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ion and functions. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GC B cell (GCB)-type diffuse large B cell lymphomas (DLBCLs) are mostly addicted to EZH2 but not the more differentiated activated B cell (ABC)-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.

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e homolog 2 (EZH2). Sanger sequencing of EZH2 in a third classically-affected proband identified a third de novo mutation in this gene. These data show that mutations in EZH2 cause Weaver syndrome.

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t-weight:700;'>EZH2, a histone modifier protein that is induced by oncogenic mutant RAS, using pancreatic cancers of transgenic rats exogenously expressing human mutant RAS. Immunohistochemical observation of preneoplastic or cancerous lesions in the animal model suggested that upregulation of Ezh2 protein is an initiating event in pancreatic carcinogenesis. MEK-inhibition or Elk-1-knockdown downregulated EZH2, and MEK-inhibition or EZH2-knockdown restored expression of a tumor suppressor, RUNX3 in human and rat pancreatic cancer cells activated by the oncogenic RAS. Furthermore, Elk-1- or EZH2-knockdown inhibited growth of the cancer cells. These results strongly suggested that the oncogenic RAS upregulates EZH2 through MEK-ERK signaling, resulted in downregulation of tumor suppressors including RUNX3 in pancreatic carcinogenesis.

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is paucity reporting the expression of cancer genes at the RNA level in MCC tumors. In this study, we studied the RNA expression profiles of 26 MCC tumors, with a goal to identify prospective molecular targets that could improve the treatment strategies of MCC.
METHODS: RNA expression of 50 cancer-related genes in 26 MCC tumors was analyzed by targeted amplicon based next-generation sequencing using the Ion Torrent technology and the expression compared with that of normal, non-cancerous skin samples. Sequencing data were processed using Torrent Suite™ Software. Expression profiles of MCV-negative and MCV-positive tumors were compared. Fluorescence in situ hybridization was performed to study ALK rearrangements and immunohistochemistry to study ALK expression in tumor tissue.
RESULTS: ALK, CDKN2A, EZH2 and ERBB4 were overexpressed, and EGFR, ERBB2, PDGFRA and FGFR1 were underexpressed in MCC tumors compared to normal skin. In the MCV-negative tumors, MET, NOTCH1, FGFR3, and SMO were overexpressed and JAK3 and NPM1 were under-expressed compared to the MCV-positive tumors.
CONCLUSIONS: High expression of ALK, CDKN2A and EZH2 was recorded in MCC tumors. No ALK fusion was seen by FISH analysis. Overexpression of EZH2 suggests its potential as a drug target in MCC.

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ostic impact in MDS, we assessed EZH2 RNA expression in primary bone marrow CD34+ cells from 78 patients. We found that 47% of patients have reduced EZH2 expression compared to normal controls. Further analyses revealed that EZH2 is significantly underexpressed in patients bearing chromosome 7 or 7q deletions (7-alt) when compared to controls, diploid patients, and patients with other cytogenetic alterations (p<0.05). In survival analysis, we found a non-significant trend toward overall survival (OS) being better among patients with EZH2 underexpression (median OS 55 vs. 36 months; p=0.71). Importantly, this trend became significant when the analysis was restricted to the subset of cases without alterations in chromosome 7 (62 vs. 36 months; p=0.033). Furthermore, our previous work has identified a spectrum of innate immune genes in MDS CD34+ cells that are deregulated via abnormal promoter histone methylation. Because EZH2 is a key regulator of histone methylation, we assessed the relationship between deregulation of these genes and EZH2 underexpression. We observed that the mRNA levels of 11 immune genes were higher in the EZH2 underexpression group and that immune gene expression was significantly higher in patients with concomitant EZH2 underexpression and KDM6B (also known as JMJD3, an H3K27 demethylase) overexpression. Taken together, these data indicate that EZH2 underexpression may have unique impact on the molecular pathogenesis and prognosis in MDS and be an important marker for patients without chromosome 7 alteration.

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ask, and a reliable marker of malignancy would be extremely helpful. METHODS: EZH2 expression was investigated in 54 malignant and 40 benign salivary gland tumors of various histological types by standard immunohistochemistry. RESULTS: The majority (n = 52) of the malignant tumors stained positively, while all the investigated benign tumors were negative for EZH2. CONCLUSIONS: EZH2 expression in salivary gland tumors, similarly to the tumors of other organs is not characteristic for any tumor type, but is a solid marker of the malignant nature of the tumors.

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fied 48 individuals with EZH2 mutations. The mutations were primarily missense mutations occurring throughout the gene, with some clustering in the SET domain (12/48). Truncating mutations were uncommon (4/48) and only identified in the final exon, after the SET domain. Through analyses of clinical data and facial photographs of EZH2 mutation-positive individuals, we have shown that the facial features can be subtle and the clinical diagnosis of Weaver syndrome is thus challenging, especially in older individuals. However, tall stature is very common, reported in >90% of affected individuals. Intellectual disability is also common, present in ~80%, but is highly variable and frequently mild. Additional clinical features which may help in stratifying individuals to EZH2 mutation testing include camptodactyly, soft, doughy skin, umbilical hernia, and a low, hoarse cry. Considerable phenotypic overlap between Sotos and Weaver syndromes is also evident. The identification of an EZH2 mutation can therefore provide an objective means of confirming a subtle presentation of Weaver syndrome and/or distinguishing Weaver and Sotos syndromes. As mutation testing becomes increasingly accessible and larger numbers of EZH2 mutation-positive individuals are identified, knowledge of the clinical spectrum and prognostic implications of EZH2 mutations should improve.

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re detected in 35 patients (23%), ASXL1 in 33 patients (22%) and EZH2 in 8 (5%). ASXL1 mutations were associated with increased colony formation of BFU-E, CFU-E and CFU-GM (P-values, 0.049, 0.011 and 0.006). EZH2 mutations were common in patients with poor IPSS cytogenetics (P=0.001) and in patients in the IPSS intermediate-2/high-risk cohorts (P=0.06). In uni- but not multi-variate analyses, mutated TET2 was associated with longer survival (P=0.044) whereas EZH2 mutations were associated with an increased risk of transformation to acute myeloid leukemia (AML; P=0.039). These data suggest ASXL1 mutations might results in dominance of the mutant clone in Chinese with MDS whereas EZH2 mutations might predict an increased risk of transformation to AML.

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utations in SWI/SNF subunits result in shared dependencies is unknown. Here we show that EZH2, a catalytic subunit of the polycomb repressive complex 2 (PRC2), is essential in all tested cancer cell lines and xenografts harboring mutations of the SWI/SNF subunits ARID1A, PBRM1, and SMARCA4, which are several of the most frequently mutated SWI/SNF subunits in human cancer, but that co-occurrence of a Ras pathway mutation is correlated with abrogation of this dependence. Notably, we demonstrate that SWI/SNF-mutant cancer cells are primarily dependent on a non-catalytic role of EZH2 in the stabilization of the PRC2 complex, and that they are only partially dependent on EZH2 histone methyltransferase activity. These results not only reveal a shared dependency of cancers with genetic alterations in SWI/SNF subunits, but also suggest that EZH2 enzymatic inhibitors now in clinical development may not fully suppress the oncogenic activity of EZH2.

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ransferase subunit of the PRC2 complex, results in reduced H3K27me3 and profound transcriptional dysregulation, including that of a set of transcription factors directly involved in cerebellar neuronal cell-type specification and differentiation. Such transcriptional changes lead to increased GABAergic interneurons and decreased Purkinje cells. Transcriptional changes also inhibit the proliferation of granule precursor cells derived from the rhombic lip. The loss of both cell types ultimately results in cerebellar hypoplasia. These findings indicate Ezh2/PRC2 plays crucial roles in regulating neurogenesis from both cerebellar germinal zones.

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te (E(z)), are defined as PcG proteins and contain a highly conserved motif, called the SET (Su(var)3-9, Enhancer of Zeste, Trithorax) domain, which is required for histone methyltransferase activity. Increased expression of the transcriptional repressor EZH2 has been reported to be associated with poor prognosis in various malignancies including breast cancer and prostate cancer. Altered expression of EZH2 was also demonstrated in lung cancer, suggesting an involvement in the progression of lung cancer.
METHODS: All 41 polymorphisms in EZH2 were genotyped in 335 patients with lung cancer and 335 age- and gender-matched healthy controls. Finally, 26 polymorphisms were selected for the statistical analysis based on minor allele frequency (>0.05) and linkage disequilibrium.
RESULTS: Two polymorphisms of EZH2, rs6950683 and rs3757441, showed a statistically significant association with reduced risk of lung cancer (adjusted OR [aOR] = 0.71, p = 0.007; aOR = 0.73, p = 0.015, respectively). Two copies of a haplotype (Ht2) of EZH2 also showed a significant association with reduced lung cancer risk (aOR = 0.45, 95% confidence interval = 0.23-0.87).
CONCLUSION: This is the first study to show a significant association between polymorphisms of the PcG gene EZH2 and lung cancer risk. This study suggests a correlation between the genotype variants in EZH2 and reduced lung cancer risk and hence presents a possible marker for lung cancer susceptibility.

