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Background: Hepatocellular carcinoma (HCC) is the predominant form of liver cancer and is accompanied by a complex regulatory network. Increasing evidence suggests that an abnormal gene expression of EZH2 is associated with HCC progression. However, the molecular mechanism by which non-coding RNAs (ncRNAs) regulate EZH2 remains elusive., Methods: The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) data were used to perform differential expression analysis and prognostic analysis. We used the Encyclopedia of RNA Interactomes (ENCORI) database to predict candidate miRNAs and lncRNAs that may bind to EZH2 . Subsequently, the comprehensive analysis (including expression analysis, correlation analysis, and survival analysis) identified ncRNAs that contribute to EZH2 overexpression., Results: EZH2 was found to be upregulated in the majority of tumor types and associated with a poor prognosis. Hsa-miR-101-3p was identified as a target miRNA of EZH2 . Additionally, SNHG6 and MALAT1 were identified as upstream lncRNAs of hsa-miR-101-3p. Meanwhile, correlation analysis revealed that EZH2 expression was significantly associated with the infiltration of several immune cell types in HCC., Conclusion: SNHG6 or MALAT1/hsa-miR-101-3p/ EZH2 axis were identified as potential regulatory pathways in the progression of HCC.

Long noncoding RNAs (LncRNAs) have been reported to play a vital role in the development of oesophageal squamous cell carcinoma (OSCC). Our previous study revealed that the significant upregulation of the LncRNA small nucleolar RNA host gene 6 (SNHG6) in OSCC promotes OSCC tumourigenesis. However, the mechanisms underlying the dynamics of SNHG6 expression in OSCC have rarely been studied. In this study, we verified the tumour-promoting effect of SNHG6 through sponging miR-101-3p, and their levels were negatively correlated in human samples of OSCC. In addition, miR-101-3p overexpression reversed the effect of SNHG6. Moreover, we confirmed that SNHG6/miR-101-3p affects OSCC by regulating the expression of the enhancer of zeste 2 (EZH2). The effect of EZH2 silencing resembled closely that of SNHG6 knockdown. EZH2 silencing inhibited the expression of protein cyclin D1 and β-catenin, but in contrast, it enhanced the expression of E-cadherin. These findings demonstrated the oncogenic role of SNHG6, which promotes OSCC progression by regulating the expression of EZH2 through its interaction with miR-101-3p. These findings may help in improving the diagnosis and treatment methods of OSCC., (© 2021 Wiley Periodicals LLC.)

Background: Gliomas are common malignant tumors in the nervous system, known for poor prognosis and low survival rate., Objective: This study aims to explore functions of miR-137 in glioma progression and identify messenger RNAs (mRNA) regulated by miR-137, which provides new ideas for further exploration of glioma therapeutic targets., Methods: Gene expression data were downloaded from the Cancer Genome Atlas database, and abnormally expressed miRNAs and mRNAs in glioma were analyzed. The expression of genes in 20 pairs of clinical tissue samples and glioma cell lines were detected through qRT-PCR, and the expression of proteins was detected through Western blot. Changes in cell proliferative level after transfection were detected via CCK8 assay, and changes in cell migratory and invasive abilities were detected by Transwell assay. Besides, dual-luciferase reporter assay was employed to testify binding relationship between two genes., Results: Our study found that miR-137 was significantly and lowly expressed in glioma tissue and cell lines, and the prognoses of glioma patients with highly expressed miR-137 were more optimistic. Overexpressed miR-137 could remarkably inhibit proliferative, invasive and migratory abilities of glioma cells U87, while transfection of miR-137 inhibitor presented an opposite effect. Additionally, EZH2 was a direct target of miR-137 and overexpressed EZH2 effectively reversed the effect of miR-137 on glioma proliferation and migration., Conclusions: Our study found that miR-137 could suppress the proliferation, invasion and migration of glioma cells through regulating the expression of EZH2. So far, we have found a novel regulatory pair that influences glioma progression, providing a basis for further development of new therapeutic strategies., (© 2021. The Genetics Society of Korea.)

Purpose: Corneal fibroblast can be transformed into corneal myofibroblasts by TGF-β1. Enhancer of zeste homolog 2 (EZH2) upregulation has been observed in the occurrence of other fibrotic disorders. We investigated the role of EZH2 in the progression of corneal fibrosis and the antifibrotic effect of EZH2 inhibition in corneal fibroblasts (CFs)., Methods: Primary CFs were isolated from corneal limbi and the CFs were treated with TGF-β1 to induce fibrosis. EPZ-6438 and EZH2 siRNA were used to inhibit EZH2 expression. Myofibroblast activation and extracellular matrix (ECM) protein synthesis was detected by quantitative real-time PCR, western blotting, and immunofluorescence staining assay. The functions of myofibroblast were evaluated by cell migration and collagen gel contraction assays. Molecular mechanisms involved in EZH2 inhibition were investigated by RNA sequencing., Results: TGF-β1 activated EZH2 expression in CFs. Treatment with EPZ-6438 (5 μM) and EZH2 siRNA considerably suppressed corneal myofibroblast activation and ECM protein synthesis in CFs induced by TGF-β1 when compared to the control group. EPZ-6438 (5 μM) suppressed cell migration and gel contraction in CFs. RNA sequencing results revealed that antifibrotic genes were activated after EZH2 inhibition to suppress corneal myofibroblast activation., Conclusion: Inhibition of EZH2 suppresses corneal myofibroblast activation and ECM protein synthesis, and could serve as a novel therapeutic target for preventing corneal scarring., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Aims: Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive lymphoma. This study was designed to compare epigenetic alterations observed in Enhancer of Zeste Homolog 2 (EZH2)-target genes between plasma-derived exosomes and primary tumors in DLBCL patients., Main Methods: Exosomes were isolated from plasma of 21 DLBCL patients and 21 controls. We analyzed the methylation status of the target genes using methylation-specific PCR. We also examined whether the exosomes and the tumor samples contained transcripts of the target genes., Key Findings: We found that CDKN2A and CDKN2B were methylated in both plasma exosomes and primary tumor tissue samples. None of the transcripts were found in the exosomes except CDKN1B which was expressed in 8 (38%) of the exosome samples., Significance: This study showed that plasma exosomes might preferably package certain target molecules from primary tumors and the exosomes containing dual methylated DNAs of CDKN2A and CDKN2B, or CDKN1B transcript may contribute to DLBCL pathogenesis.

Osteoporosis (OP) behaves in different manners in different parts of the skeleton. This study aims to investigate the effects of curcumin on bone mass of the mandibular and femur from ovariectomized OP rats and to validate whether enhancer of zeste homolog 2 (EZH2)/Wnt/β-Catenin pathway is involved in this process. Curcumin was administered intragastrically into ovariectomized rats for 12 weeks. The bone parameters and the morphology of the trabecular bone of the left mandible and left femur were assessed by micro-computed tomography assay. Morphological changes of the left mandible and left femur were evaluated by hematoxylin and eosin staining. The mRNA levels of EZH2, β-Catenin, and Runx2 in the right mandible and right femur were examined by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed to assess EZH2 expression. Both the mandible and femur exhibited OP-like changes in ovariectomized rats, while the mandible bone resorption was less than the femur bone resorption. Curcumin intragastric administration improved bone microstructure and promoted bone formation in the mandible and femur. Curcumin inhibited EZH2 mRNA level and induced that of β-Catenin and Runx2 in the mandible and femur. Collectively, curcumin exerts protective effects against OP, possibly by regulating the EZH2/Wnt/β-Catenin pathway., (© 2021 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Enhancer of zeste homolog 2 (EZH2), a component of the polycomb repressive complex 2 that catalyzes trimethylation of H3K27 (H3K27me3), has been shown to promote tumor development and progression. Expression of EZH2 is associated with cell cycle regulation and cell proliferation in various neoplasms. Oral verrucous hyperplasia (OVH) and Oral verrucous carcinoma (OVC) are rare entities and share several clinical and histopathologic features. Problems distinguishing these lesions are added by a lack of adjacent normal tissue of the biopsy samples and poorly oriented tissue sections. The aim of this study was to investigate the expression of EZH2 and H3K27me3 in OVH and OVC and comparing the expression with normal oral mucosa and oral squamous cell carcinoma (OSCC). Seventy-eight samples, including 25 cases of OVC, 8 cases of OVH, 35 cases of OSCC and 10 cases of normal oral mucosa, were retrieved and submitted for immunohistochemical staining. The results demonstrated that the mean labeling indices (LIs) of EZH2 and H3K27me3 expression were highest in OSCC, followed by the OVC, OVH, and normal mucosa. Statistical differences in EZH2 LI were observed among these lesions whereas H3K27me3 LI was significantly different among OSCC, OVH and normal mucosa. EZH2 LI was found to have a sensitivity of 72.00% and specificity of 87.50% in distinguishing OVH from OVC, and a sensitivity of 57.14% and specificity of 84.00% in distinguishing OVC from OSCC. A positive correlation between EZH2 and H3K27me3 expression was significantly found in OVC but not in OVH and OSCC. These findings highlight the involvement of epigenetic regulation by EZH2-mediated H3K27me3 in the pathogenesis of OVH and OVC, and EZH2 expression indicates disease progression of these verrucous lesions. Diagnostic test analysis further suggests that EZH2 may be used as an additional test for differentiating OVH from OVC in questionable cases.

