Enhancer Of Zeste Homolog 2 Knockdown Research Articles

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.

EZH2 engages TGFβ signaling to promote breast cancer bone metastasis via integrin β1-FAK activation

Bone metastases occur in 50–70% of patients with late-stage breast cancers and effective therapies are needed. The expression of enhancer of zeste homolog 2 (EZH2) is correlated with breast cancer metastasis, but its function in bone metastasis hasn’t been well-explored. Here we report that EZH2 promotes osteolytic metastasis of breast cancer through regulating transforming growth factor beta (TGFβ) signaling. EZH2 induces cancer cell proliferation and osteoclast maturation, whereas EZH2 knockdown decreases bone metastasis incidence and outgrowth in vivo. Mechanistically, EZH2 transcriptionally increases ITGB1, which encodes for integrin β1. Integrin β1 activates focal adhesion kinase (FAK), which phosphorylates TGFβ receptor type I (TGFβRI) at tyrosine 182 to enhance its binding to TGFβ receptor type II (TGFβRII), thereby activating TGFβ signaling. Clinically applicable FAK inhibitors but not EZH2 methyltransferase inhibitors effectively inhibit breast cancer bone metastasis in vivo. Overall, we find that the EZH2-integrin β1-FAK axis cooperates with the TGFβ signaling pathway to promote bone metastasis of breast cancer.

Combining EGFR-TKI With SAHA Overcomes EGFR-TKI-Acquired Resistance by Reducing the Protective Autophagy in Non-Small Cell Lung Cancer.

Nowadays, lung cancer has the highest mortality worldwide. The emergence of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has greatly improved the survival of patients with non-small cell lung cancer (NSCLC) having EGFR-TKI-sensitive mutations. Unfortunately, acquired resistance happens for most patients. In the present research, we found that EGFR-TKIs (such as gefitinib and osimertinib) can induce autophagy in NSCLC cell lines. Compared with parental sensitive cells, drug-resistant cells have higher autophagy activity. The use of an autophagy inhibitor could enhance the toxicity of gefitinib and osimertinib, which indicates that the enhancement of protective autophagy might be one of the mechanisms of EGFR-TKI resistance in NSCLC. In addition, increased autophagy activity is associated with decreased enhancer of zeste homolog 2 (EZH2) expression. Knockdown of EZH2 or EZH2 inhibitor treatment could lead to increased autophagy in NSCLC cells, indicating that EZH2 is a negative regulator of autophagy. We revealed that the increase in autophagy caused by the reduction of EZH2 was reversed in vitro and in vivo when combining gefitinib or osimertinib with suberoylanilide hydroxamic acid (SAHA), a broad-spectrum histone deacetylase inhibitor (HDACi). In conclusion, our results indicated that the combination of EGFR-TKIs and SAHA may be a new strategy to overcome EGFR-TKIs acquired resistance.

