Enhancer Of Zeste Homolog 2 mRNA Research Articles

Bidirectional Effects of EZH2 Inhibitor in Endometriosis

Endometriosis is a complex disorder that afflicts many women with chronic pelvic pain and infertility. It is believed that polycomb group of proteins, such as enhancer of zeste homolog 2 (EZH2), which plays a major role in cancer, also might contribute to the etiology of endometriosis. We and others have shown upregulation of EZH2 in human endometriotic tissues. However, the factors responsible for this upregulation is unknown. This study hypothesizes that the components of peritoneal fluid (PF) plays a dynamic role in epigenetic pathways in endometriosis. To test this hypothesis we measured EZH2 expression and activity (activation of H3K27me3) in endometrial cells. Ishikawa (human endometrial) cells were treated with 1% PF from IRB‐approved and consented women with and without endometriosis (n=6–8/group age and cycle matched). A subset of cells was also treated with various concentrations of GSK126 (5 nM to 10 μM) either before or after PF treatment. Real‐time PCR was used to analyze EZH2 expression in these treated cells. Our results showed bidirectional effects of GSK126 on EZH2 and H3k27me3 expression, depending on the order of the drug treatment (before or after 24 hours of PF treatment) and the source of the PF (control vs endo). When cells were treated with endo PF, there was a 2–3 fold induction in EZH2 expression when cells were treated with endo PF compared to the media control. Surprisingly, there was a dose dependent increase in EZH2 mRNA expression with GSK126 pre‐treatment followed by the PF treatments. Almost a 9‐fold increase in EZH2 expression was observed when cells were pre‐treated with 2.5 μM GSK126 followed by control PF (*p<0.05), whereas pretreatment of GSK followed by endo PF had no such effects on EZH2 expression. However, when cells were pre‐treated with PF followed by the drug the EZH2 expression levels went down. Western blot of EZH2 and H3K27me3 showed similar findings. When MTT assay was performed to examine cell viability under the above experimental conditions, no significant changes in viability was observed at the 48 or 96 hour time points. These results suggest for an intermediate pathway modulated by the PF components that might be at play. The current studies are investigating the possible role for ARID1A mutation in the bidirectional effect of the EZH2 inhibitors in endometriosis.Support or Funding InformationPhRMA Foundation Pre Doctoral Fellowship in Pharmacology/ToxicologyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of NUDT21 in microRNA-binging sites of EZH2 gene increases the of risk preeclampsia.

ObjectivesPreeclampsia (PE) is a major cause of mortality and morbidity among pregnant mothers and their fetuses worldwide. Recent studies have shown that several microRNAs (miRNAs) play crucial role in pathogenesis of PE patients; however, the mechanisms responsible for differences in miRNA function in PE largely remain to be determined.Materials and MethodsWe studied that NUDT21 expression was markedly increased, whereas EZH2 was decreased in placental samples from patients with PE. We identified NUDT21 as an interaction partner of enhancer of zeste homologue 2 (EZH2). NUDT21 co‐localized with EZH2 in the human trophoblast cell line, HTR‐8/SVneo and NUDT21 was shown to bind to EZH2 in RNA immunoprecipitation assays. NUDT21 has previously been reported to be involved in alternative polyadenylation; thus, the interaction between NUDT21 and EZH2 may play an important role in the crosstalk between alternative polyadenylation (APA) and miRNA‐mediated gene silencing in PE.ResultsIn the human trophoblast cell line HTR‐8/SVneo, loss‐of‐function assays indicated that knockdown of NUDT21 suppressed cell proliferation, migration and tube formation. Furthermore, functional studies showed that NUDT21 elongated the 3'‐UTR of mRNAs thereby exposing more miRNA binding sites (including miR138 and miR363), which enhanced the efficiency of miRNA‐mediated gene silencing and promoted EZH2 binding.ConclusionsThis is the first report about the relationship of NUDT21 and EZH2. The data indicate that the aberrant expression of NUDT21 contributes to PE by targeting 3'‐UTR of EZH2 mRNA. These findings may provide novel targets for future investigations into therapeutic strategies for PE.

Aberrant Epigenetic Regulation in Head and Neck Cancer Due to Distinct EZH2 Overexpression and DNA Hypermethylation.