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on through miRNA-dependent mechanisms is largely unknown. In the current study, we identified a direct Nrf2 targeting miRNA, miR-214, and demonstrated a protective role of miR-214 in erythroid cells against oxidative stresses generated by radiation, excess iron and arsenic (As) exposure. miR-214 expression was transcriptionally repressed by Nrf2 through a canonical antioxidant response element (ARE) within its promoter region, and this repression is ROS-dependence. The suppression of miR-214 by Nrf2 could antagonize oxidative stress-induced cell death in erythroid cells by two ways. First, miR-214 directly targeted ATF4, a crucial transcriptional factor involved in anti-stress responses, down regulation of miR-214 releases the repression of ATF4 translation and leads to increased ATF4 protein content. Second, miR-214 was able to prevent cell death by targeting EZH2, the catalytic core component of PRC2 complex that is responsible for tri-methylation reaction at lysine 27 (K27) of histone 3 (H3) (H3K27me3), by which As-induced miR-214 reduction resulted in an increased global H3K27me3 level and a compromised overexpression of a pro-apoptotic gene Bim. These two pathways downstream of miR-214 synergistically cooperated to antagonize erythroid cell death upon oxidative stress. Our combined data revealed a protective role of miR-214 signaling in erythroid cells against oxidative stress, and also shed new light on Nrf2-mediated cytoprotective machinery.

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s by regulating H3K27me3 levels at the Bhlhb4 and Vsx1 loci, both of which are transcription factors essential for the maturation of BP cell subsets. In this study, we examined the role of Ezh2 in retinal development using retina-specific Ezh2 conditional knockout mice (Ezh2-CKO). The eyes of the Ezh2-CKO mice were microphthalemic, and the proliferation of retinal cells was diminished postnatally in Ezh2-CKO. Differentiation of all examined retinal subsets was observed with higher proportion of BP cell subsets, which was determined by immunostaining using specific retinal markers. The onsets of Muller glia and rod photoreceptor differentiation were accelerated. The expression of Bhlhb4 was increased in postnatal retinas, which was accompanied by the loss of H3K27me3 modifications at these genetic loci. Decreased expression of proneural genes in postnatal stage was observed. As reported previously in other Ezh2-KO tissues, increased expression of Arf/Ink4a was observed in the Ezh2-CKO retinas. The ectopic expression of Arf or Ink4a in the retina suppressed proliferation and increased apoptosis. In addition, earlier onset of Muller glia differentiation was observed in Ink4a-expressing cells. These results support an important role for histone H3K27me3 modification in regulating the proliferation and maturation of certain subsets of interneurons in the retina.

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targeted therapies for GC B cell dysfunctions, including lymphomas. The histone H3 lysine 27 methyltransferase enhancer of zeste homolog 2 (EZH2) is highly expressed in GC B cells and is often constitutively activated in GC-derived non-Hodgkin lymphomas (NHLs). The function of EZH2 in GC B cells remains largely unknown. Herein, we show that Ezh2 inactivation in mouse GC B cells caused profound impairment of GC responses, memory B cell formation, and humoral immunity. EZH2 protected GC B cells against activation-induced cytidine deaminase (AID) mutagenesis, facilitated cell cycle progression, and silenced plasma cell determinant and tumor suppressor B-lymphocyte-induced maturation protein 1 (BLIMP1). EZH2 inhibition in NHL cells induced BLIMP1, which impaired tumor growth. In conclusion, EZH2 sustains AID function and prevents terminal differentiation of GC B cells, which allows antibody diversification and affinity maturation. Dysregulation of the GC reaction by constitutively active EZH2 facilitates lymphomagenesis and identifies EZH2 as a possible therapeutic target in NHL and other GC-derived B cell diseases.

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pression levels of DAB2IP in AAA tissues and investigate whether mir-363-3p and EZH2 can be considered as potential mediators of its expression.
MATERIALS AND METHODS: 18 AAA samples and 15 non-aneurysmatic controls were collected. Relative mRNA expression levels of DAB2IP, EZH2 and mir-363-3p were measured using qPCR.
RESULTS: DAB2IP was significant up-regulated (~2.29 fold) in AAA tissues, while EZH2 and mir-363-3p were down-regulated (3.28 and 3.62-fold, respectively). A limited negative correlation was found between the DAB2IP and EZH2 expression and between DAB2IP and the mir-363-3p.
CONCLUSION: An increased expression of DAB2IP in AAA tissues was shown. We suggest 2 potential mediators of DAB2IP expression in abdominal aortic aneurysm, EZH2 and mir-363-3p.

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with myelodysplastic disorders including myelodysplastic syndromes (MDSs) and MDS/myeloproliferative neoplasm (MPN) overlap disorders (MDS/MPN). In our patient cohort, EZH2 mutations were also found and often coincided with tet methylcytosine dioxygenase 2 (TET2) mutations. Consistent with these findings, deletion of Ezh2 alone was enough to induce MDS/MPN-like diseases in mice. Furthermore, concurrent depletion of Ezh2 and Tet2 established more advanced myelodysplasia and markedly accelerated the development of myelodysplastic disorders including both MDS and MDS/MPN. Comprehensive genome-wide analyses in hematopoietic progenitor cells revealed that upon deletion of Ezh2, key developmental regulator genes were kept transcriptionally repressed, suggesting compensation by Ezh1, whereas a cohort of oncogenic direct and indirect polycomb targets became derepressed. Our findings provide the first evidence of the tumor suppressor function of EZH2 in myeloid malignancies and highlight the cooperative effect of concurrent gene mutations in the pathogenesis of myelodysplastic disorders.

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ontains EED and SUZ12 and is required for the silencing of HOX gene expression during embryonic development. Here we demonstrate that both EZH2 and EED are essential for the proliferation of both transformed and non-transformed human cells. In addition, the pRB-E2F pathway tightly regulates their expression and, consistent with this, we find that EZH2 is highly expressed in a large set of human tumors. These results raise the question whether EZH2 is a marker of proliferation or if it is actually contributing to tumor formation. Significantly, we propose that EZH2 is a bona fide oncogene, since we find that ectopic expression of EZH2 is capable of providing a proliferative advantage to primary cells and, in addition, its gene locus is specifically amplified in several primary tumors.

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METHODS: We analyzed the reciprocal expression regulation of miR-31 and SOX4 in esophageal squamous and adenocarcinoma cell lines by qRT-PCR and Western blotting using overexpression and shRNA knock-down approaches. Furthermore, methylation studies were used to assess epigenetic regulation of expression. Functionally, we determined the cellular consequences using migration and invasion assays, as well as proliferation assays. Immunoprecipitation and ChIP were used to identify complex formation of SOX4 and co-repressor components.
RESULTS: Here, we report that SOX4 promotes esophageal tumor cell proliferation and invasion by silencing miR-31 via activation and stabilization of a co-repressor complex with EZH2 and HDAC3. We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3'-UTR. Additionally, miR-31 regulates EZH2 and HDAC3 indirectly. SOX4, EZH2 and HDAC3 levels inversely correlate with miR-31 expression in ESCC cell lines. Ectopic expression of miR-31 in ESCC and EAC cell lines leads to down regulation of SOX4, EZH2 and HDAC3. Conversely, pharmacologic and genetic inhibition of SOX4 and EZH2 restore miR-31 expression. We show that SOX4, EZH2 and HDAC3 form a co-repressor complex that binds to the miR-31 promoter, repressing miR-31 through an epigenetic mark by H3K27me3 and by histone acetylation. Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues.
CONCLUSIONS: Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas.

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EZH2 mediates gene silencing through histone-H3 lysine-27 methylation. In colorectal cancer (CRC), EZH2 overexpression predicts shorter survival. Recently, four EZH2 single-nucleotide polymorphisms (SNPs) have been described. The present study was aimed at evaluating the correlation between EZH2 SNPs and outcome parameters in mCRC patients.
PATIENTS AND METHODS: DNA was extracted from blood samples of 110 mCRC patients treated with first-line 5-fluorouracil, folinic acid, irinotecan (FOLFIRI) and bevacizumab. Genotyping was carried out by real-time PCR. Genotype was used to predict objective response, progression-free survival (PFS) and overall survival (OS). EZH2 messenger RNA levels were evaluated on lymphocytes of a parallel cohort of 50 CRC patients.
RESULTS: One allelic variant (rs3757441 C/C versus C/T or T/T) was significantly associated with shorter PFS and OS (P < 0.01 and P < 0.05, respectively). At multivariate analysis, the same variant resulted an independent predictor of PFS and OS (P < 0.05). The C/C variant was associated with significantly higher EZH2 expression (P < 0.05).
CONCLUSION: An EZH2 SNP may be useful to predict clinical outcome in mCRC patients.