Background Impaired angiogenic abilities of the microvascular endothelial cell (MVEC) play a crucial role in diabetes mellitus-impaired ischemic tissue repair. However, the underlying mechanisms of diabetes mellitus-impaired MVEC function remain unclear. We studied the role of serum-derived small extracellular vesicles (ssEVs) in diabetes mellitus-impaired MVEC function. Methods and Results ssEVs were isolated from 8-week-old male db/db and db/+ mice by ultracentrifugation and size/number were determined by the Nano-sight tracking system. Diabetic ssEVs significantly impaired tube formation and migration abilities of human MVECs. Furthermore, local transplantation of diabetic ssEVs strikingly reduced blood perfusion and capillary/arteriole density in ischemic hind limb of wildtype C57BL/6J mice. Diabetic ssEVs decreased secretion/expression of several pro-angiogenic factors in human MVECs. Mechanistically, expression of enhancer of zest homolog 2 (EZH2), the major methyltransferase responsible for catalyzing H3K27me3 (a transcription repressive maker), and H3K27me3 was increased in MVECs from db/db mice. Diabetic ssEVs increased EZH2 and H3K27me3 expression/activity in human MVECs. Expression of EZH2 mRNA was increased in diabetic ssEVs. EZH2-specific inhibitor significantly reversed diabetic ssEVs-enhanced expression of EZH2 and H3K27me3, impaired expression of angiogenic factors, and improved blood perfusion and vessel density in ischemic hind limb of C57BL/6J mice. Finally, EZH2 inactivation repressed diabetic ssEVs-induced H3K27me3 expression at promoter of pro-angiogenic genes. Conclusions Diabetic ssEVs impair the angiogenic property of MVECs via, at least partially, transferring EZH2 mRNA to MVECs, thus inducing the epigenetic mechanism involving EZH2-enhanced expression of H3K27me3 and consequent silencing of pro-angiogenic genes. Our findings unravel the cellular mechanism and expand the scope of bloodborne substances that impair MVEC function in diabetes mellitus.

Chemotherapeutic insensitivity is a major obstacle for effective treatment of hepatocellular carcinoma (HCC). Recently, new evidence showed that microRNAs (miRNAs) are closely related to drug sensitivity. This study aimed to investigate the relationship between miR-138 expression and cisplatin sensitivity of HCC cells by regulation of EZH2. CCK-8, EdU, and western blotting are determining the cell viability, proliferation, EZH2, and EMT-related protein expression. It was found that compared with normal samples, miR-138 expression was lower in cancer tissue; it was also downregulated in HCC cells. Transfected with miR-138 mimic increased sensitivity of HCC cells to cisplatin. Mechanistically, Luciferase Reporter analysis verified the interaction between miR-138 and target gene EZH2. Inhibition of EZH2 enhanced cisplatin sensitivity and transfection with EZH2 mimic mirrored the function of miR-138 in cisplatin sensitivity. Furthermore, the role of miR-138 on reversed cisplatin-induced epithelial-mesenchymal transition (EMT) was attenuated when combined with EZH2 plasmid. In conclusion, all data from this study illustrate that miR-138 may as a tumor suppressor provides a potential treatment method to treating HCC., Competing Interests: We declare that we have no conflicts of interest., (Copyright © 2021 Taohui Zeng et al.)

Purpose: Enhancer of zeste homolog 2 (EZH2), the catalytic part of the Polycomb repressive complex 2 (PRC2), has a prognostic role in renal cell carcinoma (RCC) and was recently shown to modulate the immune response by reducing tumor cell immunogenicity., Methods: To investigate whether the prognostic role of EZH2 might be driven by a modified immune environment, more than 1800 RCCs were analyzed in a tissue microarray for EZH2 expression and CD8 positive lymphocytes were quantitated by automated digital imaging., Results: EZH2 positivity was found in 75.2% of 1603 interpretable tumors. In clear cell RCC, high EZH2 expression was significantly linked to high ISUP, Furmann, and Thoenes grade (p < 0.0001 each), advanced stage (p < 0.0001), nodal (p = 0.0190) and distant metastasis (p < 0.0001) as well as shortened overall (p < 0.0027) and recurrence free survival (p < 0.0001). The density of CD8+ cells varied from 0 to 5048 cells/mm 2 (Median 120 cells/mm 2 ). A high CD8+ count was significantly associated with high ISUP, Fuhrmann, and Thoenes grade (p < 0.0001 each), advanced tumor stage (p = 0.0041), distant metastasis (p = 0.0026) as well as reduced overall survival (p = 0.0373) and recurrence free survival (p = 0.0450). The density of CD8+ cells continuously increased with raising EZH2 levels (p < 0.0001)., Conclusion: Our data support a striking prognostic role of both EZH2 expression and the density of CD8+ cells in RCC. The tight relationship of EZH2 expression and CD8+ cell counts in RCC is consistent with models suggesting that EZH2 overexpression can be caused by high lymphocyte content in certain tumor types. Such a mechanism could explain the unique finding of high lymphocyte counts driving poor prognosis in RCC patients.

Pulmonary hypertension (PH) is a life‑threatening disease that often involves vascular remodeling. Although pulmonary arterial smooth muscle cells (PASMCs) are the primary participants in vascular remodeling, their biological role is not entirely clear. The present study analyzed the role of enhancer of zeste homolog 2 (EZH2) in vascular remodeling of PH by investigating the behavior of PASMCs. The expression levels of EZH2 in PASMCs in chronic thromboembolic pulmonary hypertension (CTEPH), a type of PH, were detected. The role of EZH2 in PASMC migration was investigated by wound‑healing assay following overexpression and knockdown. Functional enrichment analysis of the whole‑genome expression profiles of PASMCs with EZH2 overexpression was performed using an mRNA Human Gene Expression Microarray. Quantitative (q)PCR was performed to confirm the results of the microarray. EZH2 expression levels increased in CTEPH cell models. The overexpression of EZH2 enhanced PASMC migration compared with control conditions. Functional enrichment analysis of the differentially expressed genes following EZH2 overexpression indicated a strong link between EZH2 and the immune inflammatory response and oxidoreductase activity in PASMCs. mRNA expression levels of superoxide dismutase 3 were verified by qPCR. The results suggested that EZH2 was involved in the migration of PASMCs in PH, and may serve as a potential target for the treatment of PH.

Background: Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. In traditional anti-cancer therapy, epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKI) have been proven to be beneficial for patients with EGFR mutations. However, patients with EGFR wild-type NSCLC were usually not respond to EGFR-TKIs. Enhancer of zeste homolog 2 (EZH2) is a key molecular in the PRC2 complex and plays an important role in epigenetic regulation and is overexpressed in variant tumors. EZH2 inhibitors have been reported to sensitize variant tumor cells to anticancer drugs. This study aimed to investigate whether the EZH2 inhibitors, GSK343 and DZNep when combined with gefitinib can reverse EGFR-TKIs resistance in EGFR wild-type NSCLC cells., Methods: The RNA-sequencing data of patients with NSCLC [502 patients with lung squamous cell carcinoma, including 49 paracancerous lung tissues and 513 patients with lung adenocarcinoma (LUAD), including 59 paracancerous lung tissues] from the Cancer Genome Atlas (TCGA), were analyzed for EZH2 expression. EZH2 expression was verified in 40 NSCLC tissue cancer samples and their corresponding paracancerous tissues from our institute (TJMUGH) via RT-PCR. A549 and H1299 cells treated with siRNA or EZH2 inhibitors were subjected to cell viability and apoptosis analyses as well to EGFR pathway proteins expression analyses via western blotting., Results: EZH2 was upregulated in human NSCLC tissues and correlated with poor prognosis in patients with LUAD based on data from both TCGA and TJMUGH. Both GSK343 and DZNep sensitized EGFR wild-type LUAD cells (A549 and H1299) to gefitinib and suppressed cell viability and proliferation in vitro by downregulating the phosphorylation of EGFR and AKT and by inducing cell apoptosis. Co-administration of EZH2 inhibitors (GSK343 or DZNep) with gefitinib exerted a stronger inhibitory effect on tumor activity, cell proliferation and cell migration than single drug administration in vitro and in vivo., Conclusions: These data suggest that the combination of EZH2 inhibitors with EGFR-TKIs may be an effective method for treating NSCLC-patients with EGFR-wild type, who do not want to undergo traditional treatment with chemotherapy.

