Enhancer Of Zeste Homolog 2 Expression Research Articles

Abstract A022: Targeting of soft tissue sarcoma cancer stem cells improves doxorubicin-sensitivity in vitro

Abstract Soft tissue sarcomas (STS) are rare tumors encompassing over 70 distinct histopathological subtypes that share a common treatment strategy comprising surgical resection, radiation, and chemotherapy in certain situations. Disease progression and failure to respond to anthracycline based chemotherapy, a standard first-line agent, is associated with poor outcomes. Recurrence and chemo-resistance represent significant barriers to improving patient survival. We are interested in the contribution of STS cancer stem cells (STS-CSCs) to the phenomenon of chemo-resistance to doxorubicin. Specifically, we hypothesized the presence of a common genetic signature across unique STS subtypes involved in CSC-regulation that could be targeted to improve the efficacy of existing treatment regimens. To this end, STS-CSCs were profiled by flow cytometry using the Aldeflour assay. This is a well-established technique to measure the aldehyde dehydrogenase activity of cells which is high in the stem cell population. This fluorescently labels Aldefluor bright and dim cells as CSCs and non-CSCs, respectively. The abundance of the CSC population in several STS cell lines modeling dedifferentiated liposarcoma, leiomyosarcoma, and undifferentiated pleomorphic sarcoma were assessed by Aldeflour assay. In order to gain insight into the molecular pathways active in STS-CSCs, Aldeflour-bright and -dim populations were isolated by FACs and analyzed by RNA-sequencing. Gene-set enrichment analysis of genes upregulated in STS-CSCs identified a signature for the histone methyltransferase Enhancer of Zeste homolog 2 (EZH2), part of the polycomb repressive complex 2 (PRC2) responsible for H3K27 methylation. As an epigenetic modulator, increased EZH2 expression and activity potentiates decreased activity of genes involved in growth suppression and thereby has oncogenic activity. EZH2 can be inhibited with small molecules such as Tazemetostat, an approved treatment for metastatic and locally advanced epithelioid sarcoma. To test the effects of EZH2 inhibition on STS-CSCs, we first generated doxorubicin resistance STS cell lines by serial selection with increasing concentrations of doxorubicin. We identified a positive correlation between CSC abundance and doxorubicin IC50 in the resistant cell lines. Further, co-treatment of doxorubicin and tazemetostat was not only synergistic in the parent cell lines, but restored chemosensitivity in doxorubicin resistant lines. These data confirm the presence of shared genetic programs across distinct subtypes of STS that are unique to CSCs and amenable to therapeutic targeting. Citation Format: Edmond F. O'Donnell, Maria Muñoz, Lor Randall, Janai R. Carr-Ascher. Targeting of soft tissue sarcoma cancer stem cells improves doxorubicin-sensitivity in vitro [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A022.

FBP1 knockdown decreases ovarian cancer formation and cisplatin resistance through EZH2-mediated H3K27me3.

Worldwide, ovarian cancer (OC) is the seventh common cancer and the second most common cause of cancer death in women. Due to high rates of relapse, there is an urgent need for the identification of new targets for OC treatment. The far-upstream element binding protein 1 (FBP1) and enhancer of zeste homolog 2 (EZH2) are emerging proto-oncogenes that regulate cell proliferation and metastasis. In the present study, Oncomine data analysis demonstrated that FBP1 was closely associated with the development of OC, and The Cancer Genome Atlas (TCGA) data analysis indicated that there was a positive correlation between FBP1 and EZH2 in ovarian tissues. Moreover, we found that FBP1 knockdown suppressed tumor formation in nude mice and cisplatin resistance of OC cells, but the role of FBP1 in the cisplatin resistance of OC cells remained unclear. In addition, we verified physical binding between FBP1 and EZH2 in OC cells, and we demonstrated that FBP1 knockdown enhanced cisplatin cytotoxicity in OC cells and down-regulated EZH2 expression and trimethylation of H3K27. These results suggested that FBP1 increases cisplatin resistance of OC cells by up-regulating EZH2/H3K27me3. Thus, FBP1 is a prospective novel target for the development of OC treatment.

EZH2 Protein Expression in Estrogen Receptor Positive Invasive Breast Cancer Treated With Neoadjuvant Endocrine Therapy: An Exploratory Study of Association With Tumor Response.