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are differentially expressed during hippocampal development. We show that depletion of Ezh2 leads to increased expression of PSD-95, a critical plasticity gene, and that reduced PSD-95 gene transcription is correlated with enrichment of Ezh2 at the PSD-95 gene promoter; however, the H3K27me3 epigenetic mark is not present at the PSD-95 gene promoter, likely due to the antagonizing effects of the H3S28P and H3K27Ac marks and the activity of the H3K27 demethylases JMJD3 and UTX. In contrast, increased PSD-95 gene transcription is accompanied by the presence of Ezh1 and elongation-engaged RNA Polymerase II complexes at the PSD-95 gene promoter, while knock-down of Ezh1 reduces PSD-95 transcription. These results indicate that Ezh1 and Ezh2 have antagonistic roles in regulating PSD-95 transcription.

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e responses during inflammatory bowel disease (IBD). Here we report that suppressing EZH2 activity ameliorates experimental intestinal inflammation and delayed the onset of colitis-associated cancer. In addition, we identified an increased number of functional MDSCs in the colons, which are essential for EZH2 inhibitor activity. Moreover, inhibition of EZH2 activity promotes the generation of MDSCs from hematopoietic progenitor cells in vitro, demonstrating a previously unappreciated role for EZH2 in the development of MDSCs. Together, these findings suggest the feasibility of EZH2 inhibitor clinical trials for the control of IBD. In addition, this study identifies MDSC-promoting effects of EZH2 inhibitors that may be undesirable in other therapeutic contexts and should be addressed in a clinical trial setting.

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span>)-mediated methylation of histone H3 lysine 9 (H3K9me3) and histone H3 lysine 27 (H3K27me3) in COX-2 silencing in IPF. Chromatin immunoprecipitation (ChIP) and re-ChIP assays demonstrated marked increases in H3K9me3, H3K27me3, and DNA methylation, together with their respective modifying enzymes G9a, EZH2, and DNA methyltransferases (Dnmts) and respective binding proteins heterochromatin protein 1 (HP1), polycomb protein complex 1 (PRC1) and methyl CpG binding protein 2 (MeCP2), at the COX-2 promoter in lung fibroblasts from patients with IPF (F-IPFs) compared with fibroblasts from nonfibrotic lungs. HP1, EZH2, and MeCP2 in turn were associated with additional repressive chromatin modifiers in F-IPFs. G9a and EZH2 inhibitors and small interfering RNAs and the Dnmt1 inhibitor markedly reduced H3K9me3 (49-79%), H3K27me3 (44-81%), and DNA methylation (61-97%) at the COX-2 promoter. These reductions were correlated with increased histone H3 and H4 acetylation, resulting in COX-2 mRNA and protein reexpression in F-IPFs. Our results support a central role for G9a- and EZH2-mediated histone hypermethylation and a model of bidirectional, mutually reinforcing, and interdependent crosstalk between histone hypermethylation and DNA methylation in COX-2 epigenetic silencing in IPF.-Coward, W. R., Feghali-Bostwick, C. A., Jenkins, G., Knox, A. J., Pang, L. A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis.

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nknown. In the present study, we performed western blot analysis, qRT-PCR, luciferase reporter assay and chromatin immunoprecipitation assay to investigate the relationship between miR-26a and the enhancer of zest homologue 2 (EZH2). CCK-8 assay and colony formation assay were carried out to explore the effect of miR-26a on HCC cells proliferation. We demonstrated that miR-26a was epigenetically repressed by EZH2-mediated H3K27 trimethylation within the miR-26a promoter. Moreover, we confirmed that EZH2 was also a direct target of miR-26a in HCC cells, thus, creating a double-negative feedback loop. Furthermore, miR-26a restoration increased the expressions of its host genes (CTDSPL and CTDSP2). Overexpression of EZH2 abrogated miR-26a induction of CTDSPL and CTDSP2. Restoring the balance of the double-negative feedback loop by miR-26a overpression or EZH2 silence significantly inhibited HCC cell growth. Overexpression of EZH2 rescued the growth inhibition effect of miR-26a. These findings suggest that an imbalanced double-negative feedback loop between EZH2 and miR-26a exists in HCC cells, which contributes to miR-26a deregulation and regulates tumor cells proliferation.

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come. In the present study, we immunohistochemically investigated the expression of EZH2 and BMI1 in 136 surgically resected ESCCs and adjacent epithelium. We also analyzed associations between aberrant expression of EZH2 and BMI1, and both clinicopathological findings and outcome. MYC, RB, and P53 expression was examined in selected cases and analyzed in relation to EZH2 expression. Mutations in the P53 gene were evaluated by direct sequencing. EZH2 mRNA expression was investigated in ESCC cell lines with and without P53 transcriptional activity. The results showed that EZH2 protein and BMI1 protein were upregulated in ESCC tissue in comparison with adjacent non-neoplastic epithelium. Aberrant EZH2 and BMI1 protein expression was observed in 19 (14.0%) and 23 (16.9%), respectively, of the 136 ESCCs. Aberrant EZH2 expression was significantly associated with larger size, greater depth of invasion, presence of distant metastasis, and shorter disease-free survival time. Aberrant BMI1 expression was inversely associated with lymph node metastasis and venous invasion, but not associated with survival of the patients. In addition, aberrant EZH2 expression was associated with P53 alteration in ESCC tissue. EZH2 mRNA expression in ESCC cell lines was reduced by activation of P53. In conclusion, P53 alteration may be involved in dysregulated EZH2 expression, and aberrant expression of EZH2 may play a role in ESCC progression.

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. We herein examined the significance of 14-3-3 σ and EZH2 in HCC (n = 167) by immunohistochemistry, RT-PCR and qRT-PCR. The correlation between 14-3-3σ and EZH2 expression and patients' clinicopathologic features were examined, as was the correlation between 14-3-3σ and EZH2 expression and the prognosis of HCC patients. We found that 14-3-3σ and EZH2 were highly expressed in HCC (71% and 90%), the expression of EZH2, but not 14-3-3σ, is associated with vascular invasion and tumor differentiation (p<0.01). The coexistence of 14-3-3σ and EZH2 overexpression is associated with a relatively unfavorable prognosis (p<0.01), suggesting that aberrant upregulation of 14-3-3σ and EZH2 expression serves as an inferior prognostic biomarker for HCC.

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s was performed to find E2F1-related genes associated with tumor progression and aggressiveness in the gene expression data from 165 primary patients with bladder cancer. The biologic activity of E2F1-related genes in tumor progression and aggressiveness was confirmed with experimental assays using bladder cancer cells and tumor xenograft assay. RESULTS: The expression of E2F1 was significantly associated with EZH2 and SUZ12. The overexpression of E2F1, EZH2, and SUZ12 enhanced cancer progression including cell colony formation, migration, and invasiveness. Knockdown of these genes reduced motility, blocked invasion, and decreased tumor size in vivo. E2F1 bound the proximal EZH2 and SUZ12 promoter to activate transcription, suggesting that E2F1 and its downstream effectors, EZH2 and SUZ12, could be important mediators for the cancer progression. In addition, we confirmed an association between these genes and aggressive characteristics. Interestingly, the treatment of anticancer drugs to the cells overexpressing E2F1, EZH2, and SUZ12 induced the expression of CD44, KLF4, OCT4, and ABCG2 known as cancer stem cell (CSC)-related genes. CONCLUSIONS: The link between E2F1, EZH2, and/or SUZ12 revealed that E2f1 directly regulates transcription of the EZH2 and SUZ12 genes. The signature of E2F1-EZH2-SUZ12 shows a predictive value for prognosis in bladder tumors and the E2F1-EZH2-SUZ12-driven transcriptional events may regulate the cancer aggressiveness and chemo-resistance, which may provide opportunity for development of new treatment modalities.