Enhancer of zeste homolog 2 (EZH2), an oncogene, is a commonly up-regulated epigenetic factor in human cancer. Hepatocellular carcinoma deletion gene 1 (DLC1) is an antioncogene that is either expressed at low levels or not expressed in many malignant tumours. Curcumin is a promising anticancer drug that has antitumour effects in many tumours, but its mechanism of action is unclear. Our research demonstrated that EZH2 was up-regulated in breast cancer (BC) tissues and cells, whereas DLC1 was down-regulated, and the expression of EZH2 and DLC1 was negatively correlated in BC. By analysing the characteristics of clinical cases, we found that positive expression of EZH2 and negative expression of DLC1 may be predictors of poor prognosis in patients with triple-negative breast cancer (TNBC). Moreover, knockdown of EZH2 expression restored the expression of DLC1 and inhibited the migration, invasion and proliferation, promoted the apoptosis, and blocked the cell cycle of MDA-MB-231 cells. Furthermore, we found that curcumin restored the expression of DLC1 by inhibiting EZH2; it also inhibited the migration, invasion and proliferation of MDA-MB-231 cells, promoted their apoptosis and blocked the cell cycle. Finally, xenograft tumour models were used to demonstrate that curcumin restored DLC1 expression by inhibiting EZH2 and also inhibited the growth and promoted the apoptosis of TNBC cells. In conclusion, our results suggest that curcumin can inhibit the migration, invasion and proliferation, promote the apoptosis, block the cycle of TNBC cells and restore the expression of DLC1 by inhibiting the expression of EZH2., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Introduction: MiR-124, a tumor suppressor, is involved in regulating various cellular processes. The purpose of this study was to investigate the possible function of miR-124 in LA (lung adenocarcinoma) cells., Aims: MiR-124 expression levels in the 54 pairs of LA tissues (and corresponding non-tumor tissues) obtained at the Sixth People's Hospital of Yancheng City and in LA cells were assessed by qRT-PCR. Colony formation assay, wound healing assay, transwell assays, attachment/detachment, western blotting and immunofluorescence assays were performed to assess the function of miR-124 on proliferation, migration and epithelial-to-mesenchymal (EMT) phenotypes in LA cells in vitro. Enhancer of zeste homolog 2 (EZH2) is identified as a target of miR-124 by bioinformatics analysis and luciferase reporter assays. Rescue assays were applied to verify the relationship between miR-124 and EZH2., Results: MiR-124 was down-regulated in LA tissues (compared to adjacent non-tumor tissues), and was down-regulated in 3 out of 4 lung cancer cell lines compared to immortalized, non-tumorigenic bronchial epithelial cells. Forced expression of miR-124 significantly suppressed tumor cell proliferation, migration and inhibited the EMT process. On the contrary, deletion of miR-124 could obviously promote cell proliferation, migration and facilitate the formation of EMT phenotype. Bioinformatics analysis and luciferase reporter assays confirmed that EZH2 was a target gene of miR-124 and was negatively correlated with the level of miR-124 in cancer tissues., Conclusion: Our current study suggested that miR-124 was a tumor suppressor in LA, and miR-124 was associated with LA cell EMT phenotype formation via targeting EZH2., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

KL (klotho) levels decline with age, which is an important mechanistic driver of aging. KL gene deficiency is associated with hypertension. The purpose of this study is to investigate the potential role of H3K27me3 (histone 3 lysine [K] 27 trimethylation) in the regulation of KL gene expression and examine the related molecular pathways that may drive kidney cell aging. Kidneys were collected from 6-month-old WT (wild type; young WT), 30-month-old WT (aged WT), and 6- (young) and 20-month-old (aged) KL mutant mice, respectively. We demonstrated that the H3K27me3 level was increased in kidneys of aged WT and KL mutant mice versus young WT mice. Elevation of H3K27me3 levels was likely due to downregulation of the H3K27 (histone H3 Lys 27)-specific demethylase JMJD3 (the Jumonji domain containing-3) in the aged kidneys. Inhibition of PRC2 (polycomb repressive complex C2; histone trimethyltransferase) decreased the H3K27me3 levels leading to an increase in the expression of KL in cultured primary renal tubule cells assessed by Western blot and KL promoter activity assays. The chromatin immunoprecipitation qPCR assay revealed that H3K27me3 was physically associated with the KL promoter region. Furthermore, aging impaired the SGK1 (serum- and glucocorticoid-induced protein kinase 1)/FOXO3a (the forkhead box class O 3a) signaling leading to upregulation of p53 and p16 (aging markers) in the kidney of aged WT mice. KL may regulate the SGK1/FOXO3 signaling, which was decreased due to KL deficiency. Thus, aging-associated downregulation of KL gene expression may be partly attributed to upregulation of H3K27me3 levels. Downregulation of KL may impair the SGK1/FOXO3 signaling contributing to kidney cell aging.

The epigenetic silencing mechanism of suppressor 3 of cytokine signaling (SOCS3) in cancers has not been fully elucidated. Polycomb repressive complexes 2 (PRC2), an important epigenetic regulatory factors, exerts a critical role in repressing the initial phase of gene transcription. Whether PRC2 participates the down- regulation of SOCS3 in Hepatocellular carcinoma (HCC) remains unclear and how does PRC2 be recruited target gene still needs to explore. In this study, Using TCGA HCC dataset, and detecting HCC tissue specimens and cell lines, we found that SOCS3 expression in HCC was inversely related to that of EZH2, and depended on its promoter methylation status. CTCF, vigilin, EZH2 and H3K27me3 were enriched at CTCF and EZH2 binding sites on the methylated SOCS3 gene promoter. The depletion of CTCF did not affect expression of EZH2 and DNMT1, but decrease recruitment of CTCF, vigilin, EZH2 and H3K27me3. Further, knockdown of CTCF led to a loss of methylation of the methylated SOCS3 promoter, which sequentially increased the expression of SOCS3 and decreased the expression of pSTAT3, the downstream effector. These findings suggest that the CTCF dependent recruitment of EZH2 to the SOCS3 gene promoter is likely to participate in the epigenetic silencing of SOCS3 and in regulating its gene expression., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: The aim of this study was to investigate whether microRNA-625-3p participated in the malignant progression of gastric cancer and inhibited GCa metastasis by regulating EZH2 (Enhancer of zeste homolog 2)., Patients and Methods: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the expression of microRNA-625-3p in 36 pairs of GCa tissues and para-cancerous tissues. The interplay between microRNA-625-3p level and clinical indexes or prognosis of GCa patients was analyzed. MicroRNA-625-3p mimics and inhibitors, as well as their negative controls, were transfected into GCa cell lines to establish microRNA-625-3p overexpression and down-regulation models in vitro, respectively. QRT-PCR was applied to further verify the transfection efficiency. Cell counting kit-8 (CCK-8), colony formation, and transwell assays were performed to analyze the impact of microRNA-625-3p on the proliferative and invasiveness abilities of GCa AGS and SGC-7901 cells. Finally, the regulatory mechanism of microRNA-625-3p on the downstream gene EZH2 was explored by cell reverse experiment., Results: QRT-PCR results revealed that microRNA-625-3p expression level in GCa tissues was remarkably lower than that of adjacent tissues, and the difference was statistically significant (p<0.05). Compared with patients with high expression of microRNA-625-3p, the incidence of lymph node or distant metastasis was significantly higher in patients with low expression of miR-625-3p, whereas the overall survival rate was lower (p<0.05). Compared with GCa cells in NC inhibitor group, the proliferative ability and invasiveness of cells in microRNA-625-3p inhibitor group were remarkably promoted (p<0.05). However, the opposite results were observed in microRNA-625-3p mimics group. Our findings further demonstrated that the expression of EZH2 increased remarkably in GCa cell lines and tissues (p<0.05). Meanwhile, its expression level was negatively correlated with microRNA-625-3p level. Cell reverse experiment showed that EZH2 could offset the influence of microRNA-625-3p on the proliferation and metastasis GCa cells, thereby affecting the malignant progression of GCa., Conclusions: MicroRNA-625-3p was remarkably correlated with lymph node or distant metastasis and poor prognosis of GCa patients. In addition, microRNA-625-3p might inhibit the malignant progression of GCa via modulating EZH2.

Objectives: Enhancer of zeste homolog 2 (EZH2) is a histone methyl transferase 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. We investigated patterns of EZH2 expression in endometrial cancer., Methods: Evaluation of EZH2 expression was completed on both early and advanced stage endometrioid adenocarcinoma tissues and a subset of matched normal mullerian tissue samples, from participants enrolled in Gynecologic Oncology Group (GOG) protocol 210, using real time reverse transcription polymerase chain reaction (RT-PCR) and western blot (WB) analysis. Non-parametric methods were used to assess differences in mRNA and protein expression respectively with known clinical/pathologic prognostic factors. Survival analysis was performed using techniques including Cox proportional hazards (PH) model to evaluate differences in progression free survival (PFS) and overall survival (OS) based on EZH2 expression., Results: Eighty-seven patient samples were analyzed that included 60 tumors and 27 matched-normal tissue specimens. EZH2 mRNA (p < .0001) and protein expression (p < .0001) in tumor specimens were significantly higher than in matched-normal tissue. In primary tumors, EZH2 protein expression was associated with lympho-vascular space invasion (LVSI, p = .044), and EZH2 mRNA expression was associated with age (p = .037). Differences in EZH2 expression between primary tumors and matched normal tissue were not associated with other known clinical and pathologic factors. However, there did appear to be a trend toward decreased progression-free survival among patients with high EZH2 expression levels., Conclusions: Our results confirm the differential expression of EZH2 in uterine cancers compared to normal tissues. However, there were no statistically significant differences in survival associated with EZH2 expression in patients with endometrial cancer. NCT #: NCT00340808., Competing Interests: Declaration of competing interest The authors have no competing interests to disclose., (Copyright © 2019. Published by Elsevier Inc.)

Background: Loss of blood group ABO antigens on red blood cells (RBCs) is well known in patients with leukemias, and such decreased ABO expression has been reported to be strongly associated with hypermethylation of the ABO promoter. We investigated the underlying mechanism responsible for A-antigen reduction on RBCs in a patient with myelodysplastic syndrome., Study Design and Methods: Genetic analysis of ABO was performed by PCR and sequencing using peripheral blood. RT-PCR were carried out using cDNA prepared from total bone marrow (BM) cells. Bisulfite genomic sequencing was performed using genomic DNA from BM cells. Screening of somatic mutations was carried out using a targeted sequencing panel with genomic DNA from BM cells, followed by transient transfection assays., Results: Genetic analysis of ABO did not reveal any mutation in coding regions, splice sites, or regulatory regions. RT-PCR demonstrated reduction of A-transcripts when the patient's RBCs were not agglutinated by anti-A antibody and did not indicate any significant increase of alternative splicing products in the patient relative to the control. DNA methylation of the ABO promoter was not obvious in erythroid cells. Targeted sequencing identified somatic mutations in ASXL1, EZH2, RUNX1, and WT1. Experiments involving transient transfection into K562 cells showed that the expression of ABO was decreased by expression of the mutated RUNX1., Conclusion: Because the RUNX1 mutation encoded an abnormally elongated protein without a transactivation domain which could act as dominant negative inhibitor, this frame-shift mutation in RUNX1 may be a genetic candidate contributing to A-antigen loss on RBCs., (© 2019 AABB.)

