Enhancer Of Zeste Homolog 2 Knockdown Research Articles

EZH2 Activates HTLV-1 bZIP Factor-Mediated TGF-β Signaling in Adult T-Cell Leukemia.

Adult T-cell leukemia (ATL) is an aggressive malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) infection. Enhancer of zeste homolog 2 (EZH2) has been implicated in the development and progression of multiple cancers, including virus-induced malignancies. However, the potential function of EZH2 in HTLV-1-induced oncogenesis has not been clearly elucidated. In the present study, we showed that EZH2 was overexpressed and activated in HTLV-1-infected cell lines, potentially due to the activation of EZH2 promoter by HTLV-1 Tax and NF-κB p65 subunit. In addition, we found that EZH2 enhanced the HBZ-induced activation of TGF-β signaling in a histone methyltransferase-independent manner. As a mechanism for these actions, we found that EZH2 targeted Smad3/Smad4 to form a ternary complex, and the association between Smad3 and Smad4 was markedly enhanced in the presence of EZH2. Knockdown of EZH2 in ATL cells indeed repressed the expressions of the TGF-β target genes. In particular, EZH2 synergistically enhanced the HBZ/TGF-β-induced Foxp3 expression. Treatment of 3-Deazaneplanocin A, a specific inhibitor of EZH2 significantly inhibited the Foxp3 expression. Taken together, our results suggest that EZH2 may be involved in the differentiation of regulatory T cells through activating the HBZ-Smad3-TGF-β signaling axis, which is considered to be a key strategy for viral persistence.

MiR-4448/Girdin/Akt/AMPK axis inhibits EZH2-mediated EMT and tumorigenesis in small-cell lung cancer.

Small-cell lung cancer (SCLC) shows high enhancer of zeste homolog 2 (EZH2) expressions. EZH2-mediated epigenetics promote epithelial-mesenchymal transition (EMT), enhancing invasive and metastatic potential in malignancies. MicroRNAs (miRNAs), small noncoding RNAs, modulate EMT, determining tumor phenotypes. However, the association between miRNAs and EZH2 in SCLC remains to be clarified-we aimed to identify a novel tumorigenic mechanism through miRNAs, EZH2, and EMT in SCLC, leading to future therapeutic applications. We analyzed EZH2 and E-cadherin expressions in lung cancer cell lines and tumor tissues from 34 SCLC patients and confirmed EZH2 siRNA-mediated EMT inhibition. miRNA expression profiles were compared between EZH2 knockdown SCLC cells and negative control SCLC cells using miRNA array. We identified a target miRNA of EZH2 showing expressional differences in EZH2-knockdown cells and analyzed the impact of the miRNA on EZH2-mediated EMT and tumorigenesis. All SCLC cells showed increased EZH2 and decreased E-cadherin expressions. SCLC tissues had higher EZH2 and lower E-cadherin expressions than other lung cancer tissues. miRNA array revealed that miR-4448 expression increased in EZH2-knockdown SCLC cells. miR-4448 overexpression reduced tumor cell growth and prevented EMT. miR-4448 bound to the 3'UTR of the girdin gene and suppressed its expression, thereby decreasing Akt phosphorylation at Ser473. Attenuated Akt phosphorylation resulted in AMP-activated protein kinase (AMPK) phosphorylation at Thr172 and 183, enhancing EZH2 phosphorylation at Thr311. SCLC characterized high EZH2 expression and promoted EMT, compared with non-small cell lung cancer. miR-4448 inhibited Girdin expression, reducing Akt phosphorylation, and enhancing AMPK and EZH2 phosphorylation. Eventually, miR-4448 prevented EZH2-mediated EMT and tumorigenesis by modulating the Girdin/Akt/AMPK axis in SCLC. miR-4448 might be a potential SCLC inhibitor.

Estrogen receptor alpha mediated repression of PRICKLE1 destabilizes REST and promotes uterine fibroid pathogenesis.

