Increased EZH2 Expression Research Articles

Polyploid Giant Cancer Cells: A Distinctive Feature in the Transformation of Epithelial Cells by High-Risk Oncogenic HCMV Strains.

Human cytomegalovirus (HCMV) infection is common in tumor tissues across different types of cancer. While HCMV has not been recognized as a cancer-causing virus, numerous studies hint at its potential role in cancer development where its presence in various cancers corresponds with the hallmarks of cancer. Herein, we discuss and demonstrate that high-risk HCMV-DB and BL strains have the potential to trigger transformation in epithelial cells, including human mammary epithelial cells (HMECs), ovarian epithelial cells (OECs), and prostate epithelial cells (PECs), through the generation of polyploid giant cancer cells (PGCCs). A discussion is provided on how HCMV infection creates a cellular environment that promotes oncogenesis, supporting the continuous growth of CMV-transformed cells. The aforementioned transformed cells, named CTH, CTO, and CTP cells, underwent giant cell cycling with PGCC generation parallel to dedifferentiation, displaying stem-like characteristics and an epithelial-mesenchymal transition (EMT) phenotype. Furthermore, we propose that giant cell cycling through PGCCs, increased EZH2 expression, EMT, and the acquisition of malignant traits represent a deleterious response to the cellular stress induced by high-risk oncogenic HCMV strains, the latter being the origin of the transformation process in epithelial cells upon HCMV infection and leading to adenocarcinoma of poor prognosis.

Potential mechanism of Luoshi Neiyi prescription in endometriosis based on serum pharmacochemistry and network pharmacology.

Endometriosis (EMs) is characterized by ectopic growth of active endometrial tissue outside the uterus. The Luoshi Neiyi prescription (LSNYP) has been extensively used for treating EMs in China. However, data on the active chemical components of LSNYP are insufficient, and its pharmacological mechanism in EMs treatment remains unclear. This study aimed to explore the potential mechanism of LSNYP for EMs through network pharmacology based on the components absorbed into the blood. Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used to analyze blood components, and a series of network pharmacology strategies were utilized to predict targets of these components and EMs. Protein-protein interaction (PPI) network analysis, component-target-disease network construction, gene ontology (GO) functional enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. Additionally, molecular docking, molecular dynamics simulations, and in vitro and in vivo experiments were conducted to validate the HIF1A/EZH2/ANTXR2 pathway associated with hypoxic pathology in EMs. Thirty-four absorbed components suitable for network pharmacology analysis were identified, and core targets, such as interleukin 6, EGFR, HIF1A, and EZH2, were founded. Enrichment results indicated that treatment of EMs with LSNYP may involve the regulation of hypoxia and inflammatory-related signaling pathways and response to oxidative stress and transcription factor activity. Experimental results demonstrated that LSNYP could decrease the expression of HIF1A, ANTXR2, YAP1, CD44, and β-catenin, and increased EZH2 expression in ectopic endometrial stromal cells and endometriotic tissues. Molecular docking and molecular dynamics simulations manifested that there was stable combinatorial activity between core components and key targets of the HIF1A/EZH2/ANTXR2 pathway. LSNYP may exert pharmacological effects on EMs via the HIF1A/EZH2/ANTXR2 pathway; hence, it is a natural herb-related therapy for EMs.

HSP90-Dependent Upregulation of EZH2 Promotes Hypoxia/Reoxygenation-Induced Pyroptosis by Inhibiting miR-22 in Endothelial Cells.