Introduction: Meningiomas comprises of a wide variety of histological entities with heterogeneous biological behaviour and prognosis. The plethora of genetic data are yet to produce relevant biomarkers for routine use. In contrast, epigenetic alterations are less elucidated., Material and Methods: The expression of the key molecules involved in the two principal epigenetic systems, i.e. DNA methylation (DNA methyltransferases [DNMT-1, -3A and -3B]) and histone modification (Enhancer of Zeste homolog-2 [EZH2] and trimethyl histone-3 [H3K27me3]) were assessed in 149 cases of meningiomas (grade I - 102, grade II - 47) by immunohistochemistry., Results: Immunopositivity for EZH2 (38.3% vs. 6.0%) and negativity for H3K27me3 (10.6% vs. 1.0%) were significantly more common in grade II tumours. Both were associated with significantly higher proliferative activity. The majority of the cases of both grades showed expression of all three DNMTs. However, high expression of DNMT-1 was significantly more common in grade II tumours (87.8% vs. 66.2%). Expression of EZH2 and loss of H3K27me3 were associated with significantly shorter progression-free survival (hazard ratio [HR] = 4.07 and 0.24, respectively)., Conclusions: The key epigenetic regulators play important roles in the pathobiology of meningiomas. EZH2 positivity and H3K27me3 negativity are associated with aggressive tumour-biology and poor prognosis. Both these markers can easily be assessed by immunohistochemistry and can be incorporated in routine practice.

Background: Mesenchymal stem cells (MSCs) play a significant role in cancer initiation and metastasis, sometimes by releasing exosomes that mediate cell communication by delivering microRNAs (miRNAs). This study aimed to investigate the effects of exosomal miR-133b derived from MSCs on glioma cell behaviors., Methods: Microarray-based analysis identified the differentially expressed genes (DEGs) in glioma. The expression patterns of EZH2 and miR-133b along with interaction between them were clarified in glioma. The expression of miR-133b and EZH2 in glioma cells was altered to examine their functions on cell activities. Furthermore, glioma cells were co-cultured with MSC-derived exosomes treated with miR-133b mimic or inhibitor, and EZH2-over-expressing vectors or shRNA against EZH2 to characterize their effect on proliferation, invasion, and migration of glioma cells in vitro. In vivo assays were also performed to validate the in vitro findings., Results: miR-133b was downregulated while EZH2 was upregulated in glioma tissues and cells. miR-133b was found to target and negatively regulate EZH2 expression. Moreover, EZH2 silencing resulted in inhibited glioma cell proliferation, invasion, and migration. Additionally, MSC-derived exosomes containing miR-133b repressed glioma cell proliferation, invasion, and migration by inhibiting EZH2 and the Wnt/β-catenin signaling pathway. Furthermore, in vivo experiments confirmed the tumor-suppressive effects of MSC-derived exosomal miR-133b on glioma development., Conclusion: Collectively, the obtained results suggested that MSC-derived exosomes carrying miR-133b could attenuate glioma development via disrupting the Wnt/β-catenin signaling pathway by inhibiting EZH2, which provides a potential treatment biomarker for glioma.

Pediatric myelodysplastic syndrome (MDS) is an uncommon disease and little is known about the molecular alterations of its development and evolution to acute myeloid leukemia (AML). The Enhancer of Zeste Homolog 2 ( EZH2 ) is the catalytic subunit of Polycomb repressive complex 2 (PCR2). It is a histone methyltransferase, that targets lysine 27 of histone 3. This methylated H3-K27 is usually associated with the silencing of genes that are involved in fundamental cellular processes, such as cell proliferation and differentiation. There are only few studies showing the status of EZH2 expression in patients with MDS and they were performed in adult MDS patients. The aim of this study was to analyze the EZH2 expression in pediatric patients with MDS and its association with karyotypes and evolution to acute myeloid leukemia (AML). We conducted the first study of EZH2 expression in pediatric patients with MDS. Considering the EZH2 expression levels in 42 patients and 17 healthy pediatric donors, it was possible to define three groups of expression in patients: low, intermediate, and high. The intermediate level encompassed patients with normal karyotypes, low level included patients with monosomy 7 and del(7q) and high level included patients with trisomy 8 and del(11q) ( p < 0.0001). Comparing the leukemic evolution, the low expression group presented disease evolution in 100% (8/8) of the cases, the intermediate expression group showed disease evolution in 4.34% (1/23) and in the high expression group, 63.63% (7/11) patients showed evolution from MDS to AML ( p < 0.0001). It is important to note that low and high EZH2 expression are associated with leukemic evolution, however low expression showed a stronger association with evolution from MDS to AML than the high expression. Our results suggest a scale of measure for EZH2 expression in pediatric MDS, where aberrant EZH2 expression may be a potential biomarker of disease evolution., Competing Interests: The authors declare that there are no conflicts of interests regarding the publication of this paper., (Copyright © 2019 Teresa de Souza Fernandez et al.)

Green tea polyphenols (GTPs) and their major constituent, epigallocatechin-3-gallate (EGCG), have been reported to demonstrate many interesting biological activities, including anticancer properties. Recent studies on prostate cancer provide strong evidence that epigenetic mechanisms are major players in the regulation of matrix metalloproteinases (MMPs) and their binding partner tissue inhibitor of MMPs (TIMPs) involved in prostate cancer progression. Here we demonstrate that GTP/EGCG mediate epigenetic reactivation of TIMP-3 that plays a key role in suppressing invasiveness and cancer progression. Treatment of human prostate cancer DUPRO and LNCaP cells with 10 µg/mL GTP and 20 µM EGCG induced TIMP-3 mRNA and protein expression. This transcriptional activation of TIMP-3 was associated with the decrease in the expression of both enhancers of zeste homolog 2 (EZH2) and its catalytic product trimethylation of histone H3 at lysine 27 (H3K27me3) repressive marks at the TIMP-3 promoter with an accompanying increase in histone H3K9/18 acetylation. In addition, GTP/EGCG treatment significantly reduced class I histone deacetylase (HDAC) activity/expression and EZH2 and H3K27me3 levels in prostate cancer cells. EGCG/GTP exposure also reduced MMP-2/MMP-9 gelatinolytic activity and abrogated invasion and migration capabilities in these cells. Silencing of EZH2 and class I HDACs strikingly increased the expression of TIMP-3 independent of DNA methylation. Furthermore, clinical trials performed on patients undergoing prostatectomy consuming 800 mg EGCG (Polyphenon E) up to 6 weeks and grade-matched controls demonstrate an increase in plasma TIMP-3 levels. A marked reduction in class I HDACs activity/expression and EZH2 and H3K27me3 levels were noted in GTP-supplemented prostate tissue. Our findings highlight that TIMP-3 induction, as a key epigenetic event modulated by green tea in restoring the MMP:TIMP balance suppresses prostate cancer progression., (© 2019 Wiley Periodicals, Inc.)

Growing evidence has uncovered that SLC34A2 plays an evident role in the progression in several types of tumors. However, the biological function and underlying molecular mechanisms of SLC34A2 remain largely unknown. Here, we indicated that SLC34A2 expression was markedly increased in SW480 and HT29 cell line cells compared with that in normal colorectal epithelial cell line cells. Array analysis displayed that the expression of enhancer of zeste 2 (EZH2) decreased considerably when SLC34A2 was knocked down. We demonstrated that SLC34A2 induced EZH2 expression and activated its promoter activity. Serial 5' deletion and site-directed mutagenesis revealed that the induction of EZH2 expression by SLC34A2 was dependent upon the hypoxia-inducible factor 1 (HIF-1)-2 binding site directly within EZH2 promoter. Moreover, HIF-1 activation was proved essential for SLC34A2-induced EZH2 expression. Reactive oxygen species (ROS) generation contributed to the stabilization of HIF-1α by leading to the binding of HIF-1α to the EZH2 promoter, which resulted in increased EZH2 expression. Additionally, we showed that the inhibition of both HIF-1α expression and ROS generation by YC-1 or BHA, respectively, decreased SLC34A2-induced EZH2 overexpression. Significantly, SLC34A2-induced EZH2 overexpression promoted the proliferation and chemo-resistance to apoptosis in colorectal cancer (CRC) cells in vitro and in vivo Altogether, we conclude that the SLC34A2-ROS-HIF-1-induced overexpression of EZH2 promotes CRC cells proliferation and chemo-resistance to apoptosis. SLC34A2-ROS-HIF-1-EZH2 signaling pathway might serve as a novel therapeutic target against CRC., (© 2019 The Author(s).)

Cell differentiation is directed by extracellular cues and intrinsic epigenetic modifications, which control chromatin organization and transcriptional activation. Central to this process is PRC2, which modulates the di- and trimethylation of lysine 27 on histone 3; however, little is known concerning the direction of PRC2 to specific loci. Here, we have investigated the physical interactome of EZH2, the enzymatic core of PRC2, during retinoic acid-mediated differentiation of neuroepithelial, pluripotent NT2 cells and the dedifferentiation of neuroretinal epithelial ARPE19 cells in response to TGF-β. We identified Smad3 as an EZH2 interactor in both contexts. Co-occupation of the CDH1 promoter by Smad3 and EZH2 and the cooperative, functional nature of the interaction were established. We propose that the interaction between Smad3 and EZH2 targets the core polycomb assembly to defined regions of the genome to regulate transcriptional repression and forms a molecular switch that controls promoter access through epigenetic mechanisms leading to gene silencing.-Andrews, D., Oliviero, G., De Chiara, L., Watson, A., Rochford, E., Wynne, K., Kennedy, C., Clerkin, S., Doyle, B., Godson, C., Connell, P., O'Brien, C., Cagney, G., Crean, J. Unravelling the transcriptional responses of TGF-β: Smad3 and EZH2 constitute a regulatory switch that controls neuroretinal epithelial cell fate specification.