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ular function and regulatory networks of EZH2 in PCa. This study aims to determine whether or not the autoregulatory feedback loop of EZH2/miR-200c/E2F3 serves key functions in PCa development. Bioinformatics and integrative analytical approaches were employed to identify the relationships of EZH2 to specific cancer-related gene sets. Results indicated that the enrichment of gene sets about cell cycle progression was associated with EZH2 expression. The depletion of EZH2 in cell experiments inhibited PCa cell growth and blocked cell cycle accompanying the downregulation of E2F3 expression. Furthermore, miR-200c served as an important mediator between EZH2 and E2F3. Compared with scrambled control cells, sh-EZH2 cells showed lower H3K27me3 expression and higher miR-200c expression. Western blot and luciferase reporter assays showed that miR-200c inversely modulated E2F3 by directly targeting the binding site within 3'UTR. Moreover, decreased miR-200c expression largely abrogated the effect of sh-EZH2 on E2F3 expression and E2F3-induced cell cycle progression. EZH2 was positively regulated by E2F3 at the transcriptional level. Immunohistochemistry and in situ hybridization revealed a significant correlation among EZH2, miR-200c, and E2F3 expression in human PCa tissues. In conclusion, the autoregulatory feedback loop of EZH2/miR-200c/E2F3 served an important function in PCa development. Targeting this aberrantly activated feedback loop may provide a new therapeutic strategy against PCa.

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ator EZH2, a pharmacologic target for many solid tumors, we reasoned that EZH2 might regulate human RB tumorigenesis. Histologic and immunohistochemical analyses were performed using an EZH2 antibody in sections from 43 samples of primary, formalin-fixed, paraffin-embedded human RB tissue, cryopreserved mouse retina, and in whole cell lysates from human RB cell lines (Y79 and WERI-Rb1), primary human fetal retinal pigment epithelium (RPE) and fetal and adult retina, mouse retina and embryonic stem (ES) cells. Although enriched during fetal human retinal development, EZH2 protein was not present in the normal postnatal retina. However, EZH2 was detected in all 43 analyzed human RB specimens, indicating that EZH2 is a fetal protein expressed in postnatal human RB. EZH2 expression marked single RB cell invasion into the optic nerve, a site of invasion whose involvement may influence the decision for systemic chemotherapy. To assess the role of EZH2 in RB cell survival, human RB and primary RPE cells were treated with two EZH2 inhibitors (EZH2i), GSK126 and SAH-EZH2 (SAH). EZH2i impaired intracellular adenosine triphosphate (ATP) production, an indicator of cell viability, in a time and dose-dependent manner, but did not affect primary human fetal RPE. Thus, aberrant expression of a histone methyltransferase protein is a feature of human RB. This is the first time this mechanism has been implicated for an eye, adnexal, or orbital tumor. The specificity of EZH2i toward human RB cells, but not RPE, warrants further in vivo testing in animal models of RB, especially those EZH2i currently in clinical trials for solid tumors and lymphoma.

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EZH2 inhibitors with oral bioavailability in mice and rats. A lead compound EBI-2511 (compound 34) demonstrated excellent in vivo efficacy in Pfeiffer tumor Xenograft models in mouse and is under preclinical development for the treatment of cancers associated with EZH2 mutations.

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e generated conditional Ezh2 knock-in (Ezh2-KI) mice. Here we show that induced Ezh2 haematopoietic expression increases the number and proliferation of repopulating haematopoietic stem cells. Ezh2-KI mice develop myeloproliferative disorder, featuring excessive myeloid expansion in bone marrow and spleen, leukocytosis and splenomegaly. Competitive and serial transplantations demonstrate progressive myeloid commitment of Ezh2-KI haematopoietic stem cells. Transplanted self-renewing haematopoietic stem cells from Ezh2-KI mice induce myeloproliferative disorder, suggesting that the Ezh2 gain-of-function arises in the haematopoietic stem cell pool, and not at later stages of myelopoiesis. At the molecular level, Ezh2 regulates haematopoietic stem cell-specific genes such as Evi-1 and Ntrk3, aberrantly found in haematologic malignancies. These results demonstrate a stem cell-specific Ezh2 oncogenic role in myeloid disorders, and suggest possible therapeutic applications in Ezh2-related haematological malignancies.

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font-weight:700;'>EZH2 expression in OSCC is highly correlated with tumorigenesis, it has not been determined if specific EZH2 genetic variants are associated with OSCC risk. The aim of this study was to investigate the relationship between genetic polymorphisms of EZH2 and susceptibility to OSCC in Taiwan. Here, four SNPs of EZH2 (rs6950683, rs2302427, rs3757441, and rs41277434) were analyzed by a real-time PCR genotyping in 576 patients with oral cancer and 552 cancer-free controls. After adjusting for other co-variants, we found that carrying CC genotype at EZH2 rs6950683 and rs3757441 had a lower risk of developing OSCC than did wild-type carriers. The CCCA or CCTA haplotype among the four EZH2 sites was also associated with a reduced risk of OSCC. Furthermore, OSCC patients who carried CC genotype at EZH2 rs6950683 had a higher methylation than TC genotype. Our results suggest that the two SNPs of EZH2 (rs6950683 and rs3757441) might contribute to the prediction of OSCC susceptibility. Moreover, rs6950683 CC genotype exhibits hypermethylation in EZH2 promoter. This is the first study to provide insight into risk factors associated with EZH2 variants and epigenetic changes in carcinogenesis of OSCC in Taiwan.

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ment can reprogram estrogen-responsive gene expression in the myometrium, resulting in hyperresponsiveness to hormone in the adult uterus and promotion of hormone-dependent uterine leiomyoma. We show here that the environmental estrogens genistein, a soy phytoestrogen, and the plasticizer bisphenol A, differ in their pattern of developmental reprogramming and promotion of tumorigenesis (leiomyomas) in the uterus. Whereas both genistein and bisphenol A induce genomic estrogen receptor (ER) signaling in the developing uterus, only genistein induced phosphoinositide 3-kinase (PI3K)/AKT nongenomic ER signaling to the histone methyltransferase enhancer of zeste homolog 2 (EZH2). As a result, this pregenomic signaling phosphorylates and represses EZH2 and reduces levels of H3K27me3 repressive mark in chromatin. Furthermore, only genistein caused estrogen-responsive genes in the adult myometrium to become hyperresponsive to hormone; estrogen-responsive genes were repressed in bisphenol A-exposed uteri. Importantly, this pattern of EZH2 engagement to decrease versus increase H3K27 methylation correlated with the effect of these xenoestrogens on tumorigenesis. Developmental reprogramming by genistein promoted development of uterine leiomyomas, increasing tumor incidence and multiplicity, whereas bisphenol A did not. These data show that environmental estrogens have distinct nongenomic effects in the developing uterus that determines their ability to engage the epigenetic regulator EZH2, decrease levels of the repressive epigenetic histone H3K27 methyl mark in chromatin during developmental reprogramming, and promote uterine tumorigenesis.

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and DNMTs (DNMT1, DNMT3A and DNMT3B) in different grades of astrocytomas (n=93) was assessed by qRT-PCR and immunohistochemistry. GBM-U87MG cell line was used for functional studies. Strong immunopositivity (LI>/=25%) for EZH2, DNMT1 and DNMT3B was detected in 52%, 56% and 64% cases of GBMs respectively, which was significantly higher as compared to Grade II/III cases. Similarly, their median fold change of mRNA expression was also significantly higher in GBMs. There was also a significant positive correlation between DNMT1/DNMT3B and EZH2 mRNA and protein expression, which was in concordance with TCGA data set. Inhibition of DNMTs in cell line by Azacytidine resulted in down-regulation of EZH2, while knock-down of EZH2 by siRNA was not associated with any significant alteration of DNMTs, indicating that EZH2 expression in GBMs is possibly regulated by DNMTs, but not the reverse. Strong immunopositivity for EZH2, DNMT1 and DNMT3B were individually associated with significantly shorter survival and showed no correlation with IDH1 mutation status. In addition, the combination of these 3 markers represented an independent prognostic signature with cases having weak/negative expression of all 3 markers being associated with best prognosis. For the first time, the present study describes an epigenetic prognostic signature in GBMs based on immunohistochemical expression of EZH2, DNMT1 and 3B which can be used easily in routine neuropathology practice.

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argely undefined. We have studied the involvement of Ezh2 in a murine model of NRASQ61K-driven leukemia that recapitulates phenotypic and transcriptional features of ETP-ALL. Homozygous inactivation of Ezh2 cooperated with oncogenic NRASQ61K to accelerate leukemia onset. Inactivation of Ezh2 accentuated expression of genes highly expressed in human ETP-ALL and in normal murine early thymic progenitors. Moreover, we found that Ezh2 contributes to the silencing of stem-cell- and early-progenitor-cell-associated genes. Loss of Ezh2 also resulted in increased activation of STAT3 by tyrosine 705 phosphorylation. Our data mechanistically link Ezh2 inactivation to stem-cell-associated transcriptional programs and increased growth/survival signaling, features that convey an adverse prognosis in patients.