Uterine fibroids (leiomyomas), benign tumors of the myometrial smooth muscle layer, are present in over 75% of women, often causing severe pain, menorrhagia and reproductive dysfunction. The molecular pathogenesis of fibroids is poorly understood. We previously showed that the loss of REST (RE-1 Silencing Transcription factor), a tumor suppressor, in fibroids leads to activation of PI3K/AKT-mTOR pathway. We report here a critical link between estrogen receptor alpha (ERα) and the loss of REST, via PRICKLE1. PRICKLE1 expression is markedly lower in leiomyomas, and the suppression of PRICKLE1 significantly down regulates REST protein levels. Conversely, overexpression of PRICKLE1 resulted in the restoration of REST in cultured primary leiomyoma smooth muscle cells (LSMCs). Crucially, mice exposed neonatally to environmental estrogens, proven risk factors for fibroids, expressed lower levels of PRICKLE1 and REST in the myometrium. Using mice that lack either endogenous estrogen (Lhb -/- mice) or ERα (Esr1 -/- mice), we demonstrate that Prickle1 expression in the myometrium is suppressed by estrogen through ERα. Enhancer of zeste homolog 2 (EZH2) is known to participate in the repression of specific ERα target genes. Uterine leiomyomas express increased levels of EZH2 that inversely correlate with the expression of PRICKLE1. Using chromatin immunoprecipitation, we provide evidence for association of EZH2 with the PRICKLE1 promoter and for hypermethylation of H3K27 within the regulatory region of PRICKLE1 in leiomyomas. Additionally, siRNA mediated knockdown of EZH2 leads to restoration of PRICKLE1 in LSMCs. Collectively, our results identify a novel link between estrogen exposure and PRICKLE1/REST-regulated tumorigenic pathways in leiomyomas.

EZH2-H3K27me3-Mediated Epigenetic Silencing of DKK1 Induces Nucleus Pulposus Cell Pyroptosis in Intervertebral Disc Degeneration by Activating NLRP3 and NAIP/NLRC4.

Nucleus pulposus (NP) cell pyroptosis is crucial for intervertebral disc degeneration (IDD). However, the precise mechanisms underlying pyroptosis in IDD remain elusive. Therefore, this study aimed to investigate how dickkopf-1 (DKK1) influences NP cell pyroptosis and delineate the regulatory mechanisms of IDD. Behavioral tests and histological examinations were conducted in rat IDD models to assess the effect of DKK1 on the structure and function of intervertebral discs. Detected pyroptosis levels using Hoechst 33,342/propidium iodide (PI) double staining, and determined pyroptosis-related protein expression via western blotting. The cellular mechanisms of DKK1 in pyroptosis were explored in interleukin (IL)-1β-induced NP cells transfected with or without DKK1 overexpression plasmids (oe-DKK1). In addition, IL-1β-treated NP cells transfected with sh-EZH2 and/or sh-DKK1 were utilized to clarify the interplay between the enhancer of zeste homologue 2 (EZH2) and DKK1 in pyroptosis. Additionally, the epigenetic regulation of DKK1 by EZH2 was explored in NP cells treated with the EZH2 inhibitors GSK126/DZNep. DKK1 expression decreased in IDD rats. Transfection with oe-DKK1 reduced pro-inflammatory factors and extracellular matrix markers in IDD rats. In IL-1β-induced NP cells, DKK1 overexpression suppressed pyroptosis and inhibited the NLRP3 and NAIP/NLRC4 inflammasome activation. EZH2 knockdown increased DKK1 expression and reduced pyroptosis-related proteins. Conversely, DKK1 downregulation reversed the inhibitory effects of EZH2 knockdown on pyroptosis. Furthermore, EZH2 suppressed DKK1 expression via H3K27 methylation at the DKK1 promoter. EZH2 negatively regulates DKK1 expression via H3K27me3 methylation, promoting NP cell pyroptosis in IDD patients. This regulatory effect involves the activation of NLRP3 and NAIP/NLRC4 inflammasomes.

MAPRE3 as an epigenetic target of EZH2 restricts ovarian cancer proliferation in vitro and in vivo

Ovarian cancer (OC) is a lethal gynecologic cancer and the common cause of death within women worldwide. The polycomb group protein enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase highly expressed in various tumors, including OC. However, the mechanistic basis of EZH2 oncogenic activity in OC remain incompletely understood. Bioinformatics analysis showed that the expression of MAPRE3 was lower in OC tissues than in normal tissues, and was positively correlated with the overall survival. MAPRE3 overexpression decreased cell growth, inducing cell cycle arrest and apoptosis in OC cells, whereas MAPRE3 silencing promoted proliferation and accelerated cell cycle progression of OC cells. The in vivo study validated that overexpression of MAPRE3 impeded tumor formation and growth of OC xenografts in nude mice. In addition, knockdown of EZH2 in OC cells downregulated H3K27me3 expression and increased MAPRE3 expression. Inhibiting EZH2 in OC cells reduced the enrichment of H3K27me3 on the promoter of MAPRE3. Furthermore, MAPRE3 silencing significantly reversed changes in the expression of cell cycle and apoptosis-related markers and cell growth mediated by EZH2 knockdown in OC cells. MAPRE3 functions as a suppressor of OC and is epigenetic repressed by EZH2, suggesting a potential therapeutic strategy for OC by targeting EZH2/MAPRE3 axis.