Enhancer of Zeste homologue 2 (EZH2) overexpression is associated with tumor proliferation, metastasis, and poor prognosis. Targeting and inhibition of EZH2 is a potentially effective therapeutic strategy for head and neck squamous cell carcinoma (HNSCC). We analyzed EZH2 mRNA expression in a well-characterized dataset of 230 (110 original and 120 validation cohorts) human head and neck cancer samples. This study aimed to investigate the effects of inhibiting EZH2, either via RNA interference or via pharmacotherapy, on HNSCC growth. EZH2 upregulation was significantly correlated with recurrence (p < 0.001) and the methylation index of tumor suppressor genes (p < 0.05). DNMT3A was significantly upregulated upon EZH2 upregulation (p = 0.043). Univariate analysis revealed that EZH2 upregulation was associated with poor disease-free survival (log-rank test, p < 0.001). In multivariate analysis, EZH2 upregulation was evaluated as a significant independent prognostic factor of disease-free survival (hazard ratio: 2.085, 95% confidence interval: 1.390–3.127; p < 0.001). Cells treated with RNA interference and DZNep, an EZH2 inhibitor, showed the most dramatic changes in expression, accompanied with a reduction in the growth and survival of FaDu cells. These findings suggest that EZH2 upregulation is correlated with tumor aggressiveness and adverse patient outcomes in HNSCC. Evaluation of EZH2 expression might help predict the prognosis of HNSCC patients.

High expression of EZH2 as a marker for the differential diagnosis of malignant and benign myogenic tumors

Overlap in morphologic features between malignant and benign myogenic tumors, such as leiomyosarcoma (LMS) vs. leiomyoma as well as rhabdomyosarcoma (RMS) vs. rhabdomyoma, often makes differential diagnosis difficult and challenging. Here the expressions of Enhancer of Zeste Homolog 2 (EZH2), Suppressor of Zeste 12 (SUZ12), retinoblastoma protein associated protein 46 (RbAp46), Embryonic Ectoderm Development (EED) and ki-67 protein were detected by immunohistochemistry to evaluate their values in differential diagnosis. The expression of EZH2 mRNA was investigated by analyzing the Gene Expression Omnibus Datasets. The results demonstrated that EZH2 protein was detected in 81.25% (26/32) of LMS and 70.58% (36/51) of RMS, whereas none of leiomyoma (n = 16), rhabdomyoma (n = 15) and normal tissues (n = 31) showed positive immunostaining (p < 0.05). EZH2 protein was found to have a sensitivity of 91.30% and specificity of 100% in distinguishing well-differentiated LMS from cellular leiomyoma, and a sensitivity of 92.86% and specificity of 100% in distinguishing well-differentiated embryonal rhabdomyosarcoma (ERMS) from fetal rhabdomyoma. Besides, the expression of EZH2 mRNA was higher in LMS and RMS than in benign tumors (p < 0.05). The expressions of SUZ12 and RbAp46 protein were higher in RMS than in rhabdomyoma (p < 0.05). Conclusively, the high expression of EZH2 is a promising marker in distinguishing well–differentiated LMS from cellular leiomyoma, or well–differentiated ERMS from fetal rhabdomyoma, and the upregulation of EZH2 protein expression may occur at transcriptional level.

MiR‑92b promotes autophagy and suppresses viability and invasion in breast cancer by targeting EZH2.

MicroRNAs (miRs) are a small non-coding RNA family with a length of 18–22 nucleotides. They are able to regulate gene expression by either triggering target messenger RNA degradation or by inhibiting mRNA translation. Enhancer of zeste homolog 2 (EZH2) is the core enzymatic subunit of polycomb repressor complex 2 and is responsible for the trimethylation of histone 3 on lysine 27 (H3K27me3); it is also able to silence a bundle of tumor suppressor genes through promoter binding. However, little is known regarding the effect of miR-92b on cell autophagy, viability and invasion as well as how it interacts with EZH2. The present study investigated the major role of miR-92b in the autophagy, viability and invasion of breast cancer. It was revealed that in MCF-7 and MDA-MB-453 cells, the expression of miR-92b promoted autophagy induced by starvation and rapamycin treatment. The results of in vitro experiments results demonstrated that miR-92b inhibited breast cancer cell viability, invasion and migration. To further elucidate the regulatory mechanisms of miR-92b in autophagy, a dual luciferase reporter assay was performed to determine whether miR-92b targeted the EZH2 gene. The expression of miR-92b was negatively correlated to EZH2 mRNA expression in breast cancer. Depletion of EZH2 induced phenocopied effects on miR-92b overexpression, thereby demonstrating its importance in autophagy. These results indicated that miR-92b may serve an important role in breast cancer in controlling autophagy, viability and invasion. The present study indicated that miR-92b and EZH2 may serve as potential biomarkers for cancer detection and highlighted their possible therapeutic implications.