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mas. Hence, the present study was conducted to evaluate EZH2 expression in astrocytic and oligodendroglial tumors and correlate with CDKN2A/p16 status as well as MIB-1 labeling index (LI). Gliomas of all grades (n = 118) were studied using immunohistochemistry to assess EZH2, p16 and MIB-1 LI and fluorescence in situ hybrization to evaluate CDKN2A gene status. EZH2 expression and CDKN2A homozygous deletion (HD) were both significantly more frequent in high-grade gliomas (HGG). Further, strong EZH2 expression (LI >/= 25%) was significantly more common in HGGs without CDKN2A HD (48.7%; 19/39) as compared to cases with deletion (15.8%; 3/19). Loss of p16 expression was noted in 100% and 51.3% of CDKN2A deleted and non-deleted tumors, respectively. Notably, 80% (16/20) of the CDKN2A non-deleted HGGs with p16 loss had strong EZH2 expression, in contrast to only 15.8% (3/19) in the deleted group. Loss of p16 expression significantly correlated with MIB-1 LI, irrespective of EZH2 status. Thus, this study shows that EZH2 expression correlates with tumor grade in both astrocytic and oligodendroglial tumors and hence can be used as a diagnostic marker to differentiate between low and HGGs. Further, this is the first report demonstrating an inverse correlation of strong EZH2 expression with CDKN2A HD in HGGs. Loss of p16 protein expression is mostly attributable to CDKN2A HD and correlates significantly with MIB-1 LI. Notably, our study for the first time suggests a possible epigenetic mechanism of p16 loss in CDKN2A non-deleted HGGs mediated by strong EZH2 expression. A hypothetical model for control of proliferative activity in low versus HGGs is therefore proposed.

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ets for therapy in cancer including MM. Recently, we suggested the polycomb repressive complex 2 (PRC2) as a common denominator of gene silencing in MM and presented the PRC2 enzymatic subunit enhancer of zeste homolog 2 (EZH2) as a potential therapeutic target in MM. Here we further dissect the anti-myeloma mechanisms mediated by EZH2 inhibition and show that pharmacological inhibition of EZH2 reduces the expression of MM-associated oncogenes; IRF-4, XBP-1, PRDM1/BLIMP-1 and c-MYC. We show that EZH2 inhibition reactivates the expression of microRNAs with tumor suppressor functions predicted to target MM-associated oncogenes; primarily miR-125a-3p and miR-320c. ChIP analysis reveals that miR-125a-3p and miR-320c are targets of EZH2 and H3K27me3 in MM cell lines and primary cells. Our results further highlight that polycomb-mediated silencing in MM includes microRNAs with tumor suppressor activity. This novel role strengthens the oncogenic features of EZH2 and its potential as a therapeutic target in MM.

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We herein demonstrated that the hematopoietic-specific deletion of Ezh2 in mice induced heterogeneous hematopoietic malignancies. Myelodysplasia was detected in mice following the deletion of Ezh2, and resulted in the development of MDS and MDS/MPN. Thrombocytosis was induced by Ezh2 loss and sustained in some mice with myelodysplasia. Although less frequent, Ezh2 loss also induced T-cell acute lymphoblastic leukemia and the clonal expansion of B-1a B cells. Gene expression profiling showed that PRC2 target genes were derepressed upon the deletion of Ezh2 in hematopoietic stem and progenitor cells, but were largely repressed during the development of MDS and MDS/MPN. Chromatin immunoprecipitation-sequence analysis of trimethylation of histone H3 at lysine 27 (H3K27me3) revealed a compensatory function of Ezh1, another enzymatic component of PRC2, in this process. The deletion of Ezh1 alone did not cause dysplasia or any hematologic malignancies in mice, but abolished the repopulating capacity of hematopoietic stem cells when combined with Ezh2 loss. These results clearly demonstrated an essential role of Ezh1 in the pathogenesis of hematopoietic malignancies induced by Ezh2 insufficiency, and highlighted the differential functions of Ezh1 and Ezh2 in hematopoiesis.

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in breast cancer subtypes, we evaluated EZH2 expression in 226 invasive breast carcinomas with four distinct immunophenotypes and in association with clinicopathological features. RESULTS: Of these cases, 138 (61.1 %) were defined as EZH2-overexpressing with a multiplicative score > 3. EZH2 expression was inversely related to the status of ER and PR (Chi-square, p < 0.001), and it was significantly associated with HER2 positivity, high proliferative index, and high histologic grade (Chi-square, p < 0.05). ER-positive breast carcinoma with low proliferative index (Ki67 < 14 %) showed the lowest expression and triple-negative breast carcinoma showed the highest overexpression of EZH2, 18.5 % (10/54) versus 90.9 % (50/55) (Chi-square, p < 0.001). Intriguingly, 88 % (44/50) cases of grade 3 triple-negative breast carcinoma showed uniformly strong EZH2 expression with a multiplicative score of 9. The percentage of EZH2 overexpression in ER-positive breast carcinoma with a high proliferative index or HER2-positive cases were 61.2 and 74 %, respectively. Furthermore, EZH2 expression was significantly elevated in high-grade DCIS compared to benign lesions (90 % versus 0, p < 0.001). However, there is no association between EZH2 expression and the status of histone 3 lysine 27 trimethylation or other clinicopathologic features. CONCLUSION: In summary, triple-negative breast carcinoma showed the highest overexpression of EZH2. EZH2 overexpression is associated aggressive pathologic features including high nuclear grade, high proliferative index, and positivity of HER2 of breast carcinoma.

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he aim of our study was to investigate a potential link between EZH2 mRNA expression and the extent of APAF-1, DAPK-1 and IGFBP-3 methylation in urothelial transitional cell carcinoma (TCC) and to correlate the data with histopathological parameters and follow-up data. EZH2 mRNA expression was measured by real-time reverse transcription polymerase chain reaction, and the methylation analysis was performed using methylation-specific real-time polymerase chain reaction. Tissue specimens were obtained from 35 patients with TCC. EZH2 mRNA expression was detected in all tumor specimens investigated. The EZH2 expression levels correlated well with the differentiation grade of the tumor specimens (p = 0.03), and the APAF-1 methylation correlated with tumor stage (p = 0.0001) and grade (p = 0.004). Matched pair analysis demonstrated a statistically significant correlation between elevated EZH2 mRNA expression and higher methylation levels of APAF-1 in superficial (p = 0.024) and well- differentiated (p = 0.04) TCC. In patients with recurrent TCC, APAF-1 and IGFBP-3 methylation levels were significantly higher (p = 0.03 and p = 0.01, respectively), which was not observed when EZH2 mRNA expression or DAPK-1 methylation levels were related to the clinical outcome. In conclusion, our data show that EZH2 expression and APAF-1 methylation are related to tumor progression and invasiveness. Moreover, these data present first evidence that APAF-1 methylation is related to transcriptional activity of EZH2 expression in early-stage tumor disease of the bladder.

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H2) in oral tumorigenesis and its clinical implication as an OSCC risk predictor. Immunohistochemistry was used to measure EZH2 expression in OLs from 76 patients, including 37 who later developed OSCC and 39 who did not. EZH2 expression was associated with clinicopathologic parameters and clinical outcomes. To determine the biological role of EZH2 in OL, EZH2 level was reduced using EZH2 siRNAs in Leuk-1 cells, its impact on cell cycle, anchorage-dependent/independent growth, and invasion was assessed. We observed strong EZH2 expression in 34 (45%), moderate expression in 26 (34%), and weak/no expression in 16 (21%) of the OLs. The higher EZH2 levels were strongly associated with dysplasia (P < 0.001) and OSCC development (P < 0.0001). Multivariate analysis indicated that EZH2 expression was the only independent factor for OSCC development (P < 0.0001). At 5 years after diagnosis, 80% of patients whose OLs expressed strong EZH2 developed OSCC whereas only 24% patients with moderate and none with weak/no EZH2 expression did so (P < 0.0001). In Leuk-1 cells, EZH2 downregulation resulted in G(1) arrest; decreased invasion capability, decreased anchorage-independent growth; downregulation of cyclin D1 and upregulation of p15(INK4B). Our data suggest that EZH2 plays an important role in OL malignant transformation and may be a biomarker in predicting OSCC development in patients with OLs.

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nvestigate the impact of histone methylation repressive marks (H3 Lys27 trimethylation; H3K27(me3)) on early NK cell differentiation. We demonstrate that selective loss of the histone-lysine N-methyltransferase Ezh2 (enhancer of zeste homolog 2) or inhibition of its enzymatic activity with small molecules unexpectedly increased generation of the IL-15 receptor (IL-15R) CD122(+) NK precursors and mature NK progeny from both mouse and human hematopoietic stem and progenitor cells. Mechanistic studies revealed that enhanced NK cell expansion and cytotoxicity against tumor cells were associated with up-regulation of CD122 and the C-type lectin receptor NKG2D. Moreover, NKG2D deficiency diminished the positive effects of Ezh2 inhibitors on NK cell commitment. Identification of the contribution of Ezh2 to NK lineage specification and function reveals an epigenetic-based mechanism that regulates NK cell development and provides insight into the clinical application of Ezh2 inhibitors in NK-based cancer immunotherapies.