The SOX4/EZH2/SLC7A11 signaling axis mediates ferroptosis in calcium oxalate crystal deposition-induced kidney injury

Epigenetic regulation is reported to play a significant role in the pathogenesis of various kidney diseases, including renal cell carcinoma, acute kidney injury, renal fibrosis, diabetic nephropathy, and lupus nephritis. However, the role of epigenetic regulation in calcium oxalate (CaOx) crystal deposition-induced kidney injury remains unclear. Our study demonstrated that the upregulation of enhancer of zeste homolog 2 (EZH2)-mediated ferroptosis facilitates CaOx-induced kidney injury. CaOx crystal deposition promoted ferroptosis in vivo and in vitro. Usage of liproxstatin-1 (Lip-1), a ferroptosis inhibitor, mitigated CaOx-induced kidney damage. Single-nucleus RNA-sequencing, RNA-sequencing, immunohistochemical and western blotting analyses revealed that EZH2 was upregulated in kidney stone patients, kidney stone mice, and oxalate-stimulated HK-2 cells. Experiments involving in vivo EZH2 knockout, in vitro EZH2 knockdown, and in vivo GSK-126 (an EZH2 inhibitor) treatment confirmed the protective effects of EZH2 inhibition on kidney injury and ferroptosis. Mechanistically, the results of RNA-sequencing and chromatin immunoprecipitation assays demonstrated that EZH2 regulates ferroptosis by suppressing solute carrier family 7, member 11 (SLC7A11) expression through trimethylation of histone H3 lysine 27 (H3K27me3) modification. Additionally, SOX4 regulated ferroptosis by directly modulating EZH2 expression. Thus, this study demonstrated that SOX4 facilitates ferroptosis in CaOx-induced kidney injury through EZH2/H3K27me3-mediated suppression of SLC7A11.Graphical

EZH2-H3K27me3-mediated silencing of mir-139-5p inhibits cellular senescence in hepatocellular carcinoma by activating TOP2A

BackgroundEpigenetic alterations play an important role in hepatocellular carcinoma (HCC) development. Enhancer of zeste homolog 2 (EZH2) is a well-known epigenetic modifier that functions as an oncogene in tumors by promoting the H3K27me3-mediated transcriptional repression of tumor suppressor genes. “Senescent cells” has been proposed as a possible core component of the hallmarks of cancer conceptualization. Induction of cell senescence and targeted elimination of these senescent tumor cells are new strategies for tumor therapy. However, the role of EZH2 in regulating cellular senescence remains poorly understood.MethodsBioinformatics analyses suggested that EZH2 and DNA topoisomerase II alpha (TOP2A) are coexpressed in tumors, including HCC. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analyses and gene set enrichment analyses (GSEA) suggests a correlation of EZH2 and TOP2A expression with cellular senescence in HCC. MicroRNA (miRNA) inhibitor and mimics, siRNA, PLKO-shRNA, and plenti6.3-miR-139 were used to upregulate or downregulate the expression of target genes. CCK8, EdU, clone formation, and senescence-associated β-galactosidase (SA-β-gal) staining assays were performed to assess cell proliferation and cellular senescence phenotypes. Dual-luciferase reporter and chromatin immunoprecipitation assays were performed to investigate the targeted binding and inhibition of TOP2A 3′ untranslated region (UTR) by miR-139-5p and the DNA enrichment of miR139-5p by EZH2 and H3K27me3. BALB/c nude mice were used to establish a xenograft tumor model and verify the phenotypes upon EZH2 and TOP2A silencing and miR-139 overexpression in vivo. In addition, tissue microarrays were used to analyze the expression patterns and correlations among EZH2, TOP2A, and miR-139-5p expression in HCC.ResultsBioinformatics analysis revealed that EZH2 and TOP2A are coexpressed in HCC. In vitro gain- and loss-of-function experiments showed that inhibition of EZH2 and TOP2A induces cellular senescence and inhibits proliferation of HCC cells. In vivo tumorigenesis assays indicated that EZH2 and TOP2A knockdown inhibits tumorigenesis by inducing cellular senescence. Mechanistically, EZH2 promotes TOP2A expression by regulating the H3K27me3-mediated epigenetic silencing of miR-139-5p. TOP2A is a direct target of miR-139-5p, and inhibition of miR-139-5p can reverse the promotion by EZH2 of TOP2A expression. The overexpression of miR-139-5p induces cellular senescence and inhibits proliferation of HCC cells both in vitro and in vivo. Clinically, expression of EZH2 and TOP2A are higher in HCC tissues than in normal tissues, and this high coexpression indicates a worse outcome of patients with HCC. Moreover, expression of EZH2 and TOP2A is significantly correlated with tumor differentiation grade, tumor invasion, and TNM stage in HCC. miR-139-5p expression is lower in HCC tumors than in normal tissues and is correlated with better prognosis of HCC patients.ConclusionsOur study revealed the role of the EZH2/miR-139-5p/TOP2A axis in regulating cellular senescence and cell proliferation in HCC, enriching the molecular mechanisms of EZH2-mediated epigenetic regulation in HCC. Therefore, our results provide insight into the therapeutic potential of targeting EZH2 to induce cellular senescence and then destroy senescent cells for HCC.Graphic