Endothelial cell pyroptosis induced by hypoxia/reoxygenation (H/R) plays a key role in the pathogenesis of myocardial infarction (MI). However, the underlying mechanism is not clearly elucidated. Human umbilical vein endothelial cells (HUVECs) exposed to H/R acted as in vitro model to investigate the mechanism of H/R-induced endothelial cell pyroptosis. CCK-8 assays were performed to investigate the viability of HUVECs. Calcein-AM/PI staining was carried out to quantify the death of HUVECs. The expression level of miR-22 was measured by RT-qPCR. The protein expression levels of zeste 2 polycomb repressive complex 2 subunit (EZH2), NLRP3, cleaved caspase-1 (c-caspase-1), GSDMD-N and heat shock protein 90 (HSP90) were measured by Western blot. Levels of IL-1β and IL-18 in culture medium were detected by ELISA. The intracellular localization of EZH2 was detected by immunofluorescence staining. Chromatin immunoprecipitation (ChIP) assay was used to detect the enrichment of EZH2 and H3K27me3 in the miR-22 promoter region. The binding between miR-22 and NLRP3 in HUVECs was confirmed by the dual luciferase assay. Reciprocal coimmunoprecipitation was conducted to detect the direct interaction between HSP90 and EZH2. H/R increased EZH2 expression, and the EZH2 siRNA could inhibit H/R-induced pyroptosis in HUVECs. H/R reduced miR-22 expression, which was reversed by EZH2 siRNA. Silencing of miR-22 by its inhibitor reversed EZH2 siRNA-induced pyroptosis inhibition in H/R-exposed HUVECs. Upregulation of miR-22 by its mimic suppressed EZH2 overexpression-enhanced pyroptosis in H/R-exposed HUVECs. ChIP assay confirmed that EZH2 bound to the miR-22 promoter region and repressed miR-22 expression through H3K27me3. Furthermore, luciferase reporter assay indicated that NLRP3 was a direct target of miR- 22 in HUVECs. Finally, HSP90 siRNA inhibited H/R-induced EZH2 expression, miR-22 downregulation, and pyroptosis in HUVECs. H/R induces pyroptosis via the HSP90/EZH2/miR-22/NLRP3 signaling axis in endothelial cells.

Ezh2 Knockout in B Cells Impairs Plasmablast Differentiation and Ameliorates Lupus-like Disease in MRL/lpr Mice.

EZH2 regulates B cell development and differentiation. We previously demonstrated increased EZH2 expression in peripheral blood mononuclear cells from lupus patients. The goal of this study was to evaluate the role of EZH2 expression in B cells in the pathogenesis of lupus. We generated an MRL/lpr mouse with floxed Ezh2, which was crossed with CD19-Cre mice to examine the effect of B cell EZH2 deficiency in MRL/lpr lupus-prone mice. Differentiation of B cells was assessed using flow cytometry. Single-cell RNA sequencing and single-cell B cell receptor sequencing were performed. In vitro B cell culture with an X-box binding protein 1 (XBP1) inhibitor was performed. EZH2 and XBP1 messenger RNA levels in CD19+ B cells isolated from lupus patients and healthy controls were analyzed. We show that Ezh2 deletion in B cells significantly decreased autoantibody production and improved glomerulonephritis. B cell development was altered in the bone marrow and spleen of EZH2-deficient mice. Differentiation of germinal center B cells and plasmablasts was impaired. Single-cell RNA sequencing showed that XBP1, a key transcription factor in B cell development, is down-regulated in the absence of EZH2. Inhibiting XBP1 in vitro impairs plasmablast development similar to EZH2 deficiency in mice. Single-cell B cell receptor RNA sequencing revealed defective immunoglobulin class-switch recombination in EZH2-deficient mice. In human lupus B cells, we observed a strong correlation between EZH2 and XBP1 messenger RNA expression levels. EZH2 overexpression in B cells contributes to disease pathogenesis in lupus.

Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer. It is characterized by the loss of androgen receptor (AR) signaling in neuroendocrine transdifferentiation, and finally, resistance to AR-targeted therapy. With the application of a new generation of potent AR inhibitors, the incidence of NEPC is gradually increasing. The molecular mechanism of neuroendocrine differentiation (NED) after androgen deprivation therapy (ADT) remains largely unclear. In this study, using NEPC-related genome sequencing database analyses, we screened RACGAP1, a common differentially expressed gene. We investigated RACGAP1 expression in clinical prostate cancer specimens by IHC. Regulated pathways were examined by Western blotting, qRT-PCR, luciferase reporter, chromatin immunoprecipitation, and immunoprecipitation assays. The corresponding function of RACGAP1 in prostate cancer was analyzed by CCK-8 and Transwell assays. The changes of neuroendocrine markers and AR expression in C4-2-R and C4-2B-R cells were detected in vitro. We confirmed that RACGAP1 contributed to NE transdifferentiation of prostate cancer. Patients with high tumor RACGAP1 expression had shorter relapse-free survival time. The expression of RACGAP1 was induced by E2F1. RACGAP1 promoted neuroendocrine transdifferentiation of prostate cancer by stabilizing EZH2 expression in the ubiquitin-proteasome pathway. Moreover, overexpression of RACGAP1 promoted enzalutamide resistance of castration-resistant prostate cancer (CRPC) cells. Our results showed that the upregulation of RACGAP1 by E2F1 increased EZH2 expression, which drove NEPC progression. This study explored the molecular mechanism of NED and may provide novel methods and ideas for targeted therapy of NEPC.