Neoadjuvant endocrine therapy (NET) can be used to treat estrogen receptor positive (ER+) invasive breast cancer (IBC). Tumors with Ki67>10% after 2 to 4 weeks of NET are considered resistant to endocrine therapy. Enhancer of Zeste Homolog 2 (EZH2) is a targetable oncoprotein and overexpression in ER+ IBC has been linked to resistance to endocrine therapy. We examined whether EZH2 expression levels in ER+ IBC could be used to predict response to NET. We retrospectively identified 46 patients with localized ER+ HER2/neu negative IBC treated with a minimum of 4 weeks of NET. We quantified EZH2 nuclear expression in pretherapy core biopsies using a score that included intensity and percent of cells staining. Ki67 was evaluated in both pretherapy core biopsies and posttherapy tumor resections and scored according to the guidelines of the International Ki67 Working Groups, with a global weighted score. Ki67≤10% after NET was considered endocrine responsive. Logistic regression analysis was performed to determine the association between EZH2 expression and response to NET. We found significant associations of tumor grade ( P =0.011), pretherapy Ki67 ( P =0.003), and EZH2 ( P <0.001), with response to NET. On logistic regression adjusted for tumor grade and pretherapy Ki67, increased EZH2 scores were associated with decreased odds of endocrine responsiveness, defined as posttreatment Ki67≤10% (odds ratio=0.976, 95% CI, 0.956 to 0.997; P =0.026). In addition, with EZH2 score in the model, associations of tumor grade and pretreatment Ki67 with posttreatment Ki67≤10% response to NET became not significant. Our results suggest that EZH2 might be a useful biomarker to predict response to NET.

Clinical Significance of EZH2 in Acute Myeloid Leukemia.

In this paper, we have focused on the investigation of the expression level of the enhancer of zeste homolog 2 (EZH2) gene in bone marrow mononuclear cells of acute myeloid leukemia (AML) patients and analyze the relationship between EZH2 gene expression and EMI. The expression of EZH2mRNA in bone marrow mononuclear cells of 26 patients with incipient AML was detected by qRT-PCR, and the relationship between EZH2mRNA expression and clinical characteristics was analyzed. EZH2 mRNA expression was increased in 26 AML patients. EZH2 gene expression in male patients was significantly higher than that in female patients. The expression of EZH2 in the group with extramedullary infiltration (EMI) was significantly higher than that in the group without EMI. The patients were divided into different groups according to the chromosomal karyotype and prognosis. Statistical analysis showed that the expression level of the medium-risk group was significantly higher than that of the low-risk group, while there was no statistical difference in other groups (P > 0.05). The expression of EZH2 gene in AML patients was closely related to EMI, and the expression of EZH2 in AML cells was closely related to cell migration ability. EZH2 is expected to be one of the indicators of disease recurrence.

Inhibition of EZH2 suppresses peritoneal angiogenesis by targeting a VEGFR2/ERK1/2/HIF-1α-dependent signaling pathway.

The catalytic subunit of polycomb repressive complex 2 (PRC2), enhancer of zeste homolog 2 (EZH2), has been reported to be involved in angiogenesis in some tumors and autoimmune diseases. However, the mechanisms by which EZH2 regulates peritoneal angiogenesis remain unclear. We detected the expression of EZH2 in clinical samples and the peritoneal tissue of a mouse peritoneal fibrosis model induced by chlorhexidine gluconate (CG). In addition, we further investigated the mechanisms by which inhibition of EZH2 by 3-deazaneplanocin A (3-DZNeP) alleviated the CG-induced peritoneal fibrosis mouse model in vivo and 3-DZNeP or EZH2 siRNA treatment in cultured human peritoneal mesothelial cells (HPMCs) and human umbilical vein endothelial cells (HUVECs). The expression of EZH2 in the peritoneum of long-term peritoneal dialysis (PD) patients and the CG-induced peritoneal fibrosis mouse model was remarkably increased and this was positively associated with higher expression of vascular markers (CD31, CD34, VEGF, p-VEGFR2). Peritoneal injection of 3-DZNeP attenuated angiogenesis in the peritoneum of CG-injured mice; improved peritoneal membrane function; and decreased phosphorylation of STAT3, ERK1/2, and activation of Wnt1/β-catenin. In in vitro experiments, we demonstrated that inhibition of EZH2 by 3-DZNeP or EZH2 siRNA decreased tube formation and the migratory ability of HUVECs via two pathways: the Wnt1/β-catenin pathway and the IL-6/STAT3 pathway. Suppression of the Wnt1/β-catenin pathway and the IL-6/STAT3 pathway subsequently reduced VEGF production in HPMCs. Using specific inhibitors of VEGFR2, ERK1/2, and HIF-1α, we found that a VEGFR2/ERK1/2/HIF-1α axis existed and contributed to angiogenesis in vitro. Moreover, phosphorylation of VEGFR2 and activation of the ERK1/2 pathway and HIF-1α in HUVECs could be suppressed by inhibition of EZH2. Taken together, the results of this study suggest that EZH2 may be a novel target for preventing peritoneal angiogenesis in PD patients. © 2022 The Pathological Society of Great Britain and Ireland.