Blood-brain barrier dysfunction mediated by the EZH2-Claudin-5 axis drives stress-induced TNF-α infiltration and depression-like behaviors

Growing evidence suggests that neurovascular dysfunction characterized by blood–brain barrier (BBB) breakdown underlies the development of psychiatric disorders, such as major depressive disorder (MDD). Tight junction (TJ) proteins are critical modulators of homeostasis and BBB integrity. TJ protein Claudin-5 is the most dominant BBB component and is downregulated in numerous depression models; however, the underlying mechanisms remain elusive. Here, we demonstrate a molecular basis of BBB breakdown that links stress and depression. We implemented an animal model of depression, chronic unpredictable mild stress (CUMS) in male C57BL/6 mice, and showed that hippocampal BBB breakdown was closely associated with stress vulnerability. Concomitantly, we found that dysregulated Cldn5 level coupled with repression of the histone methylation signature at its promoter contributed to stress-induced BBB dysfunction and depression. Moreover, histone methyltransferase enhancer of zeste homolog 2 (EZH2) knockdown improved Cldn5 expression and alleviated depression-like behaviors by suppressing the tri-methylation of lysine 27 on histone 3 (H3K27me3) in chronically stressed mice. Furthermore, the stress-induced excessive transfer of peripheral cytokine tumor necrosis factor-α (TNF-α) into the hippocampus was prevented by Claudin-5 overexpression and EZH2 knockdown. Interestingly, antidepressant treatment could inhibit H3K27me3 deposition at the Cldn5 promoter, reversing the loss of the encoded protein and BBB damage. Considered together, these findings reveal the importance of the hippocampal EZH2-Claudin-5 axis in regulating neurovascular function and MDD development, providing potential therapeutic targets for this psychiatric illness.

Lysophosphatidic Acid Induces Podocyte Pyroptosis in Diabetic Nephropathy by an Increase of Egr1 Expression via Downregulation of EzH2.

Podocyte damage and renal inflammation are the main features and pathogenesis of diabetic nephropathy (DN). Inhibition of lysophosphatidic acid (LPA) receptor 1 (LPAR1) suppresses glomerular inflammation and improves DN. Herein, we investigated LPA-induced podocyte damage and its underlying mechanisms in DN. We investigated the effects of AM095, a specific LPAR1 inhibitor, on podocytes from streptozotocin (STZ)-induced diabetic mice. E11 cells were treated with LPA in the presence or absence of AM095, and the expression of NLRP3 inflammasome factors and pyroptosis were measured. A chromatin immunoprecipitation assay and Western blotting were performed to elucidate underlying molecular mechanisms. Gene knockdown by transfecting small interfering RNA was used to determine the role of the transcription factor Egr1 (early growth response protein 1) and histone methyltransferase EzH2 (Enhancer of Zeste Homolog 2) in LPA-induced podocyte injury. AM095 administration inhibited podocyte loss, NLRP3 inflammasome factor expression, and cell death in STZ-induced diabetic mice. In E11 cells, LPA increased NLRP3 inflammasome activation and pyroptosis via LPAR1. Egr1 mediated NLRP3 inflammasome activation and pyroptosis in LPA-treated E11 cells. LPA decreased H3K27me3 enrichment at the Egr1 promoter in E11 cells by downregulating EzH2 expression. EzH2 knockdown further increased LPA-induced Egr1 expression. In podocytes from STZ-induced diabetic mice, AM095 suppressed Egr1 expression increase and EzH2/H3K27me3 expression reduction. Collectively, these results demonstrate that LPA induces NLRP3 inflammasome activation by downregulating EzH2/H3K27me3 and upregulating Egr1 expression, resulting in podocyte damage and pyroptosis, which may be a potential mechanism of DN progression.

Targeting B7‑H3 through EZH2 inhibition in MYC‑positive Group 3 medulloblastoma.