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oter regulatory elements for competency to support transcription. A critical histone mark is trimethylation of histone H3 at lysine residue 27 (H3K27me3), which is mediated by Ezh2, the catalytic subunit of the polycomb group complex PRC2 to repress transcription. Treatment of cells with the active vitamin D metabolite 1,25(OH)2 D3 , results in transcriptional activation of the CYP24A1 gene, which encodes a 24-hydroxylase enzyme, that is, essential for physiological control of vitamin D3 levels. We report that the Ezh2-mediated deposition of H3K27me3 at the CYP24A1 gene promoter is a requisite regulatory component during transcriptional silencing of this gene in osteoblastic cells in the absence of 1,25(OH)2 D3 . 1,25(OH)2 D3 dependent transcriptional activation of the CYP24A1 gene is accompanied by a rapid release of Ezh2 from the promoter, together with the binding of the H3K27me3-specific demethylase Utx/Kdm6a and thereby subsequent erasing of the H3K27me3 mark. Importantly, we find that these changes in H3K27me3 enrichment at the CYP24A1 gene promoter are highly dynamic, as this modification is rapidly reacquired following the withdrawal of 1,25(OH)2 D3 .

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ontaining protein 1 (HBP1) transcription factor is a novel activator of p21 that works as part of a complex mechanism during senescence and tumorigenesis. We found that HBP1 activates the p21 gene through enhancing p53 stability by inhibiting Mdm2-mediated ubiquitination of p53, a well known positive regulator of p21. HBP1 was also found to enhance p21 transcription by inhibiting Wnt/ß-catenin signaling. We identified histone methyltransferase EZH2, the catalytic subunit of polycomb repressive complex 2, as a target of Wnt/ß-catenin signaling. HBP1-mediated repression of EZH2 through Wnt/ß-catenin signaling decreased the level of trimethylation of histone H3 at lysine 27 of overall and specific histone on the p21 promoter, resulting in p21 transactivation. Although intricate, the reciprocal partnership of HBP1 and p21 has exceptional importance. HBP1-mediated elevation of p21 through the Mdm2/p53 and TCF4/EZH2 pathways contributes to both cellular senescence and tumor inhibition. Together, our results suggest that the HBP1 transcription factor orchestrates a complex regulation of key genes during cellular senescence and tumorigenesis with an impact on protein ubiquitination and overall histone methylation state.

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d to characterize the roles of H3K27 methyltransferase EZH2 and demethylase JMJD3 and identify their effective pathways and novel target genes in HSCs activation and liver fibrosis. Methods: In primary HSCs, we analyzed effects of pharmacological inhibitions and genetic manipulations of EZH2 and JMJD3 on HSCs activation. In HSCs cell lines, we evaluated effects of EZH2 inhibition by DZNep on proliferation, cell cycling, senescence and apoptosis. In CCl4 and BDL murine models of liver fibrosis, we assessed in vivo effects of DZNep administration and Ezh2 silencing. We profiled rat primary HSCs transcriptomes with RNA-seq, screened the pathways and genes associated with DZNep treatment, analyzed EZH2 and JMJD3 regulation towards target genes by ChIP-qPCR. Results: EZH2 inhibition by DZNep resulted in retarded growth, lowered cell viability, cell cycle arrest in S and G2 phases, strengthened senescence, and enhanced apoptosis of HSCs, decreased hepatic collagen deposition and rescued the elevated serum ALT and AST activities of diseased mice, and downregulated cellular and hepatic expressions of H3K27me3, EZH2, α-SMA and COL1A. Ezh2 silencing by RNA interference in vitro and in vivo showed similar effects. JMJD3 inhibition by GSK-J4 and overexpression of wild-type but not mutant Jmjd3 enhanced or repressed HSCs activation respectively. EZH2 inhibition by DZNep transcriptionally inactivated TGF-β1 pathway, cell cycle pathways and vast ECM components in primary HSCs. EZH2 inhibition decreased H3K27me3 recruitment at target genes encoding TGF-β1 pseudoreceptor BAMBI, anti-inflammatory cytokine IL10 and cell cycle regulators CDKN1A, GADD45A and GADD45B, and increased their expressions, while Jmjd3 overexpression manifested alike effects. Conclusions: EZH2 and JMJD3 antagonistically modulate HSCs activation. The therapeutic effects of DZNep as epigenetic drug in liver fibrosis are associated with the regulation of EZH2 towards direct target genes encoding TGF-β1 pseudoreceptor BAMBI, anti-inflammatory cytokine IL10 and cell cycle regulators CDKN1A, GADD45A and GADD45B, which are also regulated by JMJD3. Our present study provides new mechanistic insight into the epigenetic modulation of EZH2 and JMJD3 in HSCs biology and hepatic fibrogenesis.

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we investigate whether histone methylation regulation can impact renal allograft rejection in rat models.
RESULTS: Allogeneic rat renal transplantation model (Wistar to Lewis) was established, and the recipients were administrated with EZH2 inhibitor DZNep after transplantation. Renal allografts and peripheral blood were collected on day 5 after transplantation for histological examination and mechanism investigation. We found that inhibition of EZH2 by DZNep after transplantation significantly ameliorated acute rejection (AR), with decreased histological injury and reduced inflammatory infiltration in renal allografts. Attenuation of AR was due to the prohibited activation of alloreactive T cells, the subsequent impaired production of inflammatory cytokines, and also the elevated apoptosis of alloreactive T cells in both renal allografts and periphery. However, inhibition of EZH2 did not increase the regulatory T cells during the AR.
CONCLUSIONS: Disruption of EZH2 by DZNep suppressed the immune responses of alloreactive T cells and ameliorated AR of renal allografts. This suggests a therapeutic potential of targeting histone methyltransferases EZH2 in treating allograft rejection after solid organ transplantation.

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uencing data in ten cancer types were downloaded from GEO and TCGA, respectively. Integrated ChIP sequencing analysis and co-expressing analysis were conducted and both mRNA and long noncoding RNA (lncRNA) targets were detected. We detected a median of 4,672 mRNA targets and 4,024 lncRNA targets regulated by EZH2 in 19 cell lines. 20 mRNA targets and 27 lncRNA targets were found in all 19 cell lines. These mRNA targets were enriched in pathways in cancer, Hippo, Wnt, MAPK and PI3K-Akt pathways. Co-expression analysis confirmed numerous targets, mRNA genes (RRAS, TGFBR2, NUF2 and PRC1) and lncRNA genes (lncRNA LINC00261, DIO3OS, RP11-307C12.11 and RP11-98D18.9) were potential targets and were significantly correlated with EZH2. We predicted genome-wide potential targets and the role of EZH2 in regulating as a transcriptional suppressor or activator which could pave the way for mechanism studies and the targeted therapy of EZH2 in cancer.

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ne in breast cancer, but little is known about its expression pattern, biological function and underlying mechanism in NSCLC. Herein, we identified LINC00511 as an oncogenic lncRNA by driving tumorigenesis in NSCLC. We found LINC00511 was upregulated and associated with oncogenesis, tumor size, metastasis, and poor prognosis in NSCLC. Moreover, LINC00511 affected cell proliferation, invasiveness, metastasis, and apoptosis in multiple NSCLC cell lines. Mechanistically, LINC00511 bound histone methyltransferase enhancer of zeste homolog 2 ((EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2), a highly conserved protein complex that regulates gene expression by methylating lysine 27 on histone H3), and acted as a modular scaffold of EZH2/PRC2 complexes, coordinated their localization, and specified the histone modification pattern on the target genes, including p57, and consequently altered NSCLC cell biology. Thus, LINC00511 is mechanistically, functionally, and clinically oncogenic in NSCLC. Targeting LINC00511 and its pathway may be meaningful for treating patients with NSCLC.

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zh2, which catalyzes trimethylation of histone H3K27 (H3K27me3) and thereby represses gene expression. RESULTS: Herein, we show that Ezh2 plays a crucial role in the development and maintenance of the midbrain. Conditional deletion of Ezh2 in the developing midbrain resulted in decreased neural progenitor proliferation, which is associated with derepression of cell cycle inhibitors and negative regulation of Wnt/beta-catenin signaling. Of note, Ezh2 ablation also promoted ectopic expression of a forebrain transcriptional program involving derepression of the forebrain determinants Foxg1 and Pax6. This was accompanied by reduced expression of midbrain markers, including Pax3 and Pax7, as a consequence of decreased Wnt/beta-catenin signaling. CONCLUSION: Ezh2 is required for appropriate brain growth and maintenance of regional identity by H3K27me3-mediated gene repression and control of canonical Wnt signaling.