Role of hippocampal circKcnk9 in visceral hypersensitivity and anxiety comorbidity of irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by recurrent visceral pain and altered bowel habits (diarrhea or constipation). However, the molecular and pathological mechanisms are poorly understood. This study found neonatal colorectal distension to induce visceral hypersensitivity and anxiety. The expression of hippocampal circKcnk9, a novel circRNA, was significantly increased in IBS-like rats. Interestingly, CA1 shcircKcnk9 treatment inhibited long-term potentiation (LTP) and alleviated visceral hypersensitivity and anxiety in IBS-like rats, whereas overexpression of CA1 circKcnk9 induced LTP, visceral hypersensitivity, and anxiety in controls. Several experiments indicated that increased CA1 circKcnk9 acted as a miR-124-3p sponge, which resulted in the inhibitory effect of miR-124-3p on gene silencing. There was a negative correlation between circKcnk9 and miR-124-3p expression. As expected, CA1 administration of agomiR-124-3p decreased CA1 LTP, visceral hypersensitivity, and anxiety in the IBS-like rats. In contrast, CA1 treatment with antagomiR-124-3p induced LTP, visceral hypersensitivity, and anxiety in the controls. Furthermore, bioinformatic analysis and experimental data showed that EZH2 is a circKcnk9/miR-124-3p target gene, and increased EZH2 expression was involved in visceral hypersensitivity and anxiety in IBS-like rats by enhancing hippocampal synaptic plasticity. In conclusion, early life stress induces increased expression of circKcnk9 in the CA1 of IBS-like rats. Increased circKcnk9 expression regulates synaptic transmission and enhances LTP, leading to visceral hypersensitivity and anxiety in IBS-like rats. The underlying circKcnk9 signaling pathway is miR124-3p/EZH2. Increased circKcnk9 reinforces its sponging of miR124-3p and strongly suppresses miR124-3p activity, resulting in increased expression of the target gene EZH2. This study provides a new epigenetic mechanism for visceral hypersensitivity and anxiety in IBS-like rats.

CAIRE: A basket multicenter open-label phase 2 study evaluating the EZH2 inhibitor tazemetostat in combination with durvalumab in patients with advanced solid tumors.

TPS2703 Background: Recent studies have shown shown that genetic depletion of EZH2 in Tregs (using FoxP3creEZH2fl/fl mice) and pharmacological inhibition of EZH2 elicits phenotypic and functional alterations of Tregs, leading to an effector-like T cell profile. Further, EZH2 inhibition enhances the cytotoxicity of human Teffs in vitro and the proportion of tumor-infiltrating cytotoxic T cells in vivo in murine models. It has been shown that immune checkpoint inhibition (ICI) increases EZH2 expression in human T cells across various tumor types and that increased EZH2 expression in T cells inversely correlate with clinical outcome. Upregulation of EZH2 mediated by immune checkpoint inhibition in T cells modulates T cell responses and diminishes the effectiveness of immunotherapy. Consistent with this mechanism, pharmacologic inhibition of EZH2 has been shown to increase effector-like T cell responses and enhances effectiveness of immune checkpoint therapy in tumor-bearing mice [16-17]. Our group also reported first clinical data from a participant with sarcoma showing strong T cell infiltration after treatment with tazemetostat (Italiano et al Lancet Oncol 2018). Altogether, these findings pave the way for clinical trials combining ICI with the specific EZH2 inhibitor, tazemetostat in solid tumors. Methods: CAIRE (NCT04705818) is a multi-cohort, four single-arm phase 2, multicenter, open-label study investigating tazemetostat combined with durvalumab in patients with advanced cancers: pancreatic adenocarcinoma (cohort A), microsatellite stable colorectal cancer (cohort B), solid tumors with presence of tertiary lymphoid structures (assessed centrally) (cohort C), soft-tissue sarcomas (cohort D). Cohort A will enroll ̃32 patients and cohorts B, C and D ̃47 patients respectively. Eligible patients must have no standard treatment options available or a contraindication to standard treatment options, and ECOG PS 0–1. Patients with prior exposure to EZH2 inhibitor and/or PD1/PDL1 monoclonal antibodies are excluded. Tazemetostat will be administered per-os, twice daily (800 mg x 2), continuously. Treatment by Tazemetostat will start on Day 1 (of cycle 1). Durvalumab will be administered by intravenous infusion (1120 mg) on day 1, every 3 weeks. Treatment by Durvalumab will start on Day 1 of cycle 2. The primary endpoints are disease control rate within 24 weeks of treatment as per RECIST 1.1 in cohort A and B; objective response rate within 24 weeks of treatment as per RECIST 1.1 in cohort C, and 6-months non progression rate for cohort D. Secondary endpoints include adverse events (AEs)/serious AEs, duration of response, and progression-free survival. Pharmacodynamic and other biomarkers will be explored. The first patient received study drug on July, 30, 2022 and 1 site across France are currently enrolling patients. Clinical trial information: NCT04705818.