The most aggressive subtype of medulloblastoma (MB), Group 3, is characterized by MYC amplifications. However, targeting MYC has proven unsuccessful, and there remains a lack of therapeutic targets for treating MB. Studies have shown that the B7 homolog 3 (B7‑H3) promotes cell proliferation and tumor cell invasion in a variety of cancers. Similarly, it was recently revealed that B7‑H3 promotes angiogenesis in Group 3 MB and likely facilitates MB metastasis through exosome biogenesis. While therapies targeting B7‑H3 remain in the early stages of development, targeting upstream regulators of B7‑H3 expression may be more effective for halting MB progression. Notably, MYC and the enhancer of zeste homolog 2 (EZH2) are known to regulate B7‑H3 expression, and a previous study by the authors suggested that B7‑H3 amplifications present in MB are likely the result of EZH2‑MYC mediated activities. In the present study, it was reported that overexpression of EZH2 is associated with lower overall survival in Group 3 MB patients. It was also revealed that inhibition of EZH2 significantly reduces B7‑H3 and MYC transcript levels and upregulates miR‑29a, indicating that EZH2 post‑transcriptionally regulates B7‑H3 expression in Group 3 MB cells. Pharmacological inhibition of EZH2 using EPZ005687 attenuated MB cell viability and reduced the expression of B7‑H3. Similarly, pharmacological inhibition and knockdown of EZH2 led to the downregulation of MYC, B7‑H3, and H3K27me3. Further, EZH2 silencing induced apoptosis and reduced colony‑forming ability in MB cells, whereas EZH2 inhibition in MYC‑amplified C17.2 neural stem cells induced G2/M phase arrest while downregulating B7‑H3 expression. Collectively, the current study positions EZH2 as a viable target for the future development of MB treatments and that targeting EZH2 in combination with B7‑H3 immunotherapy may be an effective treatment for halting MB progression.

Dual role of enhancer of zeste homolog 2 in the regulation of ultraviolet radiation-induced matrix metalloproteinase-1 and type I procollagen expression in human dermal fibroblasts

Abnormalities in the extracellular matrix (ECM) caused by ultraviolet (UV) radiation are mediated by epigenetic mechanisms. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that is implicated in inflammation, immune regulation, and senescence. However, its role in controlling UV-induced ECM alterations in the skin remains elusive. Here, we investigated the role of EZH2 in UV-induced expression of matrix metalloproteinase (MMP)-1 and type I procollagen. We found that UV induced EZH2 expression in human skin in vivo and in human dermal fibroblasts (HDFs). EZH2 knockdown reduced the expression and promoter activity of MMP-1 and increased those of type I procollagen, whereas EZH2 overexpression had the opposite effects. Mechanistically, EZH2 increased NF-κB activity, and p65 and p50 expression and promoter activity. Intriguingly, chromatin immunoprecipitation assays revealed that the EZH2/p65/p50 complex was recruited and bound to the MMP-1 promoter after UV irradiation, independent of its histone methyltransferase activity. In contrast, EZH2-induced DNA methyltransferase 1 (DNMT1) formed a complex with EZH2 and enhanced the enrichment of H3K27me3 on the COL1A2 promoter following UV irradiation. These findings indicate that EZH2 plays a dual role in regulating MMP-1 and type I procollagen expression and improve our understanding of how this epigenetic mechanism contributes to UV-induced skin responses and photoaging. This study shows that inhibiting EZH2 is a potential anti-aging strategy for preventing UV-induced skin aging by reducing MMP-1 expression and inducing type I procollagen expression.

Stage-specific dual function: EZH2 regulates human erythropoiesis by eliciting histone and non-histone methylation.

Enhancer of zeste homolog 2 (EZH2) is the lysine methyltransferase of polycomb repressive complex 2 (PRC2) that catalyzes H3K27 tri-methylation. Aberrant expression and loss-of-function mutations of EZH2 have been demonstrated to be tightly associated with the pathogenesis of various myeloid malignancies characterized by ineffective erythropoiesis, such as myelodysplastic syndrome (MDS). However, the function and mechanism of EZH2 in human erythropoiesis still remains largely unknown. Here, we demonstrated that EZH2 regulates human erythropoiesis in a stage-specific, dual-function manner by catalyzing histone and non-histone methylation. During the early erythropoiesis, EZH2 deficiency caused cell cycle arrest in the G1 phase, which impaired cell growth and differentiation. Chromatin immunoprecipitation sequencing and RNA sequencing discovered that EZH2 knockdown caused a reduction of H3K27me3 and upregulation of cell cycle proteindependent kinase inhibitors. In contrast, EZH2 deficiency led to the generation of abnormal nuclear cells and impaired enucleation during the terminal erythropoiesis. Interestingly, EZH2 deficiency downregulated the methylation of HSP70 by directly interacting with HSP70. RNA-sequencing analysis revealed that the expression of AURKB was significantly downregulated in response to EZH2 deficiency. Furthermore, treatment with an AURKB inhibitor and small hairpin RNAmediated AURKB knockdown also led to nuclear malformation and decreased enucleation efficiency. These findings strongly suggest that EZH2 regulates terminal erythropoiesis through a HSP70 methylation-AURKB axis. Our findings have implications for improved understanding of ineffective erythropoiesis with EZH2 dysfunction.