Increasing oxaliplatin sensitivity by down-regulation the expression of EZH2 in human gastric cancer SGC7901 cells

Objective To investigate the effect of enhancer of zeste homolog 2 (EZH2) knockdown on chemosensitivity to oxaliplatin in human gastric cancer cell line SGC7901. Methods Small interfering RNA (siRNA) fragments were designed and synthesized for EZH2 mRNA sequence and divided into siRNA-1 group, siRNA-2 group and siRNA-3 group. They were transfected into gastric cancer cell line SGC7901. The negative control group and blank control group were set up at the same time. The expressions of EZH2 mRNA and protein in the SGC7901 cells were detected by quantitative real-time polyme-rase chain reaction (qRT-PCR) and Western blotting. CCK-8 assay was used to detect cell proliferation inhibition rates of SGC7901 cells treated by different concentrations of oxaliplatin. Results After transfection of EZH2 siRNA, the relative expression levels of EZH2 mRNA in siRNA-1 group, siRNA-2 group, siRNA-3 group were 0.615±0.190, 0.241±0.152 and 0.450±0.097. The relative expression levels of EZH2 mRNA of the three groups were lower than that in the negative control group (1.165±0.376), and the differences were statistically significant (P=0.028; P=0.002; P=0.007). After transfection of the best siRNA fragment into SGC7901 gastric cancer cells, the relative expre-ssion levels of EZH2 protein in interference group, blank control group and negative control group were 0.036±0.017, 0.362±0.026 and 0.398±0.036, and the diffe-rence among the three groups was statistically significant (F=157.745, P<0.001). The difference between interference group and negative control group was statistically significant (P=0.001), as compared with the blank control group (P=0.002). When oxaliplatin concentration was 2, 4, 8 and 16 μg/ml, the differences of cell proliferation inhibition rate among interference group, negative control group and blank control group were statistically significant [(18.107±2.822)%, (5.867±2.272)%, (5.333±1.883)%, F=28.185, P=0.001; (54.953±2.550)%, (22.177±1.871)%, (20.077±6.032)%, F=74.206, P<0.001; (60.337±1.641)%, (34.597±3.592)%, (30.227±5.273)%, F=54.897, P<0.001; (78.340±2.081)%, (61.857±3.507)%, (63.077±8.473)%, F=8.586, P=0.017]. There was no significant difference among groups of oxaliplatin at the concentration of 32 μg/ml [(83.450±3.715)%, (72.190±3.948)%, (70.731±17.080)%, F=1.358, P=0.326]. The median inhibitory concentration (IC50) of oxaliplatin in siRNA group, negative control group and blank control group were 5.178, 12.643, 13.601 μg/ml. Conclusion Down-regulation of EZH2 gene expression can significantly inhibit the proliferation of gastric cancer cells, and effectively enhance the sensitivity of gastric cancer cells to oxaliplatin, which indicates EZH2 may play important roles in the development of gastric cancer chemotherapy. These results provide an important theoretical basis for gene therapy of gastric cancer. Key words: Stomach neoplasms; RNA, small interfering; Enhancer of zeste homolog 2; Oxaliplatin

Ineffective erythropoiesis in myelodysplastic syndrome

Ineffective hematopoiesis is one of the hallmarks of myelodysplastic syndrome (MDS). Recently, several signaling pathways responsible for inefficient erythropoiesis in MDS have been uncovered. The p53-S100a8/S100a9-TLR4 pathway is involved in ineffective erythropoiesis in 5q minus (5q-) syndrome. Somatic mutations target multiple components of the messenger RNA (mRNA) splicing machinery including Splicing Factor 3 Subunit b1 (SF3b1) and Serine Arginine Rich Splicing Factor 2 (SRSF2) in patients with MDS. SF3b1 is the most frequently mutated spliceosome component in MDS and is mutated approximately 85% of the time in MDS with ring sideroblasts (MDS-RS). SF3b1 mutations are not simple loss-of-function or inactivating mutations but result in a change of function and cause aberrant splicing of genes that may be involved in the pathogenesis of MDS-RS. Recurrent mutations are also found in epigenetic regulator genes in MDS, including polycomb repressive complex 2 (PRC2) genes. The loss of enhancer of zeste homolog 2 (EZH2), an enzymatic component of PRC2 in mice, markedly accelerates the development of MDS in combination with representative driver mutations. The loss of EZH2 causes impaired erythropoiesis and increases ineffective hematopoiesis. Of interest, EZH2 is one of the targets of SRSF2 mutants in MDS, and mis-spliced EZH2 mRNA undergoes nonsense-mediated decay (NMD) -dependent degradation. All these data suggest that EZH2 insufficiency causes ineffective erythropoiesis.