Exosomal circPVT1 derived from lung cancer promotes the progression of lung cancer by targeting miR-124-3p/EZH2 axis and regulating macrophage polarization

ABSTRACT This study aims to probe the mechanism by which circPVT1 in lung cancer (LC)-derived exosomes (Exos) promotes proliferation, invasion and migration of LC cells by regulating macrophage polarization through miR-124-3p/EZH2 axis. The expressions of circPVT1 in blood-derived Exos extracted from lung adenocarcinoma (LA) patients were measured. Loss or gain-of-function experiments of circPVT1 and miR-124-3p were carried out to evaluate the effects of circPVT1 in LC-derived Exos and miR-124-3p on macrophage polarization toward M2 phenotype, whichi found that incubation of Exo-A with macrophages induced macrophage polarization to M2 type and M2-polarized macrophages co-incubated with A549 cells enhanced the biological function of LC cells. Co-incubation with M+ Exo-A-oecircPVT1 increased the proliferation, migration and invasion abilities of LC cells, while coculture with M+ Exo-A-si-circPVT1 reversed these abilities. The verification among circPVT1, miR-124-3p and EZH2 showed that miR-124-3p was negatively related to circPVT1 and EZH2, and EZH2 was positively related to circPVT1. CircPVT1 in LC-derived Exos increased EZH2 expression through inhibiting miR-124-3p expression level in macrophage. Taken togther, exosomal circPVT1 derived from LC mediates macrophage polarization via the miR-124-3p/EZH2 axis to potentiate LC cells’ proliferation, invasion and migration.

Intraductal prostate cancer: An aggressive subset of prostate cancers? Immunophenotypic evaluation.

Introduction:The presence of intraductal prostate cancer in a sample is often associated with large tumor volume, an advanced stage of the disease, a high Gleason score and an increased risk of recurrence, and resistance to androgen suppression and chemotherapy, which are also correlated with reduced progression-free survival and with postoperative, biochemical relapse.Methods:The aim of our study was to investigate whether carbonic anhydrase IX (CA IX) is upregulated in prostate cancer and to investigate ERG and EZH2 as potential markers for cancer aggression in aggressive acinar disease with intraductal component prostate cancer. The series consisted of 79 cases of prostate cancer. Immunohistochemical staining was performed for EZH2 ERG and CA IX.Results:The results of this study underline the fact that EZH2 protein expression is a powerful predictor of PSA relapse in prostate cancer and that this effect is stronger in ERG-positive cancers than in ERG-negative cancers. Evident EZH2 nuclear expression was found in prostatic tumor, proposing increased EZH2 expression important for the spread of prostate cancer.Conclusions:The relationship to tumor phenotype and prognosis was more considerable in ERG-positive tumors than in ERG-negative tumors. EZH2 has gained great interest as a target for epigenetic cancer therapy. Although prostate cancer is a hypoxic tumor, it does not express CA IX and cannot be used as an endogenous marker for hypoxia.

Increased prostaglandin-D2 in male STAT3-deficient hearts shifts cardiac progenitor cells from endothelial to white adipocyte differentiation.