LncRNA SLC25A21-AS1 increases the chemosensitivity and inhibits the progression of ovarian cancer by upregulating the expression of KCNK4.

Owing to high mortality rate, ovarian cancer seriously threatens women's health. Extensive abdominal metastasis and chemoresistance are the leading causes of ovarian cancer deaths. Through lncRNA sequencing, our previous study identified lncRNA SLC25A21-AS1, which was significantly downregulated in chemoresistant ovarian cancer cells. In this study, we aimed to evaluate the role and mechanism of SLC25A21-AS1 in ovarian cancer. The expression of SLC25A21-AS1 was analyzed by qRT-PCR and online database GEPIA. The biological functions of SLC25A21-AS1 and KCNK4 were analyzed by CCK-8, transwell, and flow cytometry. The specific mechanism was analyzed by RNA-sequencing, RNA binding protein immunoprecipitation, rescue experiments, and bioinformatic analysis. SLC25A21-AS1 was decreased in ovarian cancer tissues and cell lines. Overexpression of SLC25A21-AS1 enhanced the sensitivity of ovarian cancer cells to paclitaxel and cisplatin, and inhibited cell proliferation, invasion, and migration, while SLC25A21-AS1-silencing showed the opposite effect. Potassium channel subfamily K member 4 (KCNK4) was significantly up-regulated upon enforced expression of SLC25A21-AS1. Overexpression of KCNK4 inhibited cell proliferation, invasion, migration ability, and enhanced the sensitivity of ovarian cancer cells to paclitaxel and cisplatin. Meanwhile, KNCK4-overexpression rescued the promotive effect of SLC25A21-AS1-silencing on cell proliferation, invasion and migration. In addition, SLC25A21-AS1 could interact with the transcription factor Enhancer of Zeste Homolog 2 (EZH2), while EZH2 knockdown increased the expression of KCNK4 in some of the ovarian cancer cell lines. SLC25A21-AS1 enhanced the chemosensitivity and inhibited the proliferation, migration, and invasion ability of ovarian cancer cells at least partially by blocking EZH2-mediated silencing of KCNK4.

MiRNA let-7a inhibits invasion, migration, anchorage-independent growth by suppressing EZH2 and promotes mesenchymal to epithelial transition in MDAMB-231

Enhancer of zeste homolog 2 (EZH2) is a transcriptional repressor belonging to the polycomb group protein family (PRC) and is frequently linked with aggressive breast cancer. Micro RNAs (miRNAs) are single-stranded 19–25 nucleotide long RNAs that control gene expression at the post-transcriptional level. Previous reports indicated that epigenetic modulation through EZH2-mediated histone methylation can silence the miR-200 cluster. Therefore, we attempted to identify the miRNAs that regulate the expression of EZH2 in metastatic breast cancer. Our results with microarray studies showed that upon knock-down of EZH2 lead to enhanced expression of tumor-suppressive miRNAs including miR-203, miR-200a, miR-200b, and let-7a in MDAMB-231, a metastatic breast cancer cell line. Further studies to confirm the expression of EZH2 upon overexpression of the mentioned miRNAs showed that only let-7a could post-transcriptionally regulate EZH2 expression significantly. Luciferase-based 3′UTR (untranslated region) reporter assay of EZH2 confirmed a significant reduction in the reporter activity upon co-transfection of let-7a and reporter construct 3′UTR of EZH2. Interestingly, overexpression of let-7a leads to decreased expression of another PRC2 group of protein SUZ12 and produced a lowered expression of H3K27me3 methylation. Additionally, let-7a miRNA promoted mesenchymal to epithelial transition i.e., by downregulating the key metastatic markers including Vimentin, Zeb, N-cadherin, and Snail, thus leading to reduced migration and invasion ability in MDA-MB-231 cells. This data poses an impact on understanding and developing therapeutics for breast cancer.