Aims: Merkel cell carcinoma, a rare cutaneous neuroendocrine tumour of the skin, can be categorised into two groups according to Merkel cell polyomavirus (MCV) presence. MCV-negative tumours are more aggressive and frequently associated with gene mutations. Some of the genes are potential therapeutic targets. We have previously reported EGFR mutations in six of 27 MCC tumours and overexpression of ALK and EZH2 at mRNA level in MCC tumours. In this study, we sought to determine expression of ALK, EGFR and EZH2 in MCC samples and assess their correlation to MCV status and clinical parameters., Methods and Results: Tissue microarrays were utilised and stained with primary antibodies. Staining data were statistically compared to patient sex, tumour location and development of metastasis and MCC-specific death; 112 tumours and their corresponding patient data were included. We found strong expression of ALK in 51% and strong expression of EZH2 in 76% of the tumours. There was evident correlation of ALK expression with MCV-positivity. Expression of EGFR was infrequent, presenting only in seven MCV-negative tumours. None of the proteins associated with development of metastasis or MCC specific death., Conclusions: ALK and EZH2 expression are frequent in MCC and ALK expression correlates to MCV positivity. EGFR positive tumours might respond to EGFR inhibiting treatment., (© 2018 John Wiley & Sons Ltd.)

Background: Although ERG and SPINK1 molecular alterations have been studied in acinar and ductal adenocarcinoma of the prostate, EZH2 expression has not been previously evaluated in ductal adenocarcinoma., Methods: We collected cases of pure and mixed ductal adenocarcinoma of the prostate and evaluated clinical significance and EZH2, ERG, and SPINK1 protein expression., Results: We investigated 61 ductal adenocarcinomas, 22 pure and 39 mixed ductal/acinar. Except for tumor growth pattern, none of the clinical parameters studied significantly differed between pure and mixed tumors. Thirty-five percent of ductal adenocarcinomas were organ confined, 15% displayed seminal vesicle invasion. Lymph node and distal metastasis occurred in 13% and 24% of cases, respectively; 34% of patients experienced biochemical failure, 7% died of disease. Ninety-eight percent of tumors expressed EZH2; in 80% of cases >50% of tumor cells were positive. ERG and SPINK1 were expressed in 20% and 36% of cases, respectively. There was no difference in protein expression between pure and mixed ductal adenocarcinomas. ERG expression tended to be lower, and SPINK1 higher than reported for acinar tumors. Biochemical failure, metastasis and death did not differ between EZH2, ERG, and SPINK1 positive and negative patients, nor between <50% versus >50% expression of SPINK1 and EZH2, respectively., Conclusions: Pure and mixed ductal adenocarcinomas have similar clinical behavior and molecular alterations. Higher EZH2 and SPINK1 protein expression, compared to acinar prostatic adenocarcinoma, might account for the more aggressive clinical course of ductal adenocarcinoma.

Objective: To detect the expression of long non-coding ribonucleic acid (lncRNA) plasmacytoma variant translocation gene 1 (PVT1) in uveal melanoma (UM) tissues, and to investigate its influence on the proliferation and apoptosis of UM cells as well as its mechanism., Patients and Methods: 40 cases of UM tissues and 40 cases of adjacent tissues surgically resected in our hospital from October 2015 to April 2018 were collected. The expression level of lncRNA PVT1 in these tissues was determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Stable knockdown of lncRNA PVT1 was constructed in human UM cell line OCM-1 using small interfering RNA (siRNA). The impact of lncRNA PVT1 on UM cell proliferation was detected by Cell Counting kit-8 (CCK-8) and colony formation assay. Flow cytometry was applied to measure the apoptotic level of UM cells in the blank control group and lncRNA PVT1 knockdown group. Meanwhile, the expression level of enhancer of zeste homologue 2 (EZH2) was determined by Western blotting., Results: The expression level of lncRNA PVT1 in UM tissues was remarkably higher than that in the adjacent tissues (p<0.05). UM cell proliferation was notably repressed after lncRNA PVT1 knockdown by siRNA. Flow cytometry results indicated that the number of apoptotic UM cells in lncRNA PVT1 knockdown group significantly increased compared with that in the blank control group (p<0.05). The protein expression of EZH2 was suppressed after lncRNA PVT1 knockdown (p<0.05)., Conclusions: LncRNA PVT1 knockdown in UM cells can repress the proliferation of UM cells and promote their apoptosis by regulating EZH2 expression.

Triple-negative breast cancer (TNBC) commonly develops resistance to chemotherapy, yet markers predictive of chemoresistance in this disease are lacking. Here, we define WNT10B-dependent biomarkers for β-CATENIN/HMGA2/EZH2 signaling predictive of reduced relapse-free survival. Concordant expression of HMGA2 and EZH2 proteins is observed in MMTV - Wnt10b LacZ transgenic mice during metastasis, and Hmga2 haploinsufficiency decreased EZH2 protein expression, repressing lung metastasis. A novel autoregulatory loop interdependent on HMGA2 and EZH2 expression is essential for β-CATENIN/TCF-4/LEF-1 transcription. Mechanistically, both HMGA2 and EZH2 displaced Groucho/TLE1 from TCF-4 and served as gatekeepers for K49 acetylation on β-CATENIN, which is essential for transcription. In addition, we discovered that HMGA2-EZH2 interacts with the PRC2 complex. Absence of HMGA2 or EZH2 expression or chemical inhibition of Wnt signaling in a chemoresistant patient-derived xenograft (PDX) model of TNBC abolished visceral metastasis, repressing AXIN2, MYC, EZH2, and HMGA2 expression i n vivo . Combinatorial therapy of a WNT inhibitor with doxorubicin synergistically activated apoptosis in vitro , resensitized PDX-derived cells to doxorubicin, and repressed lung metastasis in vivo . We propose that targeting the WNT10B biomarker network will provide improved outcomes for TNBC. SIGNIFICANCE: These findings reveal targeting the WNT signaling pathway as a potential therapeutic strategy in triple-negative breast cancer. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/5/982/F1.large.jpg., (©2018 American Association for Cancer Research.)

The vital roles of long noncoding RNAs (lncRNAs) in the cancers have been evidenced. However, there are still numerous unsolved queries for the molecular mechanism. This study tries to investigate the role of lncRNA FOXC2-AS1 in the human prostate cancer tumorigenesis. Results stated that lncRNA FOXC2-AS1 was ectopically up-regulated in prostate cancer tissue and cells. The over-expression of FOXC2-AS1 indicates the poor prognosis of prostate cancer patients. Functionally, the gain- and loss-of-functional experiments revealed that FOXC2-AS1 promoted the proliferation and tumor growth of prostate cancer cells in vitro and in vivo. Mechanically, we found that miR-1253 targeted FOXC2-AS1 at the 3'‑untranslated regions (UTR), which in turn bind the EZH2 mRNA 3-UTR. Luciferase reporter assay and rescue experiment confirmed the FOXC2-AS1/miR-1253/EZH2 pathway. In conclusion, we confirmed that lncRNA FOXC2-AS1 accelerated the tumor progression of prostate cancer cells by regulating the proliferation and tumor growth through miR-1253/EZH2 axis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: Recently, accumulated evidence indicates that the enhancer of zeste homologue 2 (EZH2) is highly expressed in a wide range of cancer types, including NSCLC. The downstream genes regulated by EZH2 were screened using bioinformatics analysis. This study aimed to analyse the correlation between the downstream genes of EZH2 and the prognosis of lung adenocarcinoma., Methods: Expression and methylation data of lung adenocarcinoma were downloaded from The Cancer Genome Atlas (TCGA) (https://cancergenome.nih.gov/) database, and data were categorized into EZH2 overexpression and EZH2 downregulation groups according to EZH2 expression. The genes that showed opposite trends of methylation and expression changes were screened, and the association of gene expression was calculated. Based on the String database, a protein association analysis was conducted to identify genes related to EZH2, which are referred to as EZH2 regulation candidate genes. According to gene expression (GSE27262) and methylation (GSE66836) chip data in the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) database, the genes with differential expression and methylation in lung adenocarcinoma tissues were analysed, and the trends of EZH2 regulation candidate gene expression and methylation were verified to identify the EZH2 regulation candidate genes. Subsequently, MethHC (http://methhc.mbc.nctu.edu.tw/php/index.php) and UALCAN (http://ualcan.path.uab.edu/index.html) were employed to verify changes in the expression and methylation of EZH2 downstream regulation candidate genes and to analyse the correlation between these genes and the prognosis of lung adenocarcinoma., Results: Expression and methylation data of lung adenocarcinoma were downloaded from TCGA database and categorized into EZH2 overexpression and EZH2 downregulation groups according to EZH2 expression. A total of 337 genes that showed opposite trends of methylation and expression changes were obtained. The protein association analysis using the String (https://string-db.org/) database showed that 61 genes interact with EZH2 and 61 genes represent EZH2 downstream regulation candidate genes. Moreover, 222 genes obtained from GSE27262 and GSE66836 chip data were negatively correlated with methylation and expression changes, and centrosomal protein 55 (CEP55) was identified as the EZH2 downstream regulation candidate gene. CEP55 was upregulated in lung adenocarcinoma tissues and showed low methylation. According to gene expression data from TCGA database, CEP55 and EZH2 exhibit higher levels in lung adenocarcinoma tissue than in adjacent normal tissue. Finally, the survival analysis revealed that EZH2 is not associated with the prognosis of lung adenocarcinoma, while CEP55 is related to lung adenocarcinoma prognosis., Conclusion: Taken together, these results indicate that changes in EZH2 expression lead to changes in CEP55 expression in lung adenocarcinoma, and these changes are associated with its prognosis., (Copyright © 2018. Published by Elsevier GmbH.)