Cardiac levels of the signal transducer and activator of transcription factor-3 (STAT3) decline with age, and male but not female mice with a cardiomyocyte-specific STAT3 deficiency conditional knockout (CKO) display premature age-related heart failure associated with reduced cardiac capillary density. In the present study, isolated male and female CKO-cardiomyocytes exhibit increased prostaglandin (PG)-generating cyclooxygenase-2 (COX-2) expression. The PG-degrading hydroxyprostaglandin-dehydrogenase-15 (HPGD) expression is only reduced in male cardiomyocytes, which is associated with increased prostaglandin D2 (PGD2) secretion from isolated male but not female CKO-cardiomyocytes. Reduced HPGD expression in male cardiomyocytes derive from impaired androgen receptor (AR)-signaling due to loss of its cofactor STAT3. Elevated PGD2 secretion in males is associated with increased white adipocyte accumulation in aged male but not female hearts. Adipocyte differentiation is enhanced in isolated stem cell antigen-1 (SCA-1)+ cardiac progenitor cells (CPC) from young male CKO-mice compared with the adipocyte differentiation of male wild-type (WT)-CPC and CPC isolated from female mice. Epigenetic analysis in freshly isolated male CKO-CPC display hypermethylation in pro-angiogenic genes (Fgfr2, Epas1) and hypomethylation in the white adipocyte differentiation gene Zfp423 associated with up-regulated ZFP423 expression and a shift from endothelial to white adipocyte differentiation compared with WT-CPC. The expression of the histone-methyltransferase EZH2 is reduced in male CKO-CPC compared with male WT-CPC, whereas no differences in the EZH2 expression in female CPC were observed. Clonally expanded CPC can differentiate into endothelial cells or into adipocytes depending on the differentiation conditions. ZFP423 overexpression is sufficient to induce white adipocyte differentiation of clonal CPC. In isolated WT-CPC, PGD2 stimulation reduces the expression of EZH2, thereby up-regulating ZFP423 expression and promoting white adipocyte differentiation. The treatment of young male CKO mice with the COX inhibitor Ibuprofen or the PGD2 receptor (DP)2 receptor antagonist BAY-u 3405 in vivo increased EZH2 expression and reduced ZFP423 expression and adipocyte differentiation in CKO-CPC. Thus, cardiomyocyte STAT3 deficiency leads to age-related and sex-specific cardiac remodeling and failure in part due to sex-specific alterations in PGD2 secretion and subsequent epigenetic impairment of the differentiation potential of CPC. Causally involved is the impaired AR signaling in absence of STAT3, which reduces the expression of the PG-degrading enzyme HPGD.

Cross-tolerance: embryonic heat conditioning induces inflammatory resilience by affecting different layers of epigenetic mechanisms regulating IL6 expression later in life

ABSTRACT A stressor can induce resilience in another, different stressor, a phenomenon known as cross-tolerance. To learn if cross-tolerance is governed by epigenetic regulation, we used embryonic heat conditioning (EHC) in chicks, during the development of the hypothalamus, to increase the immunization response. Indeed, EHC induced a lifelong systemic antibody response to immunization, in addition to reduced hypothalamic IL6 inflammatory expression following LPS challenge. Since the outcome of EHC was long-term cross-tolerance with the immune system, we studied possible epigenetic mechanisms. We first analysed the methylation and hydroxymethylation patterns of IL6. We found reduced hydroxymethylation on IL6 intron 1 in the EHC group, a segment enriched with CpGs and NFkB-binding sites. Luciferase assay in cell lines expressing NFkB showed that IL6 intron 1 is indeed an enhancer. ChiP in the same segment against NFkB in the hypothalamus presented reduced binding to IL6 intron 1 in the EHC group, before and during LPS challenge. In parallel, EHC chicks’ IL6 intron 1 presented increased H3K27me3, a repressive translational modification mediated by EZH2. This histone modification occurred during embryonic conditioning and persisted later in life. Moreover, we showed reduced expression of miR-26a, which inhibits EZH2 transcription, during conditioning along with increased EZH2 expression. We demonstrate that stress cross-tolerance, which was indicated by EHC-induced inflammatory resilience and displayed by attenuated inflammatory expression of IL6, is regulated by different epigenetic layers.

Increased expression of EZH2 is mediated by higher glycolysis and mTORC1 activation in lupus CD4+ T cells