Expression and inhibition of BRD4, EZH2 and TOP2A in neurofibromas and malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNST) are rare, highly aggressive sarcomas that can occur spontaneously or from pre-existing plexiform neurofibromas in neurofibromatosis type1 (NF1) patients. MPNSTs have high local recurrence rates, metastasize easily, are generally resistant to therapeutic intervention and frequently fatal for the patient. Novel targeted therapeutic strategies are urgently needed. Standard treatment for patients presenting with advanced disease is doxorubicin based chemotherapy which inhibits the actions of the enzyme topoisomerase IIα (TOP2A). Recent molecular studies using murine models and cell lines identified the bromodomain containing protein 4 (BRD4) and enhancer of zeste homolog 2 (EZH2) as novel targets for MPNST treatment. We investigated the expression and potential use of BRD4, EZH2 and TOP2A as therapeutic targets in human NF1-derived MPNSTs. The transcript levels of BRD4, EZH2 and TOP2A were determined in paired formalin-fixed paraffin-embedded (FFPE) neurofibroma/MPNST samples derived from the same NF1 patient and in a set of plexiform neurofibromas, atypical neurofibromas and MPNST. We further examined the effect on cell viability of genetic or pharmacological inhibition of BRD4, EZH2 and TOP2A in an MPNST cell line panel. Our results indicated that in MPNST samples BRD4 mRNA levels were not upregulated and that MPNST cell lines were relatively insensitive to the bromodomain inhibitor JQ1. We corroborated that EZH2 mRNA expression is increased in MPNST but failed to confirm its reported pivotal role in MPNST pathogenesis as EZH2 knockdown by siRNA did not interfere with cellular proliferation and viability. Finally, the relation between TOP2A levels and sensitivity for doxorubicin was examined, confirming reports that TOP2A mRNA levels were overexpressed in MPNST and showing that MPNST cell lines exhibited relatively high TOP2A protein levels and sensitivity to doxorubicin. We tentatively conclude that the potential for effective therapeutic intervention in MPNST by targeting BRD4, EZH2 and TOP2A individually, may be limited. Clinical studies are necessary to ultimately prove the relevance of BRD4 and EZH2 inhibition as novel therapeutic strategies for MPNST.

EZH2 upregulation correlates with tumor invasiveness, proliferation, and angiogenesis in human pituitary adenomas

Enhancer of zeste homolog 2 (EZH2) is a critical component of the polycomb repressive complex 2, which epigenetically represses genes involved in tumorigenesis and is highly expressed in tumors. However, no studies have investigated EZH2 expression and its clinical significance in human pituitary adenomas (PAs). Therefore, we examined the expression pattern of EZH2 in PAs and studied the correlations between protein expression and invasiveness, proliferation, angiogenesis, hormone functioning, and some other factors. We measured EZH2 and MMP-14 protein and EZH2 mRNA expression in 62 samples of PAs by immunohistochemistry staining and quantitative real-time polymerase chain reaction and correlated protein expression relative to clinicopathologic features. The immunopositive rate of EZH2 was 88.7% (55/62). The extent of expression was associated with invasiveness, microvessel density, and proliferation (Ki-67 index). Moreover, EZH2 expression correlated with MMP-14 expression. We did not find any correlation between EZH2 overexpression and hormone-secreting function or patient age or sex. The quantitative real-time polymerase chain reaction analysis revealed that the amount of EZH2 mRNA was significantly higher in invasive than in noninvasive adenomas. This is the first report to describe EZH2 overexpression in human PAs, especially invasive adenomas. Thus, EZH2 is a potentially useful diagnostic marker and pharmacotherapeutic target for invasive PAs.

Effect of curcumol on proliferation, apoptosis and enhancer of zeste homolog 2 expression in bladder cancer EJ cells

Objective To observe the effect of curcumol on proliferation, apoptosis and enhancer of zeste homolog 2 (EZH2) expression in human bladder cancer line cell EJ, and to explore its molecular mechanism. Methods The bladder cancer EJ cells were treated with different concentrations of curcumol (12.5, 25.0, 50.0, and 100.0 mg/L) and the control group was given 1% anhydrous ethanol. The inhibitory effect of curcumol on the growth of EJ cells was evaluated by cell counting kit-8 (CCK-8) assay. Special morphological changes of apoptosis were observed by Hoechst 33258 flourescence staning. Flow cytometry technique was used to detect the apoptosis rate of each experimental group. The expression of EZH2 mRNA and protein was examined with reverse transcriptase-polymerase chain reaction (RT-PCR) and Western boltting. Results EJ cells proliferation was obviously inhibited by curcumol in a time- and concentration-dependent manner. The inhibiton rate was (32.94±0.93)%, (46.75±1.28)%, (58.16±0.76)% and (69.13±1.94)%, and the apoptosis rate was (3.60±0.46)%, (6.70±1.83)%, (16.20±1.61)% and (28.70±2.65)% in curcumol-treated (12.5, 25.0, 50.0, and 100.0 mg/L) groups respectively. A typical form of apoptosis was detected in the high concentration of curcumol by fluorescent inverted phase contrast microscope. As compared with the control group, with the increases in concentrations of curcumenol, the expression of EZH2 mRNA and protein was gradually declined. Conclusion Curcumol could significantly inhibit the growth of EJ cells and induce the apoptosis, which may be related to the down-regulation of EZH2. Key words: Curcumol; Bladder cancer; Enhancer of zeste homolog 2