Abstract PD3-03: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

Abstract Introduction: Triple negative breast cancers (TNBC) are frequently poorly differentiated with higher propensity for metastasis than all other subtypes. Enhancer of Zeste Homolog 2 (EZH2) is a lysine methyltransferase that mediates transcriptional repression of pro-differentiation genes in normal and neoplastic cells. The oncogenic role of EZH2 through H3K27me3 is well established. However, non-canonical H3K27me3-independent functions are unclear. We have reported that p38 phosphorylates T367 of EZH2, increasing TNBC metastasis. Recent reports show that EZH2 can regulate signaling pathways through direct methylation of proteins suggesting the hypothesis that EZH2 may methylate p38 in TNBC. Methods: Human tissue samples from 16 primary invasive breast carcinomas and matched distant metastasis arrayed in tissue microarrays were interrogated for pEZH2 T367 and p-p38 by immunohistochemistry. To study p38 methylation, we performed LC-MS/MS analyses GST-p38α, and GST-EZH2 were each incubated with or without recombinant PRC2 complex (EZH2/E ED/SUZ12/RbAp48/AEBP2) and S-adenosyl methionine methyl donor. MDA-MB-231, mouse and human primary TNBC cells and murine 4T1 cells were used in funcional assays in vitro and in vivo. EZH2 knockdown and rescue was carried out using lentiviral transduction of EZH2 with pBabe-myc-EZH2 (wild-type) or pBabe-myc-EZH2 (T367A). We developed p38 methylation mutants: HA-p38a K139A, HA-p38a K165A, and HA-p38a K130A/K165K. To investigate the importance of EZH2 and p38 enzymatic activities we used the EZH2 methyltransferase inhibitors GSK-343 and EPZ-6438 and the p38 inhibitor SB202190 in vitro and in vivo. For animal studies, MDA-MB-231 and 4T1 cells were orthotopically injected into the right inguinal mammary fat pad of eight-week old NOD/SCID or BALBc mice, respectively. When tumors reached 100 mm3 mice were treated 5 days/week by intraperitoneal injection with SB202190 (1mg/kg/day), EPZ-6438 (10mg/kg/day), combination of SB202190 and EPZ-6438, or control. We monitored tumor growth for 50 days, and harvested primary tumors, lungs, and sites of metastasis for histopathology, Western blot, and immunohistochemistry. Primary xenografts from the control and treatment groups were subjected to RNA sequencing. Results: EZH2 methylates p38 at lysines 139 and 165 leading to enhanced p38 stability and increased invasion, and p38 activation requires T367 phosphorylation of EZH2. Dual inhibition of EZH2 methyltransferase and p38 kinase activities downregulates pEZH2 T367, H3K27me3, and p-p38 in vivo, reduces TNBC growth and metastasis, and results in gene expression changes towards a better differentiated phenotype. pEZH2 T367 and p-p38 proteins are significantly coexpressed in human primary and metastatic breast cancer. Conclusions: We provide direct evidence that EZH2 methylates p38 with resultant p38 activation, and that EZH2 phosphorylation at T367 is important for this function. Dual targeting of EZH2 methyltransferase and p38 kinase activities with specific inhibitors reduces breast cancer growth and metastasis indicating a cooperation between EZH2 canonical and non-canonical mechanisms and suggesting therapeutic strategies. Citation Format: Maria E Gonzalez, Shilpa R. Tekula, Talha Anwar, Shoshana A. Leflein, Celina G. Kleer. EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-03.

Dysregulated lipid metabolism blunts the sensitivity of cancer cells to EZH2 inhibitor.

SummaryBackgroundSensitivity has been a key issue for Enhancer of zeste homolog 2 (EZH2) inhibitors in cancer therapy. The EZH2 inhibitor EPZ-6438 was first approved by the US Food and Drug Administration (FDA) in 2020. However, its inadequate anti-cancer activity in solid tumors limits its clinical application. In this study, we utilized the multiple cancer cell lines, which are less sensitive to the EZH2 inhibitor GSK126, combining animal model and clinical data to investigate the underlying mechanism.MethodsIncuCyte S3 was used to explore the difference in the responsiveness of hematological tumor cells and solid tumor cells to GSK126. Transcriptome and metabolome of B16F10 cells after GSK126 treatment were analyzed and the distinct changes in the metabolic profile were revealed. Real-time quantitative PCR and western blot experiments were used to further verify the multi-omics data. ChIP-qPCR was performed to detected H3K27me3 enrichment of target genes. Finally, the anti-tumor effects of combining GSK126 and lipid metabolism drugs were observed with IncuCyte S3 platform, CCK-8 and animal model respectively.FindingsWe found that although the proliferative phenotype did not show strong difference upon treatment with GSK126, the transcriptome and metabolome changed profoundly. GSK126 treatment led to broad shifts in glucose, amino acid, and lipid metabolism. Lipid synthesis was strengthened manifested by the increasing abundance of unsaturated fatty acids. SCD1 and ELOVL2 were regulated by H3K27me3 at gene regulatory region, and upregulated by EZH2 knockdown and inhibitors. SCD1 knockdown increased cellular sensitivity to GSK126. Based on the findings above, the application of the combination with SCD1 inhibitor significantly attenuated the proliferation of cancer and increased the sensitivity to GSK126 by suppressing desaturation of fatty acids.InterpretationDysregulated lipid metabolism can blunt the sensitivity of cancer cells to GSK126. These characteristics shed light on the novel combination therapy strategies to combat tumor resistance.FundingNational Natural Science Foundation of China (No. 81672091, No.91749107 and No. 81972966).