Abstract Objective EZH2 is overexpressed in lupus CD4+ T cells. Increased disease activity in lupus patients is associated with a pro-inflammatory epigenetic shift in naïve CD4+ T cells, likely mediated by EZH2. Here we aim to understand the upstream mechanisms underlying EZH2 overexpression in lupus CD4+ T cells. Methods Naïve CD4+ T cells were isolated from lupus patients and then stimulated with anti-CD3/anti-CD28. qPCR and Western blotting were used to measure mRNA and protein expression levels, respectively. 2-Deoxy-D-glucose (2-DG) was used to inhibit glycolysis. mTORC1 signaling was inhibited using rapamycin. Oxidative stress was induced by H2O2. Results Because glycolysis is increased in lupus CD4+ T cells and glycolysis regulates miR-101 and miR-26a, which target EZH2, we examined the effect of inhibiting glycolysis on EZH2 expression. 2-DG significantly inhibited EZH2 expression in lupus CD4+ T cells. In addition, 2-DG restored the expression of miR-101 and miR-26a, suggesting that suppression of EZH2 by 2-DG might be mediated by miR-101 and miR-26a. Because mTORC1 is activated in lupus CD4+ T cells in part due to increased oxidative stress, and mTORC1 activation increases glycolysis, we hypothesized that mTORC1 mediates increased EZH2 expression. Indeed, inhibiting mTORC1 increased miR-101 and miR-26a and suppressed EZH2 expression in lupus CD4+ T cells. Further, H2O2 treatment increased EZH2 expression, however, this effect appears to be independent of miR-101 and miR-26a. Conclusion Increased EZH2 is mediated by activation of mTORC1 and increased glycolysis in lupus CD4+ T cells. Therapeutic effects from mTORC1 inhibitors and glycolysis inhibitors in lupus might be in part mediated by suppression of EZH2.

Sirtuins' Deregulation in Bladder Cancer: SIRT7 Is Implicated in Tumor Progression through Epithelial to Mesenchymal Transition Promotion.

Sirtuins are emerging players in cancer biology and other age-related disorders, and their putative role in bladder cancer (BlCa) remains elusive. Further understanding of disease biology may allow for generation of more effective pathway-based biomarkers and targeted therapies. Herein, we aimed to illuminate the role of sirtuins’ family in BlCa and evaluate their potential as disease biomarkers and therapeutic targets. SIRT1-7 transcripts and protein levels were evaluated in a series of primary BlCa and normal bladder mucosa tissues. SIRT7 knockdown was performed through lentiviral transduction in MGHU3, 5637 and J82 cells and its functional role was assessed. SIRT1, 2, 4 and 5 expression levels were significantly lower in BlCa, whereas SIRT6 and 7 were overexpressed, and these results were corroborated by TCGA cohort analysis. SIRT7 transcript levels were significantly decreased in muscle-invasive vs. papillary BlCa. In vitro studies showed that SIRT7 downregulation promoted cells migration and invasion. Accordingly, increased EMT markers expression and decreased E-Cadherin (CDH1) was observed in those BlCa cells. Moreover, increased EZH2 expression and H3K27me3 deposition in E-Cadherin promoter was found in sh-SIRT7 cells. We demonstrated that sirtuins are globally deregulated in BlCa, and specifically SIRT7 downregulation is implicated in EMT, fostering BlCa invasiveness through EZH2-CDH1 axis.

Application of immunohistochemistry in diagnosis and management of malignant mesothelioma.

Immunohistochemistry plays an indispensable role in accurate diagnosis of malignant mesothelioma, particularly in morphologically challenging cases and in biopsy and cytology specimens, where tumor architecture is difficult or impossible to evaluate. Application of a targeted panel of mesothelial- and epithelial-specific markers permits correct identification of tumor lineage in the vast majority of cases. An immunopanel including two mesothelial markers (calretinin, CK5/6, WT-1, or D2-40) and two epithelial markers (MOC-31 and claudin-4) offers good sensitivity and specificity, with adjustments as appropriate for the differential diagnosis. Once mesothelial lineage is established, malignancy-specific studies can help verify a diagnosis of malignant mesothelioma. BAP1 loss, CDKN2A homozygous deletion, and MTAP loss are highly specific markers of malignancy in a mesothelial lesion, and they attain acceptable diagnostic sensitivity when applied as a diagnostic panel. Novel markers of malignancy, such as 5-hmC loss and increased EZH2 expression, are promising, but have not yet achieved widespread clinical adoption. Some diagnostic markers also have prognostic significance, and PD-L1 immunohistochemistry may predict tumor response to immunotherapy. Application and interpretation of these immnuomarkers should always be guided by clinical history, radiographic findings, and above all histomorphology.

Increased Expression of EZH2 Is Mediated by Higher Glycolysis and mTORC1 Activation in Lupus CD4+ T Cells.