Mutually exclusive expression of EZH2 and H3K27me3 in non-small cell lung carcinoma

Enhancer of zeste homolog 2 (EZH2) epigenetically represses gene expression via trimethylation of lysine 27 on histone 3 (H3K27me3). Non-small cell carcinoma (NSCLC) has been reported to show high EZH2 and low H3K27me3 expression compared to normal lung tissues, but there are no studies examining the expression of EZH2 and H3K27me3 simultaneously with immunohistochemistry. In the present study, the expression of EZH2 and H3K27me3 was examined in surgically resected NSCLC. We enrolled 27 cases of squamous cell carcinoma (SCC), 73 cases of Lepidic, 77 of Papillary/Acinar, 51 of Solid, 31 of Micropapillary, and 12 of Mucinous subtypes of adenocarcinoma. First, we examined the expression of EZH2 and H3K27me3 in normal and metaplastic bronchial epithelium adjacent to NSCLC. Normal bronchial epithelium showed EZH2 expression in a limited number of basal cells and H3K27me3 expression in surface differentiated cells with cilia or mucus. In metaplastic bronchial epithelium, the number of EZH2-positive cells increased in multilayered basal cells, and H3K27me3-positive cells were observed in the superficial layer. Then, EZH2 and H3K27me3 expression was analyzed in NSCLC. Abundant EZH2 and rare H3K27me3 expression was detected in SCC, Papillary/Acinar, Solid and Micropapillary subtypes. In Mucinous subtype, EZH2 expression was hardly detected, and H3K27me3 expression was detected in almost all tumor cells. EZH2-expressing and H3K27me3-expressing tumor cells were similarly observed in Lepidic subtype, but double immunofluorescence revealed that EZH2 and H3K27me3 expression pattern was mutually exclusive. No co-expression of EZH2 and H3K27me3 was detected in all examined subtypes. To our knowledge, there have been no reports describing mutually exclusive expression pattern of EZH2 and H3K27me3.

FAK Executes Anti-Senescence via Regulating EZH2 Signaling in Non-Small Cell Lung Cancer Cells.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase overexpressed in various cancer types that plays a critical role in tumor progression. Accumulating evidence suggests that targeting FAK, either alone or in combination with other agents, may serve as an effective therapeutic strategy for numerous cancers. In addition to retarding proliferation, metastasis, and angiogenesis, FAK inhibition triggers cellular senescence in lung cancer cells. However, the detailed mechanism remains enigmatic. In the present study, we found that FAK inhibition not only elicits DNA-damage signaling but also downregulates enhancer of zeste homolog 2 (EZH2) expression. The manipulation of FAK expression influences EZH2 expression and corresponding signaling in vitro. Immunohistochemistry shows that active FAK signaling corresponds with the activation of the EZH2-mediated signaling cascade in lung-cancer-cells-derived tumor tissues. We also found that ectopic EZH2 expression attenuates FAK-inhibition-induced cellular senescence in lung cancer cells. Our results identify EZH2 as a critical downstream effector of the FAK-mediated anti-senescence pathway. Targeting FAK-EZH2 axis-induced cellular senescence may represent a promising therapeutic strategy for restraining tumor growth.

Emerging role of EZH2 in rheumatic diseases: A comprehensive review.

Enhancer of zeste homolog 2 (EZH2) is a histone methylated enzyme. It trimethylates histone 3 lysine 27 (H3K27) to regulate epigenetic processes. Recently, studies showed excessive expression of EZH2 in rheumatic diseases, such as systemic lupus erythematosus, rheumatoid arthritis, osteoarthritis, and systemic sclerosis. Moreover, epigenetic modification of EZH2 regulates differentiation and proliferation of different immune cells. Therefore, in this review, we comprehensively discuss the role of EZH2 in rheumatic diseases. Collection of the evidence may provide a basis for further understanding the role of EZH2 and give potential for targeting these diseases.

EZH2 promotes the progression of osteosarcoma through the activation of the AKT/GSK3β pathway.

Enhancer of zeste homologue 2 (EZH2) is a clarified promoter in a list of tumours, including osteosarcoma (OS). Our research was projected to define the mechanism involved in EZH2-mediated OS progression through the protein kinase B (AKT)/glycogen synthase kinase 3β (GSK3β) pathway. EZH2 expression was tested in 66 OS tissues and five osteosarcoma cell lines (143B, SJSA-1, HOS, MG63, and U2OS). In HOS and U2OS cells, cellular malignant characteristics, and the markers of the AKT/GSK3β signalling pathway were measured when EZH2 was silenced or overexpressed. Meanwhile, rescue assays were implemented to observe whether the AKT/GSK3β signalling pathway inhibitor (MK-2206) could affect the role of overexpressed EZH2 in OS cells. EZH2 was up-regulated in tumour tissues of OS patients. OS cell lines (HOS and U2OS) showed impairments of proliferative, migratory, invasive and anti-apoptotic properties when EZH2 was silenced. Downregulated EZH2 inhibited the activation of the AKT/GSK3 signalling pathway. However, the situation in HOS and U2OS cells over-expressing EZH2 was opposite. MK-2206 erased EZH2 up-regulation-induced promotion of OS cell growth. It is demonstrated that EZH2 promotes the progression of OS via inducing the activation of the AKT/GSK3β pathway, offering a therapeutic direction for OS treatment.

Enhancer of zeste homolog 2 exerts functions in gastric cancer development via modulating microRNA-222-3p methylation and WEE1 expression.