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opment of the embryonic central nervous system. Accumulating evidence suggests that these metabolic irregularities during pregnancy may alter neurogenesis pathways, thereby predisposing the developing fetus to neurodevelopmental disorders. One primary mechanism through which such disruptions may occur involves changes in histone modifications resulting from fluctuations in the expression of histone-modifying enzymes or the availability of their substrates. Herein, we have used a rat model of maternal obesity induced by a high-fat diet before and during gestation to investigate the cellular and molecular repercussions of maternal obesity on embryonic cortical neurogenesis. Maternal obesity impairs neurogenesis by reducing cell proliferation, increasing neuronal marker expression, and shifting development toward astrogliogenesis. Differentially expressed genes revealed disruptions in key developmental signaling pathways and reduced AKT phosphorylation, particularly at E14.5. These changes were associated with epigenetic alterations, mainly the differential expression and phosphorylation of EZH2 and subsequent changes in global histone modifications. Chromatin immunoprecipitation sequencing revealed reduced H3K27me3 at genes upregulated due to maternal obesity, which could have resulted from reduced expression and increased phosphorylation of EZH2 at Thr311. Interestingly, EZH2 also showed increased O-GlcNAcylation in high-fat diet embryos along with increased association with AMPK-Thr172 in accordance with previous studies showing that Ampk catalyzes EZH2-Thr311p. These results suggest that an epigenetic gene regulatory mechanism mediated by Ampk and Ezh2 interactions resulted in reduced H3K27me3 and derepression of key developmental genes, which could have led to cell fate changes observed in the developing embryo brain cortex due to maternal obesity.

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ter assay suggested EZH2 is a direct target gene of miR-1297. Furthermore, knockdown of EZH2 have the same effect with miR-1297 overeexpression in hepatocellular carcinoma cells. These findings provide evidence that miR-1297 plays a key role in inhibition of the hepatocellular carcinoma cells proliferation, and enhancing cell apoptosis through targeting EZH2, and strongly suggest that ex ogenous miR-1297 may have therapeutic value in treating hepatocellular carcinoma.

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them to inhibitors of androgen receptor signaling. Notably, miR-124 could restore the apoptotic response of cells resistant to enzalutamide, a drug approved for the treatment of castration-resistant prostate cancer. We used xenograft models to examine the effects of miR-124 in vivo when complexed with polyethylenimine-derived nanoparticles. Intravenous delivery of miR-124 was sufficient to inhibit tumor growth and to increase tumor cell apoptosis in combination with enzalutamide. Mechanistic investigations revealed that miR-124 directly downregulated AR splice variants AR-V4 and V7 along with EZH2 and Src, oncogenic targets that have been reported to contribute to prostate cancer progression and treatment resistance. Taken together, our results offer a preclinical rationale to evaluate miR-124 for cancer treatment.

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tyle='font-weight:700;'>EZH2 in adhesion formation.
MATERIALS AND METHODS: Control lacZ, nonmodified tPA or modified tPA vectors were instilled in the peritoneal cavity after injury in de novo adhesions or after lysis of adhesions in recurrent adhesions. Adhesion severity was scored and adhesions and liver tissues were examined for adenovirus E4 gene and tPA mRNA expression. Levels of tPA, plasminogen activator inhibitor-1 (PAI-1), transforming growth factor-β1 (TGF-β1), and EZH2 expression were measured.
RESULTS: E4 transcripts were detected in adhesions of nonmodified and modified and in livers of nonmodified but not in livers of modified de novo adhesions. Both nonmodified (p = 0.021) and modified vectors (p = 0.036) reduced the severity of de novo adhesions compared to lacZ vector. Levels of tPA in nonmodified (p = 0.021) and modified adhesions (p = 0.001) were elevated while PAI-1 (p = 0.013 and p = 0.001, respectively) and TGF-β1 levels (p = 0.002 and p = 0.016, respectively) were reduced compared with lacZ group. All vectors were not expressed in recurrent adhesions and severity score were not different among groups. EZH2 levels were elevated in de novo nontreated (p = 0.001) and was further increased in recurrent (p = 0.001) nontreated adhesions compared with noninjured peritoneum.
CONCLUSION: Modified adenovirus successfully targeted de novo adhesions but not liver tissues and reduced the severity of de novo adhesions. EZH2 is involved in the development and progression of peritoneal adhesions.

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es from formalin-fixed and paraffin embedded (FFPE) tissues from a total of 37 patients with mature B-cell non-Hodgkin lymphomas were included in the study. Molecular genetic analysis was performed by direct sequencing of the DNA samples.
RESULTS: We analyzed formaldehyde fixed-paraffin embedded (FFPE) tumor tissue samples from 17 female and 20 male patients with a median age of 63.7 years at the time of diagnosis. None of the patients had previously reported hot spot mutations in EZH2 and CD79B, but previously unreported single nucleotide variations of CD79B were present in nine patients. rs779833118 was the most frequent variation (7/37 patients, 18.9%). A non-synonymous variation rs757407417, which could have a potentially damaging outcome, was detected in two patients.
CONCLUSIONS: None of the patients had well-known hot spot mutations in EZH2 and CD79B. However, we detected novel CD79B variations in mature B-cell non-Hodgkin's lymphoma patients.

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essor of zeste (SUZ) 12, and methyltransferase enhancer of zeste (EZH2)-containing complex, PRC2, which is abundant in highly proliferative cells (including cancer cells), establishes a repressive methylation mark on histone 3 (H3K27me3). From the perspective of molecular cancer pathogenesis, this effect, when directed towards a promoter of tumor suppressor genes, represents pro-tumorigenic effect. This mode of action was shown in several cancer models. However, EZH2 function extends beyond this scenario. The highly specific cellular background, related to the origin of cell and numerous external stimuli during a given time-window, may be the trigger for EZH2 interaction with other proteins, not necessarily histones. This is particularly relevant for cancer. This review provides a critical overview of the evolutional importance of PRC and discusses several important aspects of EZH2 functioning within PRC. The review also deals with mutational studies on EZH2. Due to the existence of several protein (and messenger RNA (mRNA)) isoforms, these mutations were stratified, using the protein sequence which is considered canonical. This approach showed that there is an urgent need for the uniformed positioning of currently known EZH2 mutations (somatic-in tumors, as well as germline mutations in the Weaver's syndrome). Finally, we discuss EZH2 function with respect to amount of trimethylated H3K27, in a specific cellular milieu, through presenting the most recent data related to EZH2-H3K27m3 relationship in cancer. All these points are significant in considering EZH2 as a therapeutic target.

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ysis. Fifty-one studies with a total of 9444 patients were included. The prevalence of high EZH2 expression was 0.54 (95% CI: 0.47-0.61). High EZH2 expression was significantly associated with poorer prognosis [overall survival: HR 1.54 (95% CI: 1.30-1.78), P < 0.000; disease free survival: HR 1.35 (95% CI: 1.00-1.71), P < 0.000]. In breast cancer, high EZH2 expression correlated with histological types [OR: 1.53 (95CI: 1.13-2.06); P < 0.006], histological grade [OR: 1.62 (95CI: 1.35-1.95); P < 0.000], estrogen receptor (ER) negativity [OR: 2.05 (95CI: 1.67-2.52); P < 0.000], progesterone receptor (PgR) negativity [OR: 1.42 (95CI: 1.03-1.96); P = 0.034], HER-2 positivity [OR: 1.35 (95CI: 1.08-1.69); P = 0.009], and high p53 expression [OR: 1.66 (95CI: 1.07-2.59); P = 0.024]. These results suggest that high EZH2 expression may be a promising prognostic factor to different cancers. High EZH2 expression tends to correlate with pathological types, histological grade, ER negativity, PgR negativity, HER-2 positivity and p53 high expression in breast cancer.

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t genes affected by EZH2 are poorly understood. In view of this we have used an integrated systems biology approach to analyze 471 cases of skin cutaneous melanoma (SKCM) in The Cancer Genome Atlas (TCGA) for mutations and amplifications of EZH2. Identified changes in target genes were validated by interrogation of microarray data from melanoma cells treated with the EZH2 inhibitor GSK126. We found that EZH2 activation by mutations, gene amplification and increased transcription occurred in about 20% of the cohort. These alterations were associated with significant hypermethylation of DNA and significant downregulation of 11% of transcripts in patient RNASeq data. GSK126 treatment of melanoma lines containing EZH2 activation reversed such transcriptional repression in 98 candidate target genes. Gene enrichment analysis revealed genes associated with tumor suppression, cell differentiation, cell cycle inhibition and repression of metastases as well as antigen processing and presentation pathways. The identified changes in EZH2 were associated with an adverse prognosis in the TCGA dataset. These results suggest that inhibiting of EZH2 is a promising therapeutic avenue for a substantial fraction of melanoma patients.