Objective:EZH2 is overexpressed in CD4+ T cells from patients with systemic lupus erythematosus (SLE). Increased disease activity in SLE patients is associated with a proinflammatory epigenetic shift in naïve CD4+ T cells, likely mediated by EZH2. Here we aim to understand the upstream mechanisms underlying EZH2 overexpression in SLE CD4+ T cells.Methods:Naïve CD4+ T cells were isolated from SLE patients and then stimulated with anti-CD3/anti-CD28. qPCR and Western blotting were used to measure mRNA and protein expression levels, respectively. 2-Deoxy-d-glucose (2-DG) was used to inhibit glycolysis. mTORC1 signaling was inhibited using rapamycin. Oxidative stress was induced by H2O2.Results:Because glycolysis is increased in SLE CD4+ T cells and glycolysis regulates miR-26a and miR-101, which target EZH2, we examined the effect of inhibiting glycolysis on EZH2 expression. 2-DG significantly inhibited EZH2 expression in SLE CD4+ T cells. In addition, 2-DG restored the expression of miR-26a and miR-101, suggesting that suppression of EZH2 by 2-DG occurs at the post-transcriptional level. Because mTORC1 is activated in SLE CD4+ T cells in part due to increased oxidative stress, and mTORC1 activation increases glycolysis, we hypothesized that mTORC1 mediates increased EZH2 expression. Indeed, inhibiting mTORC1 increased miR-26a and miR-101 and suppressed EZH2 expression in SLE CD4+ T cells. Further, H2O2 treatment increased EZH2 expression, however, this effect appears to be independent of miR-26a and miR-101.Conclusion:Increased EZH2 is mediated by activation of mTORC1 and increased glycolysis in SLE CD4+ T cells. Therapeutic effects from inhibiting mTOR or glycolysis in SLE might be in part mediated by suppression of EZH2.

Circular RNA circ-PRMT5 facilitates non-small cell lung cancer proliferation through upregulating EZH2 via sponging miR-377/382/498

Emerging evidence demonstrates that circular RNA (circRNA) is a novel class of non-coding RNA that plays a pivotal role in cancer. Recently, circ-PRMT5 was identified as an oncogene in bladder cancer. Nevertheless, its contribution to non-small cell lung cancer (NSCLC) is unknown. Herein, we aimed to clarify the biological role of circ-PRMT5 in NSCLC. High circ-PRMT5 expression was identified in NSCLC tissues and cell lines and positively correlated with larger tumor size, advanced clinic stage, lymph node metastasis as well as worse prognosis. Stable knockdown of circ-PRMT5 dramatically weakened the proliferative capacities of NSCLC cells both in vitro and in vivo. Mechanically, circ-PRMT5 could simultaneously effectively sponge three miRNAs (miR-377, miR-382 and miR-498) and alleviate their repression on the well-known oncogenic EZH2, resulting in increased EZH2 expression, thereby facilitating NSCLC progression. Importantly, a strong positive correlation between circ-PRMT5 and EZH2 expression was observed in NSCLC tissues. Overall, our data indicate that circ-PRMT5 is an oncogenic circRNA in NSCLC that can promote the growth of NSCLC via regulation of miR-377/382/498-EZH2 axis.

Long noncoding RNA SNHG14 facilitates colorectal cancer metastasis through targeting EZH2-regulated EPHA7

Accumulating evidence suggested the participation of long noncoding RNAs (lncRNAs) in regulating various biological processes so as to affecting cancer progression. However, the functional role of most lncRNAs in colorectal carcer (CRC) is still largely covered. In the present study, we disclosed SNHG14 as a carcinogene in CRC development, as it was low-expressed in normal colon tissues but markedly upregulated in CRC cell lines. Besides, SNHG14 contributed to CRC cell proliferation, motility and EMT in vitro, and inhibition of it confined CRC tumor growth and liver metastasis in vivo. Next, the mechanistic investigations confirmed that SNHG14-promoted CRC progression was mediated by EPHA7, which was negatively regulated by SNHG14 in CRC via an EZH2-dependent way. Importantly, EZH2 was proved as a transcription factor of EPHA7 and functioned as a repressor in EPHA7 transcription by enhancing methylation on EPHA7 promoter. Meanwhile, SNHG14 increased EZH2 expression in CRC via stabilizing its mRNA by interacting with FUS, and via freeing its mRNA from miR-186-5p-induced silence. All in all, our observations demonstrated that SNHG14 serves as a facilitator in CRC through targeting EZH2-repressed EPHA7 by enhancing EZH2 via recruiting FUS and absorbing miR-186-5p, indicating a promising new road for CRC diagnosis and treatment.