Enhancer of zeste homolog 2 (EZH2) has been studied in gastric cancer (GC), while the role of EZH2 in GC via binding to microRNA (miR)-222-3p remains obscure. This research aims to unravel the regulatory mechanism of EZH2 in GC progression via the modulation of miR-222-3p/WEE1 axis. Initially, EZH2, miR-222-3p, and WEE1 levels in GC cells and tissues were examined. Thereafter, constructs altering EZH2, miR-222-3p, or WEE1 expression were transfected into GC cells to determine the malignant behaviors involved in tumorigenesis of GC cells. Finally, the targeting relations among EZH2, miR-222-3p, and WEE1 were validated. EZH2 and WEE1 were upregulated while miR-222-3p was down-regulated in GC tissues and cells. The decreased EZH2, silenced WEE1, or restored miR-222-3p constrained the malignant behaviors involved in tumorigenesis of GC cells. Deletion of miR-222-3p could reverse the effect of silenced EZH2 on suppressing the biological functions of GC cells. EZH2 could bind to the promoter of miR-222-3p, and there was a targeting relationship between miR-222-3p and WEE1. Our study demonstrates that EZH2 promotes GC development via the modulation of miR-222-3p/WEE1 axis, thus providing promising therapeutic targets for GC therapy.

Abstract 5365: Repositioning nicardipine as a novel targeted therapy for chemoresistant prostate cancer

Abstract Background: It is imperative to develop novel therapeutics to overcome chemoresistance, a major obstacle in the clinical management of prostate cancer (PCa) and other cancers. Drug repositioning is being pursued as an attractive approach due to its potentially lower overall development costs and shorter timelines. The purpose of this study is to investigate the preclinical efficacy and mechanism of action of nicardipine, an FDA-approved hypertension drug, as a new treatment against chemoresistant prostate cancer. Methods: A panel of FDA-approved non-oncology drugs was screened for their selectivity and potency to inhibit chemoresistant PCa cells using a two-tier phenotypic screening platform (Theranostics, 2021, 11(14): 6873-6890). The mechanism of action of potential candidate(s) was evaluated using in silico docking, cellular and molecular assays in ARCaPE-shEPLIN and C4-2B-TaxR, two independent chemoresistant PCa cell lines. The in vivo efficacy was evaluated in clinically-relevant xenograft models of PCa. Results: Nicardipine exhibited high selectivity and potency against chemoresistant PCa cells via effectively inducing apoptosis and cell cycle arrest. The IC50 of nicardipine was determined as 0.5 μM and 2.1 μM in ARCaPE-shEPLIN and C4-2B-TaxR cells, respectively. Mechanically, nicardipine targeted a novel non-canonical enhancer of zeste homolog 2 (EZH2) signal pathway in chemoresistant PCa cells. Specifically, nicardipine might bind embryonic ectoderm development (EED), interrupt the integrity and stability of the polycomb repressive complex 2 (PRC2), reduce the phosphorylation and expression of EZH2, and subsequently inhibit downstream effectors including signal transducer and activator of transcription 3 (Stat3), S-phase kinase-associated protein 2 (SKP2), ATP binding cassette B1 (ABCB1) and survivin. In animal models, as a monotherapy, nicardipine significantly suppressed the intratibial growth of chemoresistant C4-2B-TaxR xenografts, with an average PSA level of 43.47 ng/ml, 36.63 ng/ml, and 28.78 ng/ml for the vehicle control, docetaxel, and nicardipine groups at the endpoint, respectively (p < 0.05 between the nicardipine group and the vehicle control or docetaxel groups). As a combination regime, nicardipine synergistically enhanced the efficacy of docetaxel against the intratibial growth of C4-2 xenografts, with an average PSA level of 79.17 ng/ml, 49.04 ng/ml, 42.80 ng/ml, and 23.23 ng/ml for the vehicle control, docetaxel, nicardipine, and combination groups at the endpoint, respectively (p < 0.0001 or 0.01 between the combination group and the vehicle control or docetaxel groups, respectively). Conclusion: These results demonstrated that, for the first time, nicardipine could be a novel EED inhibitor that has the potential to be promptly tested in PCa patients to overcome chemoresistance and improve clinical outcomes. Citation Format: Xin Li, Alira Danaher, Jason M. Wu, Yifei Wu, Zhong-Ru Xie, Nicholas Cook, Nathan Bowen, Daqing Wu. Repositioning nicardipine as a novel targeted therapy for chemoresistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5365.