Transcriptome analysis constructed the necroptosis associated prognostic signature in endometrial cancer and identified EZH2 as a potential biomarker

Endometrial cancer (EC) is one of the most common malignancies of the female reproductive system, but our understanding of the tumor microenvironment of EC remains unclear. Programmed cell death (PCD) plays an important role in the genesis and progression of tumors. Necroptosis is a novel form of PCD that does not rely on the caspase system. However, the role of necroptosis in EC is unclear. Transcriptome data of endometrial cancer were downloaded from The Cancer Genome Atlas (TCGA) database and log2 conversion was performed. Expression analysis and correlation analysis were performed to explore necroptosis gene expression and interaction in EC. Lasso regression was used to construct necroptosis-related prognostic signature. Finally, immunocorrelation analysis and single cell sequencing analysis were used to explore the significance of this signature in EC tumor microenvironment. A total of 15 of the 17 necroptosis genes were differentially expressed in EC. Subsequently, necroptosis related prognostic signature was constructed through Lasso regression. Riskscore = (−0.0999) × Toll-likereceptor4 (TLR4) + (−0.0528) × tumor necrosis factor receptor superfamily member 1A (TNFRSF1A) + (0.1208) × Enhancer of Zeste Homolog 2 (EZH2) + (−0.004) × N-myc Down-stream Regulated Gene 2 (NDRG2). EC patients can be divided into high-risk group and low-risk group based on the median riskscore and the high-risk group has a worse prognosis. Survival analysis showed a worse prognosis for patients in the high-risk group (p < 0.05). Immunomicroenvironment analysis showed a significant negative correlation between risk score and infiltration levels of B cells, CD4+ T cells, CD8+ T cells, Endothelial cells, macrophages, and NK cells. Subsequent cell experiments showed that knockdown of the key gene EZH2 in signature significantly reduced the invasion, migration and healing abilities of EC cell lines, proving that EZH2 is a promising marker of EC.

EZH2-H3K27me3 mediated KRT14 upregulation promotes TNBC peritoneal metastasis

Triple-Negative Breast Cancer (TNBC) has a poor prognosis and adverse clinical outcomes among all breast cancer subtypes as there is no available targeted therapy. Overexpression of Enhancer of zeste homolog 2 (EZH2) has been shown to correlate with TNBC’s poor prognosis, but the contribution of EZH2 catalytic (H3K27me3) versus non-catalytic EZH2 (NC-EZH2) function in TNBC progression remains elusive. We reveal that selective hyper-activation of functional EZH2 (H3K27me3) over NC-EZH2 alters TNBC metastatic landscape and fosters its peritoneal metastasis, particularly splenic. Instead of H3K27me3-mediated repression of gene expression; here, it promotes KRT14 transcription by attenuating binding of repressor SP1 to its promoter. Further, KRT14 loss significantly reduces TNBC migration, invasion, and peritoneal metastasis. Consistently, human TNBC metastasis displays positive correlation between H3K27me3 and KRT14 levels. Finally, EZH2 knockdown or H3K27me3 inhibition by EPZ6438 reduces TNBC peritoneal metastasis. Altogether, our preclinical findings suggest a rationale for targeting TNBC with EZH2 inhibitors.

EZH2 Promotes Cholangiocarcinoma Development and Progression through Histone Methylation and microRNA-Mediated Down-Regulation of Tumor Suppressor Genes

Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tree. Although studies have implicated enhancer of Zeste homolog 2 (EZH2) in CCA growth, the role of EZH2 in CCA development has not been investigated, and the mechanism for EZH2-regulated gene expression in CCA remains to be further defined. The current study used a mouse model of CCA induced by hydrodynamic tail vein injection of Notch1 intracellular domain and myristoylated-AKT plasmids. Mice with liver-specific EZH2 knockout displayed reduced CCA development. In a xenograft model, EZH2 knockdown significantly decreased CCA progression. Administration of the EZH2 inhibitor GSK126 decreased CCA tumor burden in mice. Accordingly, EZH2 depletion or inhibition reduced the growth and colony formation capability of CCA cells. Analysis of high-throughput data identified a set of 12 tumor-inhibiting genes as targets of EZH2 in CCA. The experimental results suggest that EZH2 may down-regulate these tumor-inhibiting genes through methylation of lysine 27 on histone H3 (H3K27) in the gene louses and through regulation of specific miRNAs. High mobility group box 1 was shown to facilitate the methyltransferase activity of EZH2, which is implicated in the regulation of CCA cell growth. The study shows that EZH2 promotes CCA development and progression through a complicated regulatory network involving tumor-inhibiting genes, miRNAs, and high mobility group box 1, which support targeting EZH2 as a potentially effective strategy for CCA treatment.

Dysregulation of EZH2/miR-138-5p Axis Contributes to Radiosensitivity in Hepatocellular Carcinoma Cell by Downregulating Hypoxia-Inducible Factor 1 Alpha (HIF-1α).