Epigenomic Regulation of SATB2 Links Maternal Obesity to Impaired Osteoblast Differentiation (P11-034-19)

ObjectivesNutritional status during intrauterine and/or early postnatal life has substantial influences on adult offspring health. However, evidence on the impact of high fat diet (HFD)-induced maternal obesity on regulation of fetal bone development is sparse. Thus, we investigated the effects of maternal obesity in rodent and human cells on epigenetic regulation of osteoblast differentiation. MethodsFirst, female Sprague-Dawley rats were fed either a low-fat AIN-93G control diet or a high fat diet (HFD) (45% fat calories) for 10 wk starting at 6 wk of age. Lean (from control diet) and obese (from HFD) female rats were then time-impregnated (n = 6 per group) by control diet fed male rats. At gestational day 18.5 (E18.5), all fetuses were taken and embryonic osteogenic calvarial cells (EOCCs) were isolated. Second, human osteo-progenitors of mesenchymal stem cells were isolated from umbilical cord following delivery from pregnant mothers. ResultsWe found epigenetic regulation of polycomb-regulated gene Ezh2 (Enhancer of zeste homolog 2) in embryonic rats from HFD obese rat dams. Increased enrichment of repressive histone mark H3K27me3 on the gene body of SATB2 (ChIP Seq analysis) was associated with aberrant differentiation of EOCCs to mature osteoblasts. Knocking down Ezh2 in EOCCs and ST2 cells increased SATB2 expression; on the other hand, Ezh2 overexpression in EOCCs and ST2 cells decreased SATB2 expression. These data were consistent with ChIP experimental results showing strong association between H3K27me3, Ezh2 and SATB2. Second, human mesenchymal stem cells (MSCs) from umbilical cord (UC) were isolated following delivery from obese/overweight (pre-pregnancy BMI ≥ 25 kg/m2) and control (pre-pregnancy BMI between 19–25 kg/m2) pregnant mothers. We found: 1) UC-MSCs from pregnant obese/overweight mothers showed increased Ezh2 expression and decreased SATB2 mRNA expression, which was concurrent lower osteoblastogenesis potential in EOCCs; 2) ChIP experiments using H3K27me3 IP (immune-precipitation) showed significant association between H3K27me3, Ezh2 and SATB2. ConclusionsThese findings indicate maternal HFD-induced obesity-associated decrease of fetal pre-osteoblastic cell differentiation is under epigenetic control through SATB2 expression. Funding SourcesSupported by USDA-ARS Project.

The SLC34A2-ROS-HIF-1-induced up-regulation of EZH2 expression promotes proliferation and chemo-resistance to apoptosis in colorectal cancer.

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.

Increased EZH2 expression in prostate cancer is associated with metastatic recurrence following external beam radiotherapy.

BackgroundEnhancer of zeste 2 (EZH2) promotes prostate cancer progression. We hypothesized that increased EZH2 expression is associated with postradiotherapy metastatic disease recurrence, and may promote radioresistance.MethodsEZH2 expression was investigated using immunohistochemistry in diagnostic prostate biopsies of 113 prostate cancer patients treated with radiotherapy with curative intent. Associations between EZH2 expression in malignant and benign tissue in prostate biopsy cores and outcomes were investigated using univariate and multivariate Cox regression analyses. LNCaP and PC3 cell radiosensitivity was investigated using colony formation and γH2AX assays following UNC1999 chemical probe‐mediated EZH2 inhibition.ResultsWhile there was no significant association between EZH2 expression and biochemical recurrence following radiotherapy, univariate analysis revealed that prostate cancer cytoplasmic and total EZH2 expression were significantly associated with metastasis development postradiotherapy (P = 0.034 and P = 0.003, respectively). On multivariate analysis, the prostate cancer total EZH2 expression score remained statistically significant (P = 0.003), while cytoplasmic EZH2 expression did not reach statistical significance (P = 0.053). No association was observed between normal adjacent prostate EZH2 expression and biochemical recurrence or metastasis. LNCaP and PC3 cell treatment with UNC1999 reduced histone H3 lysine 27 tri‐methylation levels. Irradiation of LNCaP or PC3 cells with a single 2 Gy fraction with UNC1999‐mediated EZH2 inhibition resulted in a statistically significant, though modest, reduction in cell colony number for both cell lines. Increased γH2AX foci were observed 24 hours after ionizing irradiation in LNCaP cells, but not in PC3, following UNC1999‐mediated EZH2 inhibition vs controls.ConclusionsTaken together, these results reveal that high pretreatment EZH2 expression in prostate cancer in diagnostic biopsies is associated with an increased risk of postradiotherapy metastatic disease recurrence, but EZH2 function may only at most play a modest role in promoting prostate cancer cell radioresistance.