Abstract 5381: Combinatory effect of EZH2 inhibitor and dopamine D1 receptor agonist enhance inhibition of triple negative breast cancer cell growth

Abstract Treatment for Triple Negative Breast Cancer remains a challenge due to its aggressive behavior; poor clinical outcome and disposition to therapeutic resistance. The diverse clinical and molecular characteristics drives high level of heterogeneity in breast cancer. Overall change in the epigenome triggers oncogenic activity of enhancer of zeste homolog 2 (EZH2) causing changes in gene expression that leads to cancer progression. EZH2 is the catalytic member of the Polycomb group protein complex that provides the active site for covalent methylation reactions. High expression of EZH2 is strongly associated with aggressive TNBC phenotypes, compared to other non-TNBCs. Development of resistance to EZH2 inhibitors limit their therapeutic use. Studies have shown that dopamine receptor (DR) agonists and antagonists alter TNBC cell growth in both in vitro and in vivo models. Our objective was to investigate if a combination of a D1R agonist with an EZH2 inhibitor was effective to attenuate TNBC cell proliferation. Docking studies were performed using Schrodinger software and visualization software to determine the affinity of GSK126 and SKF38393 towards EZH2. TNBC cells were treated individually and combined with EZH2 inhibitor, GSK126, and D1 agonist SKF38393 for cell viability and immunoprecipitation. Insilco analysis using docking studies revealed that SKF38393 actively interacts in the same region where GSK126 binds to EZH2 i.e. the catalytic site. Combination treatment of TNBC cells with GSK126 and SKF38393 caused synergistic inhibition of cell viability, suppressed the H3K27 trimethylation activity, and inhibited invasion of TNBC cells. Immunoprecipitation of EZH2 in the presence of GSK126 and SKF38393 dissociated the association between EZH2, EED, and SUZ12 in MDA-MB-231 cells. We report that combination of GSK126 and SKF38393 may have an enhanced efficacy in inhibiting the catalytic function of EZH2. The combined treatment of TNBC cells with GSK126 and SKF38393 inhibit TNBC proliferation by disrupting EZH2 functions. (This work is supported by DOD: W81XWH2010065, for Eswar Shankar) Citation Format: Prem Prakash Kushwaha, Shiv Verma, Gautham Sarathy, Anmol Kumar, Sanjay Gupta, Bhuvaneswari Ramaswamy, Sarmila Majumder, Eswar Shankar. Combinatory effect of EZH2 inhibitor and dopamine D1 receptor agonist enhance inhibition of triple negative breast cancer cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5381.

Enhancer of Zeste Homolog 2 (EZH2) Is a Marker of High-Grade Neuroendocrine Neoplasia in Gastroenteropancreatic and Pulmonary Tract and Predicts Poor Prognosis.

Simple SummaryNeuroendocrine neoplasms most frequently arise in the gastroenteropancreatic and pulmonary tract and show an increasing incidence and prevalence. The prognosis and treatment depend on tumor proliferation and clinical behavior. Highly proliferating grade 3 neoplasms especially, show a wildly divergent therapy response and prognosis. In particular, it is crucial to securely separate the more indolent G3 tumors from the more aggressive carcinomas. Currently, this distinction is based on a combination of clinical, morphologic, immunohistochemical, and molecular biomarkers. However, none of these markers allow for a reliable distinction, and additional markers are needed. EZH2 has attracted increasing interest in different tumor entities. We aimed to analyze the expression of EZH2 in different neuroendocrine neoplasms and to correlate the expression with clinical parameters and survival. We demonstrate that EZH2 is nearly exclusively expressed in highly proliferative neoplasms and is a robust biomarker for identifying aggressive G3 tumors with poor prognosis.Tumor grading is a robust prognostic predictor in patients with neuroendocrine neoplasms (NEN) and guides therapy, especially in tumors with high proliferation. NEN can be separated into well-differentiated and poorly differentiated types. The more aggressive NEN have been further separated into neuroendocrine tumors (NET G3) with a better prognosis and neuroendocrine carcinomas (NEC) with a worse prognosis. Despite this distinction’s tremendous clinical and therapeutic relevance, optimal diagnostic biomarkers are still lacking. In this study, we analyzed the protein expression and prognostic impact of Enhancer of Zeste Homolog 2 (EZH2) by immunohistochemistry in 219 tissue samples of gastroenteropancreatic (GEP-NEN) and pulmonary NEN (P-NEN). EZH2 was almost exclusively expressed in NEN with a proliferation rate above 20% (G3), while all low-grade tumors were nearly negative. Among high-grade NEN, 65% showed high and 35% low expression of EZH2. In this group, the high expression of EZH2 was significantly associated with poor overall survival and NEC histology. Interestingly, EZH2 seems to act independently of Polycomb Repressive Complex 2 (PRC2) in NEN. In conclusion, we propose EZH2 as a robust biomarker for distinguishing between NET G3 and NEC among gastroenteropancreatic and pulmonary NEN.

Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus.