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase involved in cell proliferation, invasion, angiogenesis, and metastasis in various cancers, including hepatocellular carcinoma (HCC). However, the role and molecular mechanisms of EZH2 in HCC radiosensitivity remain unclear. Here, we show that EZH2 is upregulated in HCC cells and the aberrantly overexpressed EZH2 is associated with the poor prognosis of HCC patients. Using miRNA databases, we identified miR-138-5p as a regulator of EZH2. We also found that miR-138-5p was suppressed by EZH2-induced H3K27me3 in HCC cell lines. MiR-138-5p overexpression and EZH2 knockdown enhanced cellular radiosensitivity while inhibiting cell migration, invasion, and epithelial-mesenchymal transition (EMT). Analysis of RNA-seq datasets revealed that the hypoxia-inducible factor-1 (HIF-1) signaling pathway was the main enrichment pathway for differential genes after miR-138-5p overexpression or EZH2 knockdown. Expression level of HIF-1α was significantly suppressed after miR-138-5p overexpression or silencing of EZH2. HIF-1α silencing mitigated resistance of HCC cells and inhibited EMT. This study establishes the EZH2/miR-138-5p/HIF-1α as a potential therapeutic target for sensitizing HCC to radiotherapy.

Hypoxic condition induced H3K27me3 modification of the LncRNA Tmem235 promoter thus supporting apoptosis of BMSCs

Bone marrow mesenchymal stem cells (BMSCs) have strong regenerative potential and show good application prospects for treating clinical diseases. However, in the process of BMSC transplantation for treating ischemic and hypoxic diseases, BMSCs have high rates of apoptosis in the hypoxic microenvironment of transplantation, which significantly affects the transplantation efficacy. Our previous studies have confirmed the key role of long non-coding RNA Tmem235 (LncRNA Tmem235) in the process of hypoxia-induced BMSC apoptosis and its downstream regulatory mechanism, but the upstream mechanism by which hypoxia regulates LncRNA Tmem235 expression to induce BMSC apoptosis is still unclear. Under hypoxic conditions, we found that the level of LncRNA Tmem235 promoter histone H3 lysine 27 trimethylation modification (H3K27me3) was significantly increased by CHIP-qPCR. Moreover, H3K27me3 cooperated with LncRNA Tmem235 promoter DNA methylation to inhibit the expression of LncRNA Tmem235 and promote apoptosis of BMSCs. To study the mechanism of hypoxia-induced modification of LncRNA Tmem235 promoter H3K27me3 in the hypoxia model of BMSCs, we detected the expression of H3K27 methylase and histone demethylase and found that only histone methylase enhancer of zeste homolog 2 (EZH2) expression was significantly upregulated. Knockdown of EZH2 significantly decreased the level of H3K27me3 modification in the LncRNA Tmem235 promoter. The EZH2 promoter region contains a hypoxia-responsive element (HRE) that interacts with hypoxia-inducible factor-1alpha (HIF-1α), which is overexpressed under hypoxic conditions, thereby promoting its overexpression. In summary, hypoxia promotes the modification of the LncRNA Tmem235 promoter H3K27me3 through the HIF-1α/EZH2 signaling axis, inhibits the expression of LncRNA Tmem235, and leads to hypoxic apoptosis of BMSCs. Our findings improve the regulatory mechanism of LncRNA Tmem235 during hypoxic apoptosis of BMSCs and provide a more complete theoretical pathway for targeting LncRNA to inhibit hypoxic apoptosis of BMSCs.

Targeting Triple-Negative Breast Cancer by a Novel Proteolysis Targeting Chimera Degrader of Enhancer of Zeste Homolog 2.

Enhancer of zeste homolog 2 (EZH2), a catalytic subunit of polycomb repressive complex 2 (PRC2), is overexpressed in triple-negative breast cancer (TNBC), correlating with poor prognosis. However, EZH2 catalytic inhibitors are ineffective in suppressing the growth of TNBC cells that are dependent on EZH2. Knockdown of EZH2 inhibits the proliferation of these cells, suggesting that EZH2 protein overexpression but not its catalytic activity is critical for driving TNBC progression. Several proteolysis targeting chimera (PROTAC) degraders of EZH2, including the von Hippel-Lindau (VHL)-recruiting PROTAC YM281, have been reported. However, the effects of these EZH2 PROTACs in TNBC cells were not investigated. Here, we report the discovery and characterization of a novel, potent, and selective EZH2 PROTAC degrader, MS8815 (compound 16), which induced robust EZH2 degradation in a concentration-, time-, and proteasome-dependent manner in TNBC cells. Importantly, 16 effectively suppressed the cell growth in multiple TNBC cell lines and primary patient TNBC cells.