Selective inhibition of EZH2 by a small molecule inhibitor regulates microglial gene expression essential for inflammation

Multiple studies have documented that Enhancer of zeste homolog 2 (EZH2) could play a role in inflammation and a wide range of malignancies; however, the underlying mechanisms remain largely unaddressed. Microglial activation is a key process in the production and release of numerous pro-inflammatory mediators that play important roles in inflammation and neurodegeneration in the central nervous system (CNS). Therefore, our aim was to investigate whether inhibition of EZH2 with the selective small molecule inhibitor EPZ-6438 protects against neonatal microglial activation. First, in mouse primary microglial cells and a microglial cell line, we found that LPS can rapidly increase EZH2 mRNA level and we subsequently performed gene expression profiling and constructed networks in resting, EPZ-6438-treated, LPS-treated and LPS+EPZ-6438-treated primary microglial cells and a microglial cell line using transcriptome RNA sequencing and bioinformatics analyses. By examining the RNA sequencing, we identified EPZ-6438 target genes and co-regulated modules that were critical for inflammation. We also identified unexpected relationships between the inducible transcription factors (TFs), motif strength, and the transcription of key inflammatory mediators. Furthermore, we showed that EPZ-6438 controls important inflammatory gene targets by modulating interferon regulatory factor (IRF) 1, IRF8, and signal transducer and activator of transcription (STAT) 1 levels at their promoter sites. Our unprecedented findings demonstrate that pharmacological interventions built upon EZH2 inhibition by EPZ-6438 could be a useful therapeutic approach for the treatment of neuroinflammatory diseases associated with microglial activation.

Epigenetic regulation of miRNA-124 and multiple downstream targets is associated with treatment response in myeloid malignancies.

Epigenetic regulation of microRNA (miRNA) expression has recently been implicated in the pathogenesis of myelodysplastic syndrome (MDS). Particular interest has focused on miRNA-124 expression, which is inhibited in MDS and has recently been demonstrated to be upregulated in response to epigenetic treatment (EGT). Previous studies have determined the in vitro and in vivo expression of miRNA-124 and several molecular targets, including cyclin-dependent kinase (CDK) 4, CDK6 and enhancer of zeste homolog 2 (EZH2), in order to elucidate the molecular mechanisms associated with the miRNA-124-mediated therapeutic response to EGT in MDS and identify additional potential biomarkers of early EGT treatment response in myeloid malignancies. In vitro studies in the HL60 leukemic cell line identified upregulation of miRNA-124 expression in response to single-agent EGT with either azacytidine (AZA) or the histone deacetylase inhibitor panobinostat (LBH589). Combination EGT with AZA and LBH589 resulted in significant additive induction of miRNA-124 expression. Expression of downstream targets of miRNA-124, including CDK4, CDK6 and EZH2, in response to single agent and combined EGT was determined in HL60 cells. Single and combination EGT treatment resulted in inhibition of CDK4, CDK6 and EZH2 expression with combination EGT resulting in a significant and additive inhibitory effect. In vivo studies using peripheral blood mononuclear cells from patients receiving combination EGT for high risk MDS or acute myeloid leukemia demonstrated significant induction of miRNA-124 and inhibition CDK4 and CDK6 messenger (m)RNA expression in patients that responded to combination EGT. A trend to inhibited EZH2 mRNA expression was also identified in response to combination EGT. Overall, the present observations identify a potential molecular mechanism for miRNA-124-mediated response to EGT involving regulation of CDK4, CDK6 and EZH2 expression. In addition, the present findings further qualify miRNA-124 as a possible biomarker of early response to EGT in myeloid malignancies and potentially a valid therapeutic target, together with CDK4, CDK6 and EZH2.

Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I

BackgroundPolyphyllin I (PPI), a bioactive phytochemical extracted from the Rhizoma of Paris polyphylla, has been reported to exhibit anti-cancer activity. However, the detailed mechanism underlying this remains to be elucidated.MethodsCell viability and cell cycle distribution were measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. The expression of enhancer of zeste homolog 2 (EZH2) mRNA was measured by quantitative real time PCR (qRT-PCR). Western blot analysis was performed to examine the phosphorylation and protein expression of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p65, DNA methyltransferase 1 (DNMT1) and EZH2. Exogenous expression of p65, DNMT1, and EZH2 were carried out by transient transfection assays. Promoter activity of EZH2 gene was determined using Secrete-Pair Dual Luminescence Assay Kit. A xenografted tumor model in nude mice and bioluminescent imaging system were used to further test the effect of PPI in vivo.ResultsWe showed that PPI significantly inhibited growth and induced cell cycle arrest of non-small cell lung cancer (NSCLC) cells in a dose-dependent manner. Mechanistically, we found that PPI increased the phosphorylation of SAPK/JNK, reduced protein expression of p65 and DNMT1. The inhibitor of SAPK/JNK (SP600125) blocked the PPI-inhibited p65 and DNMT1 protein expression. Interestingly, exogenously expressed p65 overcame PPI-inhibited protein expression of DNMT1. Moreover, PPI reduced EZH2 protein, mRNA, and promoter activity; overexpression of EZH2 resisted the PPI-inhibited cell growth, and intriguingly, negative feedback regulation of SAPK/JNK signaling. Finally, exogenous expression of DNMT1 antagonized the PPI-suppressed EZH2 protein expression. Consistent with this, PPI inhibited tumor growth, protein expression levels of p65, DNMT1 and EZH2, and increased phosphorylation of SAPK/JNK in vivo.ConclusionOur results show that PPI inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of p65 and DNMT1 protein levels, subsequently; this results in the reduction of EZH2 gene expression. The interactions among p65, DNMT1 and EZH2, and feedback regulation of SAPK/JNK by EZH2 converge on the overall responses of PPI. This study reveals a novel mechanism for regulating EZH2 gene in response to PPI and suggests a new strategy for NSCLC associated therapy.

Abstract 4752: Overexpression of enhancer of zeste homolog 2 (EZH2) in endometrial carcinoma: evidence from an NRG Oncology/Gynecologic Oncology Group trial

Abstract Objectives: Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that mediates epigenetic silencing of tumor suppressor genes. It is commonly over-expressed in several solid tumors and has been shown to be a prognostic biomarker in breast, renal, and gastric cancers. This study aims to investigate patterns of EZH2 expression in endometrial cancer. Methods: Evaluation of expression of EZH2 was completed on both early and advanced stage endometrioid adenocarcinoma tissues and a subset of matched normal mullerian tissue samples, from participants enrolled in GOG protocol 210, using real time reverse transcription polymerase chain reaction (RT-PCR) and western blot (WB) analysis. Investigators were blinded to clinical data. One-way ANOVA, F test and Fisher's exact test were used to assess differences in log mRNA and protein expression respectively with known clinical and pathologic prognostic factors. Kaplan Meier survival analysis was used to evaluate differences in progression free survival (PFS) based on EZH2 expression. Results: Eight-seven specimens were analyzed including 60 tumors and 27 normal tissue specimens. EZH2 mRNA and protein expression in tumor specimens was significantly higher than in matched controls (paired t test, p &amp;lt; 0.001). EZH2 expression was associated with lympho-vascular space invasion (p = 0.044) but other clinical and pathologic factors including age, stage, grade, nodal involvement or disease status were not associated with significant differences in expression. However, there does appear to be a trend towards decreased progression free survival among patients with increased EZH2 expression levels. Conclusions: Our results confirm that there is differential expression of EZH2 in uterine cancers compared to normal tissues and suggests a potential role of the EZH2 as prognostic marker of aggressive disease in patients with endometrial cancer. In view of the substantial portion of early stage low grade tumors expressing EZH2, it will be interesting to investigate if increased EZH2 protein levels may also have therapeutic implications, since targeted inhibition of EZH2 expression has been shown to repress tumor cell growth in other solid malignancies. Further investigation into this potential prognostic biomarker and therapeutic target is warranted. Citation Format: Lauren Krill, Shamshad Ali, Ramez Eskander, Meenakshi Singh, David Mutch, Susan Zweizig, Michael Birrer, Heather Lankes, Bang Hoang, Leslie Randall. Overexpression of enhancer of zeste homolog 2 (EZH2) in endometrial carcinoma: evidence from an NRG Oncology/Gynecologic Oncology Group trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4752.

BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer

BRCA1 mutation or depletion correlates with basal-like phenotype and poor prognosis in breast cancer but the underlying reason remains elusive. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with downregulation of the expression of the pleiotropic tumour suppressor FOXO3. Knockdown of BRCA1 by small interfering RNA (siRNA) resulted in downregulation of FOXO3 expression in the BRCA1-competent MCF-7, whereas expression of BRCA1 restored FOXO3 expression in BRCA1-defective HCC70 and MDA-MB-468 cells, suggesting a role of BRCA1 in the control of FOXO3 expression. Treatment of HCC70 and MDA-MB-468 cells with either the DNA methylation inhibitor 5-aza-2'-deoxycitydine, the N-methyltransferase enhancer of zeste homologue 2 (EZH2) inhibitor GSK126 or EZH2 siRNA induced FOXO3 mRNA and protein expression, but had no effect on the BRCA1-competent MCF-7 cells. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNMT1/3a/b and histone H3 lysine 27 trimethylation (H3K27me3) are recruited to the endogenous FOXO3 promoter, further advocating that these proteins interact to modulate FOXO3 methylation and expression. In addition, ChIP results also revealed that BRCA1 depletion promoted the recruitment of the DNA methyltransferases DNMT1/3a/3b and the enrichment of the EZH2-mediated transcriptional repressive epigenetic marks H3K27me3 on the FOXO3 promoter. Methylated DNA immunoprecipitation assays also confirmed increased CpG methylation of the FOXO3 gene on BRCA1 depletion. Analysis of the global gene methylation profiles of a cohort of 33 familial breast tumours revealed that FOXO3 promoter methylation is significantly associated with BRCA1 mutation. Furthermore, immunohistochemistry further suggested that FOXO3 expression was significantly associated with BRCA1 status in EZH2-positive breast cancer. Consistently, high FOXO3 and EZH2 mRNA levels were significantly associated with good and poor prognosis in breast cancer, respectively. Together, these data suggest that BRCA1 can prevent and reverse FOXO3 suppression via inhibiting EZH2 and, consequently, its ability to recruit the transcriptional repressive H3K27me3 histone marks and the DNA methylases DNMT1/3a/3b, to induce DNA methylation and gene silencing on the FOXO3 promoter.

Higher EZH2 expression is associated with extramedullary infiltration in acute myeloid leukemia.

Accumulating evidence indicates that enhancer of zeste homolog 2 (EZH2) promotes the metastatic ability of solid tumors, but the role of EZH2 in extramedullary infiltration (EMI) in acute myeloid leukemia (AML) has not been thoroughly explored. In the present study, we investigated the possible association between EZH2 and EMI. We found that the messenger RNA (mRNA) and protein expression levels of EZH2 in AML patients were both significantly higher than in idiopathic thrombocytopenic purpura (ITP) patients. Furthermore, a positive correlation between EZH2 mRNA expression and percentage of peripheral blood blasts wa s found in AML patients (r = 0.404, p = 0.009). The migratory capacities of Kasumi-1 and HL-60, which both show a high level of EZH2 expression, were markedly higher than those of U937 and KG-1α. In contrast, silencing of EZH2 resulted in reduction in proliferation and migration ability and an increase in apoptosis. The latter observation was accompanied by reduced expression of associated proteins p-ERK, p-cmyc, and matrix metalloproteinase 2 (MMP-2) and an increase in epithelial cadherin (E-cadherin). These data suggest that higher expression of EZH2 may be associated with extramedullary infiltration in acute myeloid leukemia and affect pathogenesis via activation of the p-ERK/p-cmyc/MMP-2 and E-cadherin signaling pathways.

MicroRNA-144 suppresses tumorigenesis and tumor progression of astrocytoma by targeting EZH2.

Our previous study demonstrated that enhancer of zeste homolog 2 (EZH2) overexpression may be associated with aggressive tumor progression and poor prognosis in human astrocytoma. The aim of this study was to investigate the underlying mechanisms of EZH2 on astrocytoma tumorigenesis. An online program miRWalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/) was used to predict possible microRNAs (miRNAs) that might target EZH2 messenger RNA (mRNA). Then the functions of the miRNA-EZH2 mRNA axis in astrocytoma cell proliferation, invasion, and migration were also assessed. We further evaluated the clinical value of the miRNA-EZH2 mRNA axis in astrocytomas. As a result, we identified EZH2 as a target gene of miR-144. In addition, forced expression of miR-144 suppressed astrocytoma cell proliferation, invasion, and migration by down-regulating EZH2. Moreover, miR-144 down-regulation and EZH2 mRNA up-regulation were both significantly associated with advanced World Health Organization grades and low Karnofsky performance status score of astrocytoma patients. Importantly, survival analysis identified the combined expression of miR-144 and EZH2 (miR-144/EZH2) as an independent prognostic factor for overall survival in astrocytoma patients. In conclusion, miR-144 may function as a tumor suppressor by regulating EZH2 expression, and miR-144/EZH2 expression may be a highly sensitive marker for the prognosis in astrocytoma patients.

Alterations in enhancer of zeste homolog 2, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 expression are associated with ex vivo and in vitro bone metastasis in renal cell carcinoma.