Background: Enhancer of zeste homolog 2 (EZH2) is reported to be involved in lung cancer pathogenesis via the epigenetic regulation of various genes. Recently, EZH2 was shown to control mechanisms of adaptive resistance to immunotherapy in melanoma; however, the association between EZH2 and programmed death-ligand 1 (PD-L1), which reflects the tumor microenvironment, remains poorly understood., Methods: A total of 428 patients with resected lung adenocarcinoma were analyzed for their EZH2 and PD-L1 expression by immunohistochemistry and evaluated to determine the association between the EZH2 and PD-L1 expression., Results: Among 428 patients, the EZH2 expression was identified in 219 (51.2%) patients, while the PD-L1 expression was observed in 88 (20.6%) patients. The recurrence-free and overall survival were significantly shorter in patients with the EZH2 expression than in those without it. A multivariate analysis showed that EZH2 remained an independent prognosticator for recurrence-free and overall survival. Patients with the EZH2-positive lung adenocarcinoma exhibited a significantly higher expression of PD-L1 than did those without it. A logistic regression analysis with backward elimination revealed that the presence of lymphatic and vessel invasion and PD-L1 positivity were independently associated with the EZH2 expression, while age over 70 years, the presence of vessel invasion, wild-type epidermal growth factor receptor, and EZH2 positivity were significantly associated with the PD-L1 expression., Conclusions: EZH2-expressing lung adenocarcinomas were shown to express the PD-L1 protein more frequently than were nonexpressing lesions. This study provides the first evidence of a possible association between the EZH2 and PD-L1 expression in patients with resected lung adenocarcinoma., (Copyright © 2019 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

EZH2 is a methyltransferase that plays an important tumorigenic role in various neoplasms. We previously found that EZH2 is expressed in a range of aggressive B-cell lymphomas (ABCLs), T-cell lymphomas, and histiocytic neoplasms, with differential expression of intracellular signaling molecules p-ERK, MYC, and p-STAT3, potential regulators of EZH2 expression. We studied EZH2 expression in nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), classic Hodgkin lymphoma (cHL), T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL), and B-cell Lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphomas and classic Hodgkin lymphoma (BCLu-DLBCL/cHL), as well as the coexpression of p-ERK, MYC, and p-STAT3 in these neoplasms. The neoplastic LP cells of NLPHL and Hodgkin/Reed-Sternberg cells of cHL were strongly positive for EZH2, as were the neoplastic cells in THRLBCL and BCLu-DLBCL/cHL. EZH2 expression correlated with proliferation rate, as assessed by Ki-67 staining. LP cells in NLPHL and Hodgkin/Reed-Sternberg cells in cHL were strongly positive for p-ERK, p-STAT3, and MYC, as were the neoplastic cells in THRLBCL and BCLu-DLBCL/cHL, in contrast to the differential expression of these molecules seen in ABCLs. These findings suggest that combined expression of p-ERK, MYC, and p-STAT3 is a useful immunohistochemical pattern for the diagnosis of EZH2-positive Hodgkin lymphomas and related lymphomas, in contrast to ABCLs. Furthermore, the overexpression of EZH2, in association with coexpression of tumorigenic signaling molecules, suggests an oncogenic role for this molecule in the development of Hodgkin lymphomas and related lymphomas. THRLBCL and BCLu-DLBCL/cHL appear to have a mechanism for the regulation of EZH2 expression that is similar to NLPHL and cHL and different from that of ABCLs. In addition, EZH2 and associated signaling cascades may serve as therapeutic targets for the treatment of Hodgkin lymphomas and related lymphomas.

Enhancer of zeste homolog 2 (EZH2), an H3K27-specific histone methyltransferase, has been shown to be frequently overexpressed in various human cancers including lymphoma. Here we investigate the expression and functionality of EZH2 and H3K27me3 in extranodal NK/T-cell lymphoma, nasal type (ENKTL). Results of NanoString analysis revealed that EZH2 and related histone H3 families were up-regulated genes in ENKTL tissues. Results of immunohistochemistry demonstrated that EZH2 and trimethylation of Lys-27 in histone (H3K27me3) were highly expressed in 55.2% and 78.0% of patients with ENKTL, respectively. EZH2 overexpression was significantly associated with higher tumor cell proliferation (r = 0.582, P = .000), advanced stage (P = .012), and predicted poorer overall survival (P = .016) in ENKTL. H3K27me3-positive expression was correlated with lower tumor cell proliferation (r = -0.623, P = .036), earlier stage (P = .043), and predicted better overall survival (P = .020). In addition, EZH2 and H3K27me3 showed inverse correlations (r = -0.652, P = .002) in clinical samples by immunohistochemistry. Furthermore, inhibition of EZH2 by 3-deazaneplanocin A significantly suppressed tumor cell growth. Interestingly, pharmacologic suppression of the JAK3/STAT3 pathway effectively reduced EZH2 and enhanced H3K27me3 in NK/T tumor cell lines. Our data suggest that EZH2 and H3K27me3 are important prognostic markers and potential therapeutic targets in ENKTL., (Copyright © 2018 Elsevier Inc. All rights reserved.)

During the evolution into castration or therapy resistance, prostate cancer cells reprogram the androgen responses to cope with the diminishing level of androgens, and undergo metabolic adaption to the nutritionally deprived and hypoxia conditions. AR (androgen receptor) and PKM2 (pyruvate kinase M2) have key roles in these processes. We report in this study, KDM8/JMJD5, a histone lysine demethylase/dioxygnase, exhibits a novel property as a dual coactivator of AR and PKM2 and as such, it is a potent inducer of castration and therapy resistance. Previously, we showed that KDM8 is involved in the regulation of cell cycle and tumor metabolism in breast cancer cells. Its role in prostate cancer has not been explored. Here, we show that KDM8's oncogenic properties in prostate cancer come from its direct interaction (1) with AR to affect androgen response and (2) with PKM2 to regulate tumor metabolism. The interaction with AR leads to the elevated expression of androgen response genes in androgen-deprived conditions. They include ANCCA/ATAD2 and EZH2, which are directly targeted by KDM8 and involved in sustaining the survival of the cells under hormone-deprived conditions. Notably, in enzalutamide-resistant cells, the expressions of both KDM8 and EZH2 are further elevated, so are neuroendocrine markers. Consequently, EZH2 inhibitors or KDM8 knockdown both resensitize the cells toward enzalutamide. In the cytosol, KDM8 associates with PKM2, the gatekeeper of pyruvate flux and translocates PKM2 into the nucleus, where the KDM8/PKM2 complex serves as a coactivator of HIF-1α to upregulate glycolytic genes. Using shRNA knockdown, we validate KDM8's functions as a regulator for both androgen-responsive and metabolic genes. KDM8 thus presents itself as an ideal therapeutic target for metabolic adaptation and castration-resistance of prostate cancer cells.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with high proliferative and metastatic phenotypes. CDCA7, a new member of the cell division cycle associated family of genes, is involved in embryonic development and dysregulated in various types of human cancer. However, the biological role and molecular mechanism of CDCA7 in TNBC have not been defined. Herein, we found that CDCA7 was preferentially and markedly expressed in TNBC cell lines and tissues. High expression of CDCA7 was associated with metastatic relapse status and predicted poorer disease-free survival in patients with TNBC. We observed that CDCA7 silencing in TNBC cell lines effectively impaired cell proliferation, invasion and migration in vitro. Importantly, depletion of CDCA7 strongly reduced the tumorigenicity and distant colonization capacities of TNBC cells in vivo. Furthermore, CDCA7 increased the expression of EZH2, a marker of aggressive breast cancer that is involved in tumor progression, by enhancing the transcriptional activity of its promoter. This increase in EZH2 expression was essential for the CDCA7-mediated effects on TNBC progression. Finally, our immunohistochemical analysis revealed that the CDCA7/EZH2 axis was clinical relevant. These findings suggest CDCA7 plays a crucial role in TNBC progression by transcriptionally upregulating EZH2 and might be a potential prognostic factor and therapeutic target in TNBC., (© 2018 UICC.)

Purpose: Studies on the performance of epigenetic-based biomarkers in colorectal cancer (CRC) are scarce and have shown contradictory results. Thus, we sought to examine the prognostic value of histone-modifying enzymes (EZH2, SETDB1 and LSD-1) and histone post-translational marks (H3K27me3 and H3K9me3) in CRC., Methods: A retrospective series of 207 CRC patients primarily submitted to surgery in a cancer center was included in this study. Clinicopathological data were retrieved. One representative paraffin block per case was selected for immunohistochemistry, including normal and CRC tissues whenever possible. The percentage of positive nuclear staining (digital image analysis) was used to classify patients into "low" and "high" expression groups for each biomarker. Correlations between immunoexpression levels, clinicopathological features and clinical outcomes [disease-specific (DSS) and disease-free (DFS) survival] were examined. Statistical significance was set at p < 0.05., Results: CRC tissues showed significantly lower expression of SETDB1 and higher expression of the remainder four biomarkers compared to normal mucosa. High EZH2 expression correlated with disease recurrence/progression, whereas low LSD1 expression and high H3K9me3 and H3K27me3 expression were associated with more advanced stage. In multivariable analysis, cases with high LSD1 expression displayed significantly better DSS and DFS (HR 0.477, 95% confidence interval: 0.247-0.923) adjusted for pathological TNM stage., Conclusion: EZH2, SETDB1, LSD1, H3K9me3 and H3K27me3 expression are altered in CRC and may play a role in colorectal carcinogenesis. LSD1 immunoexpression levels independently predicted patient outcome in this cohort. Further investigations, using larger series, are warranted to confirm its potential clinical value and unravel underlying molecular mechanisms.