SummaryBackgroundHuman cytomegalovirus (HCMV) infection has been actively implicated in complex neoplastic processes. Beyond oncomodulation, the molecular mechanisms that might underlie HCMV-induced oncogenesis are being extensively studied. Polycomb repressive complex 2 (PRC2) proteins, in particular enhancer of zeste homolog 2 (EZH2) are associated with cancer progression. Nevertheless, little is known about EZH2 activation in the context of HCMV infection and breast oncogenesis.MethodsHerein, we identified EZH2 as a downstream target for HCMV-induced Myc upregulation upon acute and chronic infection with high-risk strains using a human mammary epithelial model.FindingsWe detected polyploidy and CMV-transformed HMECs (CTH) cells harboring HCMV and dynamically undergoing the giant cells cycle. Acquisition of embryonic stemness markers positively correlated with EZH2 and Myc expression. EZH2 inhibitors curtail sustained CTH cells’ malignant phenotype. Besides harboring polyploid giant cancer cells (PGCCs), tumorigenic breast biopsies were characterized by an enhanced EZH2 and Myc expression, with a strong positive correlation between EZH2 and Myc expression, and between PGCC count and EZH2/Myc expression in the presence of HCMV. Further, we isolated two HCMV strains from EZH2HighMycHigh basal-like tumors which replicate in MRC5 cells and transform HMECs toward CTH cells after acute infection.InterpretationOur data establish a potential link between HCMV-induced Myc activation, the subsequent EZH2 upregulation, and polyploidy induction. These data support the proposed tumorigenesis properties of EZH2/Myc, and allow the isolation of two oncogenic HCMV strains from EZH2HighMycHigh basal breast tumors while identifying EZH2 as a potential therapeutic target in the management of breast cancer, particularly upon HCMV infection.FundingThis work was supported by grants from the University of Franche-Comté (UFC) (CR3300), the Région Franche-Comté (2021-Y-08292 and 2021-Y-08290) and the Ligue contre le Cancer (CR3304) to Georges Herbein. Zeina Nehme is a recipient of a doctoral scholarship from the municipality of Habbouch. Sandy Haidar Ahmad is recipient of a doctoral scholarship from Lebanese municipality. Ranim El Baba is a recipient of a doctoral scholarship from Hariri foundation for sustainable human development.

MO398: Salvianolic Acid C Attenuated Fibrosis Through Enhancer of Zeste Homolog 2 in Fibrotic Kidneys

Abstract BACKGROUND AND AIMS Salvianolic acid C (SAC) is a component of Danshen, a widely used herbal medicine for the treatment of renal cardiovascular diseases. Renal interstitial fibrosis is a common pathway of all kinds of chronic kidney diseases progressing to the end-stage of renal diseases. We aimed to study the effect of SAC on renal fibrosis and explore its underlining mechanisms. METHOD Mouse renal fibrosis model was induced by unilateral ureteral obstruction (UUO) or peritoneally injection of aristolochic acid I (AAI). Moreover, normal rat kidney interstitial fibroblast (NRK-49F) cells were treated with various concentrations of SAC (10 mM–100 mM). Protein samples from in vivo and in vitro experiments were collected to assess renal fibrosis. RESULTS Masson staining and Western blotting analysis showed that 2 weeks treatment with SAC attenuated renal fibrosis in UUO or AAI induced fibrotic kidneys. The expression of fibronectin, collagen-I and α smooth muscle actin (αSMA) were increased in UUO or AAI induced fibrotic kidneys, which were down-regulated in SAC treated UUO or AAI induced fibrotic kidneys. In parallel, treatment with SAC reduced the expression of fibronectin and αSMA in NRK-49F cells. Mechanistically, we found that enhancer of zeste homolog 2 (EZH2) is up-regulated in UUO or AAI induced fibrotic kidneys and Tan-I reduced the expression of EZH2 in fibrotic kidneys. Moreover, inhibition of EZH2 reduced the expression of pro-fibrotic markers and abolished the ani-fibrotic effect of SAC in rat renal fibroblasts. CONCLUSION We conclude that SAC attenuates fibrosis in fibrotic kidneys through inhibition of EZH2.

EZH2 enhances proliferation and migration of trophoblast cell lines by blocking GADD45A-mediated p38/MAPK signaling pathway

ABSTRACT Impaired activity of the trophoblasts is a major contributor to the progression of pregnancy pathologies including preeclampsia (PE). This research probed the function of enhancer of zeste homolog 2 (EZH2) in activity of trophoblast cells and its correlation with growth arrest and DNA damage inducible alpha (GADD45A). EZH2 was predicted to be downregulated in placental tissues in PE according to a gene chip analysis, and reduced expression of EZH2 was detected in the placental tissues of patients with PE. Overexpression of EZH2 augmented proliferation and invasiveness of two trophoblast cell lines HTR-8/SVneo and JEG3 cells. EZH2 catalyzed trimethylation of lysine 27 on histone 3 (H3K27me3) in GADD45A promoter to suppress its transcription. GADD45A silencing increased the activity of the trophoblast cell lines and inactivated the p38/mitogen-activated protein kinase (MAPK) signaling pathway. Rescue experiments confirmed that either inhibition of GADD45A or p38 restored the proliferation, migration, and invasiveness of the trophoblast cell lines suppressed by EZH2 silencing. In conclusion, this work suggests that EZH2 enhances activity of trophoblast cell lines by suppressing GADD45A-mediated p38/MAPK signaling pathway.

Genome-wide gain-of-function screening identifies EZH2 mediating resistance to PI3Kα inhibitors in oesophageal squamous cell carcinoma.