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gression by inactivating tumor suppressor genes. In this study we have targeted EZH2 with a specific inhibitor (GSK126) or depleted EZH2 protein by stable shRNA knockdown. We show that inhibition of EZH2 has potent effects on the growth of both wild-type and EZH2 mutant human melanoma in vitro particularly in cell lines harboring the EZH2Y646 activating mutation. This was associated with cell cycle arrest, reduced proliferative capacity in both 2D and 3D culture systems, and induction of apoptosis. The latter was caspase independent and mediated by the release of apoptosis inducing factor (AIFM1) from mitochondria. Gene expression arrays showed that several well characterized tumor suppressor genes were reactivated by EZH2 inhibition. This included activating transcription factor 3 (ATF3) that was validated as an EZH2 target gene by ChIP-qPCR. These results emphasize a critical role for EZH2 in the proliferation and viability of melanoma and highlight the potential for targeted therapy against EZH2 in treatment of patients with melanoma.

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ved at the epigenetic level remains elusive. We hypothesized that Polycomb methyltransferase EZH2 is involved in the effects of FSS in human endothelial cells. We showed that FSS decreases the expression of the Polycomb methyltransferase EZH2. Despite simultaneous activation of MAPK7, MAPK7 pathway does not directly influence the transcription of EZH2. Interestingly though, the knockdown of EZH2 activates the protective MAPK7 signaling in endothelial cells, even in the absence of FSS. To understand the influence of the FSS-decreased expression of EZH2 on endothelial transcriptome, we performed RNA-seq and differential gene expression analysis. We identified candidate groups of genes dependent on both EZH2 and FSS. Among those, Gene Ontology overrepresentation analysis revealed highly significant enrichment of the cell cycle-related genes, suggesting changes in proliferation. Indeed, the depletion of EZH2 strongly inhibited endothelial proliferation, indicating cell cycle arrest. The concomitant decrease in CCNA expression suggests the transition of endothelial cells into a quiescent phenotype. Further bioinformatical analysis suggested TXNIP as a possible mediator between EZH2 and cell cycle-related gene network. Our data show that EZH2 is a FSS-responsive gene. Decreased EZH2 levels enhance the activation of the atheroprotective MAPK7 signaling. Decrease in EZH2 under FSS mediates the decrease in the expression of the network of cell cycle-related genes, which allows the cells to enter quiescence. EZH2 is therefore important for the protective effects of FSS in endothelium.

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and tumorigenic in some mouse mammary cancer models, and these tumor-initiating cells have been isolated from human breast cancers. MSC expansion is associated with aggressive biological behavior in human breast cancer. The LP population is tumorigenic in some mouse mammary cancer models, and is the progenitor population of basal breast cancer in humans. The enhancer of zeste homolog 2 (EZH2) is a methyltransferase which catalyzes lysine 27 methylation in histone H3 resulting in suppression of target gene expression. The histone demethylase JMJD3 opposes the activity of EZH2 by demethylating histone H3 lysine 27. EZH2 is a member of the polycomb group of proteins which regulates cell type identity. EZH2 expression was found to be increased in histologically normal human breast tissue among women with high breast cancer risk, and was elevated in ductal hyperplasia and ductal carcinoma in situ. EZH2 overexpression is associated with poorly differentiated and aggressive breast cancer in humans. However, the mechanisms by which EZH2 results in increased breast cancer risk and aggressive tumors are not completely characterized. Using in vivo transplantation of mammary cancer stem cells transduced with EZH2 or JMJD3 shRNAs, we demonstrated that EZH2 promotes mammary stem and LP cell expansion, metastasis and inhibits ER-positive cellular differentiation.

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tions, rearrangements, translocations, as well as mutations, have been found in UFs. These genetic anomalies suggest that low DNA damage repair capacity may be involved in UF formation. The objective of this study was to determine whether expression levels of DNA damage repair-related genes were altered, and how they were regulated in the pathogenesis of UFs. Expression levels of DNA repair-related genes RAD51 and BRCA1 were deregulated in fibroid tissues as compared to adjacent myometrial tissues. Expression levels of chromatin protein enhancer of zeste homolog 2 (EZH2) were higher in a subset of fibroids as compared to adjacent myometrial tissues by both immunohistochemistry and Western blot analysis. Treatment with an inhibitor of EZH2 markedly increased expression levels of RAD51 and BRCA1 in fibroid cells and inhibited cell proliferation paired with cell cycle arrest. Restoring the expression of RAD51 and BRCA1 by treatment with EZH2 inhibitor was dependent on reducing the enrichment of trimethylation of histone 3 lysine 27 epigenetic mark in their promoter regions. This study reveals the important role of EZH2-regulated DNA damage-repair genes via histone methylation in fibroid biology, and may provide novel therapeutic targets for the medical treatment of women with symptomatic UFs.

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ssociated with BRAF mutation and poor prognosis in colorectal cancer. EZH2 is functionally considered to suppress miR-31 expression in human cancers; however, no study has reported its relationship with colon cancer. We therefore evaluated EZH2 expression using immunohistochemistry and assessed miR-31 and epigenetic alterations using 301 colorectal carcinomas and 207 premalignant lesions. Functional analysis was performed to identify the association between EZH2 and miR-31 using cancer cell lines. In the current study, negative, weak, moderate, and strong EZH2 expressions were observed in 15%, 19%, 25%, and 41% of colorectal cancers, respectively. EZH2 was inversely associated with miR-31 (P < 0.0001), independent of clinicopathological and molecular features. In a multivariate stage-stratified analysis, high EZH2 expression was related to favorable prognosis (P = 0.0022). Regarding premalignant lesions, negative EZH2 expression was frequently detected in sessile serrated adenomas/polyps (SSA/Ps) (76%; P < 0.0001) compared with hyperplastic polyps, traditional serrated adenomas, and non-serrated adenomas (25-36%). Functional analysis demonstrated that the knockdown of EZH2 increased miR-31 expression. In conclusion, an inverse association was identified between EZH2 and miR-31 in colorectal cancers. Our data also showed that upregulation of EZH2 expression may be rare in SSA/Ps. These results suggest that EZH2 suppresses miR-31 in colorectal cancer and may correlate with differentiation and evolution of serrated pathway.

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tumor progression. However, the expression pattern and biological function of lncRNAs in non-small-cell lung cancer (NSCLC) remain largely unknown. In this study, we performed comprehensive analysis of lncRNA expression in human NSCLC samples by using microarray data from Gene Expression Omnibus. After validation in a cohort of 80 pairs of NSCLC tissues, we identified a differentially expressed novel oncogenic lncRNA termed as AGAP2-AS1. The AGAP2-AS1 expression level was significantly upregulated in NSCLC tissues and negatively correlated with poor prognostic outcomes in patients. In vitro loss- and gain-of-function assays revealed that AGAP2-AS1 knockdown inhibited cell proliferation, migration and invasion, and induced cell apoptosis. In vivo assays also confirmed the ability of AGAP2-AS1 to promote tumor growth. Furthermore, mechanistic investigation showed that AGAP2-AS1 could bind with enhancer of zeste homolog 2 and lysine (K)-specific demethylase 1A, and recruit them to KLF2 and LATS2 promoter regions to repress their transcription. Taken together, our findings indicate that AGAP2-AS1 may act as an oncogene by repressing tumor-suppressor LATS2 and KLF2 transcription. By clarifying the AGAP2-AS1 mechanisms underlying NSCLC development and progression, these findings might promote the development of novel therapeutic strategies for this disease.

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EZH2) to be highly overexpressed in lung and other cancers, and show that EZH2 is integral to proliferation in cancer cells. Quantitative real-time PCR analysis revealed higher expression of EZH2 in clinical bladder cancer tissues than in corresponding non-neoplastic tissues (P < 0.0001), and we confirmed that a wide range of cancers also overexpress EZH2, using cDNA microarray analysis. Immunohistochemical analysis showed positive staining for EZH2 in 14 of 29 cases of bladder cancer, 135 of 292 cases of non-small-cell lung cancer (NSCLC), and 214 of 245 cases of colorectal cancer, whereas no significant staining was observed in various normal tissues. We found elevated expression of EZH2 to be associated with poor prognosis for patients with NSCLC (P = 0.0239). In lung and bladder cancer cells overexpressing EZH2, suppression of EZH2 using specific siRNAs inhibited incorporation of BrdU and resulted in significant suppression of cell growth, even though no significant effect was observed in the normal cell strain CCD-18Co, which has undetectable EZH2. Because EZH2 expression was scarcely detectable in all normal tissues we examined, EZH2 shows promise as a tumor-specific therapeutic target. Furthermore, as elevated levels of EZH2 are associated with poor prognosis of patients with NSCLC, its overexpression in resected specimens could prove a useful molecular marker, indicating the necessity for a more extensive follow-up in some lung cancer patients after surgical treatment.