BACKGROUND Brain glioma is a type of common primary intracranial malignant tumor, the prognosis of which is frequently unfavorable. Enhancer of zeste homolog 2 (EZH2) belongs to poly-sulfur protein family and can mediate cell proliferation and differentiation via the modulation of various genes expressions. In addition, it is further related with occurrence and metastasis of malignant tumors. This study investigated the effect of EZH2 expression on proliferation and tumorigenesis of brain glioma cells. MATERIAL AND METHODS Glioma tumor tissues were collected from 3 patients who received surgery, and the glioma stem cells were then separated, cultured, and identified by flow cytometry. RNA interference approach was used to suppress EZH2 expression, which was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Clonal formation assay analyzed the change of cell proliferation potency. The effect on tumorigenesis potency of glioma stem cells was determined by mouse transplantation assay. Western blot investigated the effect of EZH2 on levels of oncogenes such as HER-2, c-myc, PI3K, and Akt. RESULTS Flow cytometry revealed cancer stem cells in glioma tissues took up 39.4%, and qRT-PCR showed that EZH2 expression was decreased by 72% after the treatment of RNA interference in glioma cells (P<0.05). Both cell clonal formation and xenograft assays showed that the downregulation of EZH2 inhibited glioma cell proliferation (P<0.05) and weakened tumorigenesis potency (P<0.05). Western blot results showed that the reduction of EZH2 also suppressed expressions of oncogenes including c-myc and Akt (P<0.05). CONCLUSIONS Our data demonstrated that in brain glioma cells, the decrease of EZH2 level could suppress cell proliferation and tumorigenesis potency, and meanwhile inhibit the expressions of oncogenes including c-myc and Akt.

Lymph node involvement of chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL) is characterised by the diffuse infiltration of small neoplastic lymphocytes, which is accompanied by the presence of proliferation centres (PCs) comprising prolymphocytes and paraimmunoblasts. There is increasing evidence of accumulation of various molecular alterations in the tumour cells of PCs, which may explain why extended PCs are related to a less favourable prognosis. To further characterize PCs, we compared the expression level of EZH2 protein, the overexpression of which has recently been recognized as poor prognostic factor in CLL/SLL, in the PCs and the intervening small cell areas in lymph nodes of 15 patients with CLL/SLL. We also investigated the mutational profile of EZH2 and the expression of its upstream regulators c-Myc, E2F1, pRB and miR-26a. Our results showed a significantly increased expression of EZH2 in the PCs. No EZH2 mutations were detected, however, overexpression of c-Myc, E2F1 and pRb proteins as well as reduced expression of the tumor suppressor miR-26a were demonstrated in the PCs. In summary our findings indicate that EZH2 pathway is significantly upregulated in the PCs of CLL/SLL lymph nodes, providing further evidence for the distinguished biological features of the PCs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Honey bee larvae products have been widely used as traditional daily supplements and complementary medicine for health promotion. However, there is little scientific evidence about their bioactivities. This study was designed to examine the anti-tumor and anti-metastasis effects of honey bee larvae powder (HLP) and explore the underlying mechanism. A subcutaneous transplantation model (murine breast cancer cell 4T1-LUC) and lung metastasis model (murine melanoma cell B16-F10) were established to evaluate the anti-tumor and anti-metastasis effects of HLP. Honey bee larvae powder extract (HLE) was obtained by 70% ethanol extraction, and its chemical composition was determined according to physiochemical methods. Cell Counting Kit-8 assay was performed to test the cytotoxicity of HLE, and qRT-PCR assays were conducted to examine the mRNA levels of tumor marker EZH2 in HLE-treated tumor cells. In vivo xenograft tumor assays in BALB/c mice revealed dose-dependent suppression of tumor growth and lung metastasis showing an inhibition rate of 37.5% and 70.4% at 6 g/kg HLP-administered group with no toxicity to the animals. In vitro studies indicated that HLE showed no cytotoxicity to cancer cells at doses up to 1000 μg/mL, however, it significantly decreased EZH2 mRNA levels in HLE (1000 μg/mL)-treated B10-F10 cells (28.49%) and 4T1-LUC cells (26.75%). Further studies to elucidate the mechanisms involved and to isolate the active components of honey bee larva may provide more valuable information for its development and application in cancer treatment., (Copyright © 2018. Published by Elsevier Masson SAS.)

To investigate effect of long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on epithelial-to-mesenchymal transition (EMT) of esophageal cancer (EC) and role of enhancer of zeste homolog 2 (Ezh2)-Notch1 signaling pathway in the process. The expression of MALAT1 was determined in four EC cell lines by real-time PCR. TE-1 and EC109 cells were transfected with sh-MALAT1 to inhibit expression of MALAT1 or transfected with pcDNA3.1-Ezh2 to overexpress Ezh2. Invasion and migration assays were conducted to analyze cell metastasis, and expressions of Ezh2-Notch1 signaling-related proteins as well as EMT related proteins were determined using both real-time PCR and western blot. MALAT1 was significantly up-regulated in all EC cell lines compared with the normal cells. Silencing MALAT1 using shRNA could significantly inhibit cell viability (reduced almost 30% of cell viability compared with the control), invasion (reduced almost 30% of cell migration compared with the control), and migration (reduced almost 50% of cell migration compared with the control) of both TE-1 and EC109 cells (P<0.05). Meanwhile, expression of Ezh2, Notch1, Hes1, MMP-9, and Vimentin was significantly decreased and expression of E-cadherin was significantly increased when cells were transfected with sh-MALAT1 compared with the nontransfected cells (P<0.05). However, when cells were cotransfected with both sh-MALAT1 and pcDNA3.1-Ezh2, the protein expression changes induced by sh-MALAT1 were recovered. MALAT1 could affect EMT and metastasis of EC cells through Ezh2-Notch1 signaling pathway. This study can give deeper understandings of the role of MALAT1 in EC and may provide some new directions for treatment of patients with EC.

Context: CD4 + CD25+ regulatory T (Treg) lymphocytes are critical for immune homeostasis. Foxp3 (Forkhead Box protein P3) is always considered as a marker of function and identities determination of Treg cells because of special occurring in Treg cell. People who lack Treg cells or have a low expression of Foxp3 gene will suffer fatal autoimmunity. Scientists are trying to use Treg cells as a treatment for autoimmune disease, such as systemic lupus erythematosus., Objective: Our objective was to induce Foxp3 + CD4+ T cells from naïve CD4 + T cells isolated from C57 mice spleen in vitro using stimuli that include the short chain fatty acid sodium butyrate. Furthermore, to explore the relationship between Foxp3+ T cells induction and epigenetic modification, by observing the changes of Foxp3, Ezh2 (Enhancer of Zeste Homolog 2) and phosphorylated Ezh2 in the induced Treg cells., Materials and Methods: The naïve CD4+ T cells were separated from C57 mice spleen by immunomagnetic separation. Anti-CD28, anti-CD3, IL-2, TGF-β1, and sodium butyrate were added with proper concentration to induce Foxp3 expression during 72 hours. Then, we observed the effect of GSK126 (Ezh2 inhibitor) on the induction within the same over 72 hours duration. Then, western blot and Q-PCR were used to see the changes in gene/protein expression of Foxp3, Ezh2, and phosphorylated Ezh2., Results: According to our results, group 3 that received full stimulus had a significant higher level of Foxp3 and Ezh2 expression (p < .05, comparing with group 1,2) and adding 5 mM sodium butyrate to the full stimulus (group 5) increased significantly the induction of Foxp3 and Ezh2 than control group and higher concentration group (p < .05, comparing with group 3,4, 6). The gene and protein expression of Foxp3 and Ezh2 both were enhanced in group 5 (p < .05 comparing with group 3). However, phosphorylated Ezh2 decreased in group 5 (p < .05 comparing with group3). Sodium butyrate removed part inhibition of GSK126, result in Foxp3 and Ezh2 expression (p < .05, p < .01, comparing with group7)., Conclusion: In this study, we were able to transform CD4 + T cells into CD4 + Foxp3 + T cell by using stimulus like antibodies (anti-CD28, anti-CD3) and cytokines (IL-2, TGF-β1). Sodium butyrate contributes to CD4 + Foxp3 + T cell induction in vitro and at an optimum concentration of 5 mM. Sodium butyrate promotes expression of Ezh2 and Fxop3 of T cells in vitro; in addition, to lowering relative expression of phosphorylated Ezh2 probably be influencing some pathways like PI3K-Akt. Epigenetic modification is also thought to take essential part into the upregulation of Foxp3 from naïve CD4 + Tcells.