Phosphoinositide‐3 kinase alpha (PI3Kα) has been confirmed to be a potential therapeutic target for esophageal squamous cell carcinoma (ESCC), while the potency of PI3Kα inhibitors is often attenuated by concurrent oncogenic signalling pathways. We performed genome‐wide gain‐of‐function screening with a CRISPR‐SAM library and identified enhancer of zeste homolog 2 (EZH2) rendering ESCC cells resistant to the PI3Kα inhibitor CYH33. Enhanced expression of EZH2 frequently occurs in ESCC and is related to poor prognosis. Overexpression of full‐length EZH2 but not methyltransferase‐deficient EZH2 conferred resistance to CYH33, while downregulating EZH2 expression restored sensitivity. EZH2 expression was negatively related to the activity of CYH33 against the proliferation of ESCC cell lines and patient‐derived cells. Transcriptomic analysis revealed that EZH2 abrogated CYH33‐mediated cell cycle regulation. EZH2 epigenetically suppressed the transcription of CDKN1A, promoting RB phosphorylation and cell cycle progression. Concurrently targeting EZH2 significantly potentiated CYH33 to inhibit the growth of ESCC cells and patient‐derived xenografts accompanied by enhanced cell cycle arrest. Taken together, our study demonstrated that an EZH2‐p21‐RB axis remodeled cell cycle regulation and rendered resistance to PI3Kα inhibitors in ESCC. Simultaneously targeting PI3Kα and EZH2 may provide an effective strategy for ESCC therapy with high expression of EZH2.

Progranulin regulates the development and function of NKT2 cells through EZH2 and PLZF.

T helper 2 (Th2) cytokine production by invariant natural killer T (iNKT) cells is involved in the development of asthma, but the regulation of Th2 cytokines in iNKT cells remains unknown. Although it is known that progranulin (PGRN) induces the production of Th2 cytokines in iNKT cells in vivo, the underlying mechanism is not clear. This study aims to investigate the role of PGRN in iNKT cells. The effects of PGRN on the differentiation of iNKT cells was detected by flow cytometry. Then stimulation of iNKT cells and airway resistance were carried out to evaluate the function of PGRN on iNKT cells. Furthermore, the mechanisms of PGRN in regulating iNKT cells was investigated by RT-PCR, WB, confocal and luciferase reporter assays. The absolute number of iNKT cells decreased in PGRN KO mice despite an increase in the percentage of iNKT cells. Furthermore, analyzing the subsets of iNKT cells, we found that NKT2 cells and their IL-4 production were reduced. Mechanistically, the decrease in NKT2 cells in the PGRN KO mice was caused by increased expression of enhancer of zeste homolog 2 (EZH2), that in turn caused increased degradation and altered nuclear localization of PLZF. Interestingly, PGRN signaling decreased expression of EZH2 and treatment of the PGRN KO mice with the EZH2 specific inhibitor GSK343 rescued the defect in NKT2 differentiation, IL-4 generation, and PLZF expression. Altogether, We have revealed a new pathway (PGRN-EZH2-PLZF), which regulates the Th2 responses of iNKT cells and provides a potentially new target for asthma treatment.

Effects and molecular mechanism of histone methyltransferase enhancer of zeste homolog 2 on regulating sepsis-induced T cell dysfunction

To investigate the effect and mechanism of histone methyltransferase enhancer of zeste homolog 2 (EZH2) on sepsis-induced T cell dysfunction. Twenty-four male C57BL/6 mice were divided into three groups randomly: sham operated group, sepsis model group [cecum ligation and puncture (CLP)+dimethyl sulfoxide (DMSO) group] and EZH2 selective inhibitor treated group (CLP+GSK126 group), with 8 mice in each group. Sepsis murine model was reproduced by CLP. CLP+DMSO group and CLP+GSK126 group were treated with DMSO or GSK126 (10 mg/kg) respectively right after surgery through intraperitoneal injection. The mice were sacrificed 24 hours after operation, and the mesenteric lymph nodes were collected. The expression of EZH2, apoptosis rates, cell proliferation marker ki-67 antigen positive T lymphocytes (ki-67+ cell), interferon-γ positive T lymphocytes (IFN-γ + cell), programmed death receptor-1 positive T lymphocytes (PD-1+ cell) and programmed death-ligand 1 positive T lymphocytes (PD-L1+ cell) were determined by flow cytometry. Compared with sham operated group, the expression of EZH2 in T lymphocytes was up-regulated on mesenteric lymph nodes of CLP+DMSO group. Compared with CLP+DMSO group, the ratio of CD3+ T lymphocytes in CLP+GSK126 group was up-regulated (0.70±0.02 vs. 0.50±0.07, P < 0.01), indicating that the EZH2 inhibitor could increase the number of T lymphocytes in lymph nodes of septic mice; the ratio of ki-67+ cells in CD4+ and CD8+ T lymphocytes in CLP+GSK126 group was increased (CD4+: 0.74±0.05 vs. 0.63±0.04, CD8+: 0.82±0.06 vs. 0.70±0.04, both P < 0.05), indicating that the EZH2 inhibitor could increase the ratio of T lymphocytes with high proliferative activity in lymph nodes of septic mice. However, no significant difference was found on both CD4+ and CD8+ T lymphocytes apoptosis rates in the mesenteric lymph nodes of mice between CLP+GSK126 group and CLP+DMSO group [CD4+: (21.53±2.87)% vs. (20.48±3.21)%, CD8+: (8.34±1.02)% vs. (7.71±1.38)%, both P > 0.05], indicating that no extra T lymphocytes apoptosis was induced by EZH2 inhibitor. Compared with CLP+DMSO group, the ratios of IFN-γ + CD4+ and IFN-γ + CD8+ T lymphocytes were increased in CLP+GSK126 group (IFN-γ +CD4+: 0.31±0.11 vs. 0.14±0.06, IFN-γ +CD8+: 0.30±0.10 vs. 0.13±0.06, both P < 0.05), suggesting that secretion of IFN-γ in lymph nodes by sepsis T lymphocytes was augmented after EZH2 inhibitor administration. Furthermore, compared with CLP+DMSO group, the ratio of PD-1+ cell in CD8+ T lymphocyte was down-regulated in CLP+GSK126 group (0.092±0.006 vs. 0.135±0.004, P < 0.01), suggesting that EZH2 inhibitor restrained the PD-1 expression on sepsis lymphoid node CD8+ T lymphocytes, however, it had no significant effect on PD-L1+ cells. EZH2, regulates sepsis-induced T lymphocyte dysfunction, possibly through modulating the expression of PD-1.

