH2 and RUNX3 gene expression in five cancer cell lines, i.e. gastric, breast, prostate, colon, and pancreatic cancer cell lines. Chromatin immunoprecipitation assay showed an association between EZH2 bound to the RUNX3 gene promoter, and trimethylated histone H3 at lysine 27, and HDAC1 (histone deacetylase 1) bound to the RUNX3 gene promoter in cancer cells. RNA interference-mediated knockdown of EZH2 resulted in a decrease in H3K27 trimethylation and unbound HDAC1 and an increase in expression of the RUNX3 gene. Restoration of RUNX3 expression was not associated with any change in DNA methylation status in the RUNX3 promoter region. RUNX3 was repressed by histone deacetylation and hypermethylation of a CpG island in the promoter region and restored by trichostatin A or/and 5-aza-2′-deoxycytidine. Immunofluorescence staining confirmed restoration of expression of the RUNX3 protein after knockdown of EZH2 and its restoration resulted in decreased cell proliferation. In vivo, an inverse relationship between expression of the EZH2 and RUNX3 proteins was observed at the individual cell level in gastric cancer patients in the absence of DNA methylation in the RUNX3 promoter region. The results showed that RUNX3 is a target for repression by EZH2 and indicated an underlying mechanism of the functional role of EZH2 overexpression on cancer cell proliferation.

Enhancer of zeste homolog 2 downregulates E‐cadherin by mediating histone H3 methylation in gastric cancer cells

Overexpression of enhancer of zeste homolog 2 (EZH2), an epigenetic repressor, occurs in various malignancies and is associated with poor prognosis; however, the functional role of EZH2 overexpression in cancer versus non-cancerous tissue remains unclear. In this study, we found an inverse correlation between EZH2 and E-cadherin gene expression in gastric cancer cells. Knockdown of EZH2 by short interfering RNA in gastric cancer cells resulted in a restoration of the E-cadherin gene. We showed that the EZH2 complex existed with histone H3 and Lys27, which were methylated on E-cadherin promoter regions in gastric cancer cells. The restoration of E-cadherin was not involved in the change of the DNA methylation status in the E-cadherin promoter region. Immunofluorescence staining confirmed the expression of E-cadherin protein present in the cell membrane was restored after knockdown of EZH2, resulting in changing the cancer phenotype, such as its invasive capacity. In vivo, the relationship of inverse expression between EZH2 protein and E-cadherin protein was observed at the individual cellular level in gastric cancer tissue. This study provides into the mechanisms underlying the functional role of EZH2 overexpression in gastric cancer cells and a new modality of regulation of E-cadherin expression in silencing mechanisms of tumor suppressor genes. Our present study paves the way for exploring the blockade of EZH2 overexpression as a novel approach to treating cancer.

Proteomic analysis of EZH2 downstream target proteins in hepatocellular carcinoma

Enhancer of zeste homolog 2 (EZH2) is suggested to be a potential therapeutic target and a diagnostic marker for cancer. Our previous study also showed the critical role of EZH2 in hepatocellular carcinoma (HCC) tumorigenesis. The present study is aimed at revealing the comprehensive downstream pathways of EZH2 by functional proteomic profiling. Lentivirus mediated RNA interference (RNAi) was employed to knockdown EZH2 in HCC cells. The 2-DE was employed to compare the expression profile difference between parental and EZH2-knockdown HCC cells. In total, 28 spots were differentially expressed during EZH2 inhibition. Among all, 18 proteins were identified by PMF with MALDI-TOF MS. Western blotting further validated upregulation of 60S acidic ribosomal protein P0 (L10E), and downregulation of two proteins with EZH2 inhibition: stathmin1 and probable protein disulfide isomerase (PDI) ER-60 precursor (ERp57). Moreover, L10E was downregulated with overexpression of EZH2 in hepatocytes, and L10E reversed the effect of EZH2 on cell proliferation, suggesting it a downstream target of EZH2. The comprehensive and comparative analyses of proteins associated with EZH2 could further our understanding on the downstream signal cascade of EZH2 leading to tumorigenesis.

Integration of Estrogen and Wnt Signaling Circuits by the Polycomb Group Protein EZH2 in Breast Cancer Cells

Essential for embryonic development, the polycomb group protein enhancer of zeste homolog 2 (EZH2) is overexpressed in breast and prostate cancers and is implicated in the growth and aggression of the tumors. The tumorigenic mechanism underlying EZH2 overexpression is largely unknown. It is believed that EZH2 exerts its biological activity as a transcription repressor. However, we report here that EZH2 functions in gene transcriptional activation in breast cancer cells. We show that EZH2 transactivates genes that are commonly targeted by estrogen and Wnt signaling pathways. We demonstrated that EZH2 physically interacts directly with estrogen receptor alpha and beta-catenin, thus connecting the estrogen and Wnt signaling circuitries, functionally enhances gene transactivation by estrogen and Wnt pathways, and phenotypically promotes cell cycle progression. In addition, we identified the transactivation activity of EZH2 in its two N-terminal domains and demonstrated that these structures serve as platforms to connect transcription factors and the Mediator complex. Our experiments indicated that EZH2 is a dual function transcription regulator with a dynamic activity, and we provide a mechanism for EZH2 in tumorigenesis.