Primary myelofibrosis (PMF) is one of the BCR/ABL-negative myeloproliferative neoplasms (MPNs), characterized by the diffuse fibrous hyperproliferation, bone marrow osteosclerosis, extramedullary hematopoiesis, and marked splenomegaly. The patients with PMF have an insidious onset, a long duration of clinical course, and the deteriorated quality of life. It has been reported that the CALR gene 9 exon mutations were detected in 25-30% PMF patients, particularly as high as 80% in the JAK2/MPL-negative ones. As the second most common mutation in BCR/ABL-negative MPNs, CALR mutation has been included in the latest World Health Organization (WHO) classification criteria as one of the main diagnostic criteria for both essential thrombocythemia (ET) and PMF. Moreover, the CALR mutations indicated a favorable prognosis, which the mechanism is still under investigation. It was demonstrated that a characterized high expression of EZH2 and SUZ12 in CALR-mutated patients. Taking EZH2 as the research entry point, we initially discussed the mechanism that the CALR-positive patients with PMF exhibited a better prognosis in the current study.

Background/aim: Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that is deeply involved in cancer pathogenesis. Although clinicopathological significance of EZH2 in non-small cell lung cancer has been gradually elucidated, such significance in small cell lung cancer (SCLC) has yet to be fully investigated., Patients and Methods: Forty patients with resected SCLC were analyzed for EZH2. EZH2 expression was evaluated using the Allred score (0-8) and was classified into negative (0-6) and positive (7 and 8). We evaluated the association between EZH2 and the clinicopathological characteristics and postoperative survivals., Results: Among 40 patients, 15 (37.5%) and 25 (62.5%) were classified as being negative and positive for EZH2, respectively. Fisher's exact test demonstrated no significant associations between the positivity for EZH2 and clinicopathological characteristics. No significant differences were observed in recurrence-free and overall survivals between EZH2-negative/low and EZH2-high patients., Conclusion: EZH2 was frequently observed in patients with resected SCLC, but no significant associations were found between its expression and the clinicopathological characteristics and postoperative survivals., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Polyphyllin I (PPI), one of the steroidal saponins in paris polyphylla, has been reported to exhibit antitumor effects. However, the detailed molecular mechanism underlying this has not been elucidated., Methods: Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Flow cytometry assays, respectively. Cell invasion and migration were examined by Transwell invasion and wound healing assays. Western blot analysis was performed to examine the protein expressions of zeste homolog 2 (EZH2), DNA methyltransferase 1 (DNMT1). QRT-PCR was used to examine the levels of long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR). Small interfering RNAs (siRNAs) method was used to knockdown HOTAIR. Exogenously expressions of HOTAIR, DNMT1 and EZH2 were carried out by Transient transfection assays. EZH2 promoter activity was measured by Secrete-Pair Dual Luminescence Assay Kit. A nude mice xenograft model was used to confirm the findings in vitro., Results: We showed that PPI significantly inhibited growth, induced cell cycle arrest of castration-resistant prostate cancer (CRPC) cells. In addition, PPI also reduced the migration and invasion in CRPC cells. In mechanism, we found that PPI decreased the protein expressions of EZH2, DNMT1 and levels of HOTAIR. Interestingly, silenced HOTAIR reduced EZH2 and DNMT1 protein expressions. On the contrary, exogenously expressed HOTAIR resisted PPI-inhibited EZH2 and DNMT1 protein expressions, EZH2 promoter activity and cell growth. Moreover, excessive EZH2 antagonized PPI-suppressed DNMT1 protein expression or vice versa. Consistent with this, PPI inhibited tumor growth, HOTAIR, the protein expressions of DNMT1 and EZH2 in vivo., Conclusion: Our results show that PPI inhibits growth of CRPC cells through inhibition of HOTAIR expression, subsequently; this results in the repression of DNMT1 and EZH2 expressions. The interactions among HOTAIR, DNMT1 and EZH2, and reciprocal regulation of DNMT1 and EZH2 contribute to the overall responses of PPI. This study reveals a novel mechanism for HOTAIR-mediated regulating DNMT1 and EZH2 in response to PPI in inhibition of the growth of CRPC cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Polycomb repressive complex 2 (PRC2; formed by EZH2, SUZ12, and EED protein subunits) and PRC1 (BMI1 protein) induce gene silencing through histone modification by H3K27me3. In the present study, we characterized the PRC expression pattern and its clinical implication in sarcoma., Methods: Using immunohistochemistry, we analyzed PRC expression in 105 sarcoma patients with 5 subtypes: synovial sarcoma (n = 18), rhabdomyosarcoma (n = 28), Ewing sarcoma (n = 15), osteosarcoma (n = 30), and others (n = 14)., Results: The median age at diagnosis in the patient cohort was 26.8 years (range: 1-78 years) and the male-to-female ratio was 1:4. Initial disease presentation was locoregional disease in 83% of patients and initial metastatic disease in the remaining 17%. PRC expression was not significantly different according to histologic subtype (P = 0.400). Overall survival (OS) was significantly poor for SUZ12 high (P = 0.001), EED1 high (P = 0.279), and H3K27me3 high (P = 0.009). Ultimately, patients with PRC2 high had significantly inferior OS than the no expression group (P = 0.009). In the Cox proportional hazard model adjusted for stage, histologic grade, surgery, margin and initial metastasis, SUZ12 expression (P = 0.020, HR 29.069, 95% CI 1.690-500.007), H3K27me3 (P = 0.010, HR 3.743, 95% CI 1.370-10.228) expression was significantly associated with shorter OS., Conclusion: We detected PRC expression in various sarcomas and demonstrated its independent negative prognostic role, suggesting the PRC axis as promising therapeutic target for treating sarcoma.

Background/aim: Enhancer of zeste homolog 2 (EZH2) is a member of the polycomb group of genes, which are key factors in the regulation of cell proliferation and differentiation. EZH2 is overexpressed in many malignancies. We analyzed EZH2 protein expression levels in different histological subtypes of thyroid cancer to examine its utility as a prognostic factor., Materials and Methods: We examined EZH2 protein expression by immunohistochemistry in tissue samples from 67 cases of poorly differentiated (PDTC) and 48 cases of anaplastic thyroid carcinoma (ATC), and in samples of adjacent normal and differentiated thyroid carcinoma (DTC). We examined differences in expression of EZH2 among various histological types of thyroid cancer, and the relationship between EZH2 expression and patient outcome., Results: EZH2 protein was expressed in PDTC and ATC, but not in normal thyroid gland or DTC. EZH-positivity increased in the order of DTC, PDTC, and ATC (p<0.01). Higher EZH2 expression correlated with poorer survival in PDTC (p=0.004), and a similar but non-significant trend was observed in ATC (p=0.166). Multivariate analysis identified EZH2 as an independent prognostic factor similar to metastatic status in the Japanese Society of Thyroid Surgery (JSTS) classification of PDTC., Conclusion: EZH2 overexpression is associated with malignant potential in thyroid cancer, and may thus be a useful prognostic marker of aggressive thyroid cancer., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objective: Cervical cancer has an increasing incidence in developing countries with a predominance of squamous cell carcinoma. In this work, we aimed to analyze the role of EZH2, Endothelin-1, and CD34 as indicators of the aggressiveness in cervical squamous cell carcinoma., Material and Method: Immunohistochemical expression of EZH2, Endothelin-1, and CD34 was studied in 54 paraffin-embedded tissue specimens of cervical squamous cell carcinoma. Their correlation to the clinicopathologic features and the potential angiogenic role were analyzed., Results: High EZH2 expression was noted in 78% of cervical squamous cell carcinoma with a significant relation with tumor grade, FIGO stage and lymph node metastasis (p= < 0.001, p=0.007, p=0.03 respectively). Endothelin-1 overexpression was detected in 63% of the studied cases with a significant association with tumor size, FIGO stage and lymph node metastasis (p=0.009, p=0.002, p=0.02 respectively). High CD34 expression (MVD) was noted in 56% of the cases and associated with the tumor size, FIGO stage and lymph node metastasis (p < 0.001, p < 0.001, p=0.04 respectively). The three markers were significantly associated (p < 0.05)., Conclusion: EZH2, ET-1, and CD34 may act as biomarkers of aggressive cervical squamous cell carcinoma. They may contribute to the signaling pathway of angiogenesis. Therefore, they could potentially be used in targeted therapy.

Synovial sarcoma (SS) is a type of soft-tissue sarcoma, often linked to poor survival. Although overexpression of enhancer of zeste homologue 2 (EZH2) has been associated with poor prognosis in different tumors, a few studies investigated this link in SS. Here, we analyzed the relationship between EZH2 expression and prognostic factors in SS. We included 29 patients with SS. Immunostaining of EZH2 was performed with (D2C9) XPTM Rabbit mAb antibody, and the results were classified as low EZH2 expression (negative or weak expression) and high EZH2 expression category (moderate or strong expression). Analysis of survival in relation to prognostic factors was performed with Kaplan-Meier survival curves and Cox proportional hazard regression analysis. Our sample included 19/29 female and 10/29 male patients, with age range 16-63 years. The tumor diameter ranged from 2 to 15 cm. Necrosis was observed in 15/29 cases. Sixteen cases had >10 mitoses per 50 high-power fields (HPFs). Out of 29 cases, 14 showed low and 15 had high EZH2 expression. Statistically significant results were obtained for the association between the presence of metastasis and necrosis (p = 0.042), high EZH2 expression and distant metastasis (p = 0.018), high EZH2 expression and necrosis (p = 0.016), and high EZH2 expression and the tumor size >5 cm versus tumor size ≤5 cm (p = 0.014). Patients with all of the following: the tumor size ≤5 cm, low EZH2 expression, and without necrosis and distant metastasis had significantly longer survival time. Our results are consistent with previous studies, suggesting that EZH2 overexpression is an indicator of poor prognosis in SS.