Combined Detection of RUNX3 and EZH2 in Evaluating Efficacy of Neoadjuvant Therapy and Prognostic Value of Middle and Low Locally Advanced Rectal Cancer.

ObjectiveThis article investigated whether Runt-Related Transcription Factor 3 (RUNX3) and enhancer of zeste homolog 2 (EZH2) can be used to evaluate the clinical efficacy of neoadjuvant therapy and prognosis of locally advanced rectal cancer (LARC). MethodsEighty LARC patients admitted to the Tianjin Medical University Cancer Institute/Hospital and First Affiliated Hospital of Hebei North University from Jan 2015 to Jan 2016 were enrolled. The patients were followed up for 60 months through hospital visits. All patients received neoadjuvant chemoradiotherapy (long range radiotherapy + oral capecitabine) + total mesorecta excision (TME) surgery. The clinical efficacy of the treatments was evaluated through endoscopic, radiography, and tumor regression grade (TRG). In addition, expression level of RUNX3 and EZH2 was quantified via immunohistochemistry. The association of RUNX3 and EZH2 with clinicopathological characteristics of advanced tumors and efficacy of neoadjuvant therapy was explored. Logistic regression analysis was performed to identify predictors of efficacy of neoadjuvant chemoradiotherapy. Survival curve was used to evaluate the impact of RUNX3 and EZH2 on the prognosis of LARC patients.ResultsA total of 80 patients diagnosed with LARC were enrolled in the study. Expression of RUNX3 was elevated in 25 (31.25%) patients, whereas expression of EZH2 was upregulated in 44 (55.00%) patients. Analysis of tumor regression identified 10 cases with TRG grade 0 (pathologic complete response, PCR), 24 cases with TRG grade 1, 35 cases with TRG grade 2, and 11 cases with TRG grade 3. Furthermore, 38 cases had significant down-staging, and 42 cases showed no significant down-staging as revealed by endoscopy and imaging. Patients with high expression of RUNX3 showed better tumor regression response and down-staging compared with those with low expression of RUNX3 (P < 0.001, P < 0.001). Moreover, patients with low EZH2 expression achieved TRG grade 0 and 1 response and down-staging effect compared with those with high expression of EZH2 (P < 0.001, P < 0.001). Logistic regression analysis showed that high expression of RUNX3, low expression of EZH2, and clinical N (cN) stage were good predictors of tumor regression response and down-staging. The 5-year disease free survival (DFS) and overall survival (OS) were 48.75 (39/80) and 58.75% (47/80), respectively. The 5-year DFS and OS of patients with high RUNX3 expression were significantly higher than low RUNX3 expression, whereas the 5-year DFS and OS of patients with high EZH2 expression were significantly lower than low EZH2 expression (P < 0.001). Univariate survival analysis showed that RUNX3 expression, EZH2 expression, cN, clinical T (cT), pathological T (pT) and pathological N (pN) were significantly correlated with the 5-year DFS and 5-year OS. Multivariate survival analysis showed that EZH2 expression and PN were good predictors of 5-year DFS and 5-year OS, whereas RUNX3 was a good predictor of 5-year DFS but not 5-year OS.ConclusionsExpression level of RUNX3 and EZH2 accurately predicts clinical efficacy of neoadjuvant chemoradiotherapy and the prognosis of LARC patients, suggesting that RUNX3 and EZH2 can be used as pivotal clinical predictors for LARC.