Renal cell carcinoma (RCC) has a high potential for bone metastasis; however, the molecular mechanisms underlying this metastasis have remained to be elucidated. The present study aimed to explore the expression levels of enhancer of zeste homolog 2 (EZH2), matrix metalloproteinase-2 (MMP2) and tissue inhibitor of metalloproteinase-2 (TIMP2) as determinants of RCC-associated bone metastasis. Their expression was evaluated in a newly generated RCC cell subline that has a high potential for bone metastasis, in tissue specimens from metastasized bone tissues from patients with RCC and in RCC tissues without metastasis. A total of 25 RCC tissue specimens without metastasis and 13 RCC tissue specimens with bone metastasis were acquired for immunohistochemical analysis of EZH2, MMP2 and TIMP2 protein expression. The expression levels of EZH2, MMP2 and TIMP2 mRNA and protein were analyzed in the ACHN and ACHN-BO5 cell lines using western blot and reverse transcription polymerase chain reaction (PCR) analyses. Methylation-specific PCR was also used to analyze TIMP2 promoter methylation. EZH2 and MMP2 proteins were found to be expressed at higher levels in tissues from patients where RCC had metastasized to the bone as compared with those in RCC patients without metastasis, whereas there was no significant difference in the expression of TIMP2 protein between the two tissues. Furthermore, the expression of EZH2 protein was correlated with MMP2 expression, but there was no significant correlation between the expression of EZH2 and TIMP2 proteins. The in vitro results using cell lines confirmed the ex vivo findings, indicating that the expression levels of EZH2 and MMP2 protein and mRNA were higher in ACHN-BO5 cells than those in ACHN cells. By contrast, TIMP2 protein and mRNA expression levels were lower in ACHN-BO5 cells than those in the parental ACHN cells. The TIMP2 promoter was highly methylated in ACHN-BO5 cells compared with that in ACHN cells. Upregulation of EZH2, MMP2 and TIMP2 expression was correlated with metastasis of RCC to bone tissues ex vivo and in vitro. Further studies are required in order to elucidate the mechanism underlying the altered expression of these genes.

MiR-26a Promoted by Interferon-Alpha Inhibits Hepatocellular Carcinoma Proliferation and Migration by Blocking EZH2

Interferon (IFN)-α has been used to treat hepatocellular carcinoma (HCC). Here, we report that the IFN-α-induced microRNA-26a (miR-26a) can inhibit HCC proliferation and invasion by suppressing enhancer of zeste homologue 2 (EZH2) expression in tumor cells. First, the miR-26a transcription level was quantified by real-time quantitative PCR in the HCC specimens from IFN-α-treated HCC patients. Next, we transfected HepG2 cells with miR-26a mimics and miR control, and then we investigated the influence of miR-26a mimic transfection on HepG2 cell proliferation and invasion. It was shown that there was increased miR-26a accompanied with downregulated EZH2 expression in the HCC specimens, and EZH2 mRNA levels were inversely correlated with miR-26a expression. There was a dose-response correlation between the IFN-α dosage and EZH2 expression. In addition, the miR-26a mimic transfection decreased the EZH2 expression level significantly in the transfected HepG2 cells and inhibited HepG2 cell proliferation and invasion effectively. Our results indicate that miR-26a exerts growth inhibition in HCC and that its inhibitory effect is mediated briefly by blocking EZH2 expression.

Effects of triptolide on prostate cancer cell line DU145 in vitro and and possible mechanisms

Objective To detect the curative effectiveness and its possible mechanisms of triptolide (TPL) on prostate cancer DU145 cells.Methods After DU145 cells were treated with 0,5,25,50,and 100 mmol/L TPL for 24 and 48 h,methyl thiazol tetrazolium (MTT) assay was used to observe the proliferation inhibition.Apoptosis was evaluated by Annexin V-fluoresceine isothiocyanate (FITC)/propidirum iodide (PI) double staining with flow cytometry and visualized using PI staining of apoptotic nuclei.Protein histone H3 containing the trimethylated lysine 27 (H3K27me3) and enhancer of zeste homolog 2 (EZH2) expression levels were detected by Western blotting,and EZH2 gene expression by real-time quantitative polymerase chain reaction (FQ-PCR).Results Treatment with TPL for 24 h or 48 h inhibited proliferation of DU145 cells with 50％ inhibitory dose (IC50) being (68.71 ±0.15) and (36.87 ±5.80)nmol/L respectively,with the difference being statistically significant in comparison to the normal control group (P ＜ 0.05).As compared with the normal control group,with the increases of TPL concentrations (25,50 and 100 nmol/L),the apoptosis rate was gradually increased,and the early apoptosis rate was (10.9 ± 1.3) ％,(22.4 ± 1.3) ％ and (31.3 ± 2.4) ％,respectively.There was statistically significant difference between each two groups (P ＜ 0.05).PI staining results showed typical apoptotic characteristics.After DU145 cells were treated with TPL at different concentrations for 24 h,Western blotting showed the expression levels of trimethylated H3K27me3 and EZH2 were significantly reduced (P ＜ 0.05).Using FQ-PCR,we verified that EZH2 mRNA was decreased by TPL in a concentration-dependent manner (P ＜0.05).Conclusion TPL inhibited the proliferation of DU145 cells in a concentration-and time-dependent manner and induced epigenetic alterations probably by regulating histone methylation. Key words: Triptolide ; Prostate cancer; Epigenetic ; Histone modification