Enhancer Of Zeste Homolog 2 Target 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.

Assessment of the anti-tumor activity of cyanidin-3-O-arabinoside from apple against APN, JAK, and EZH2 target proteins

This study aimed to evaluate the anti-tumor activity and the mechanism of apple polyphenols on aminopeptidase N (APN), janus kinase 3 (JAK3) and enhancer of zeste homolog 2 (EZH2) using molecular docking and in vitro human colon (Caco-2) cells. The 88 apple polyphenols were docked with targets, i.e., APN, JAK3, and EZH2, and the potential polyphenol cyanidin-3-O-arabinoside was selected. CCK-8 assay results showed that cyanidin-3-O-arabinoside exerted a significant inhibitory effect on the proliferation of Caco-2 cells, with an IC50 value of 112 ± 4.36 μg mL−1 at 72 h. Results indicated that cyanidin-3-O-glucoside can block S phase to induce cell apoptosis by influencing S phase and the expression of Bcl-2 and Bax. In summary, cyanidin-3-O-glucoside from apple polyphenols may be used as a potential inhibitor to prevent colon cancer.

LncRNA MEG3 up-regulates SIRT6 by ubiquitinating EZH2 and alleviates nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a global health threat. Here, we presented the significant role of a novel signaling axis comprising long non-coding RNA maternally expressed gene 3 (MEG3), enhancer of zeste homolog 2 (EZH2), and sirtuin 6 (SIRT6) in controlling lipid accumulation, inflammation, and the progression of NAFLD. Mice fed with high-fat diet (HFD) were established as in vitro and in vivo NAFLD models, respectively. Lipid accumulation was measured by oil red O staining and assays for triglycerides or cholesterol. Inflammation was examined by ELISA for pro-inflammatory cytokines. Gene expressions were examined by RT-qPCR or Western blot. Interactions between key signaling molecules were examined by combining expressional analysis, RNA immunoprecipitation, cycloheximide stability assay, co-immunoprecipitation, and chromatin immunoprecipitation. MEG3 level was reduced in FFA-challenged hepatocytes or liver from HFD-fed mice, and the reduction paralleled the severity of NAFLD in clinic. Overexpressing MEG3 suppressed FFA-induced lipid accumulation or inflammation in hepatocytes. By promoting the ubiquitination and degradation of EZH2, MEG3 upregulated SIRT6, an EZH2 target. SIRT6 essentially mediated the protective effects of MEG3 in hepatocytes. Consistently, overexpressing MEG3 alleviated HFD-induced NAFLD in vivo. By controlling the expressions of genes involved in lipid metabolism and inflammation, the MEG3/EZH2/SIRT6 axis significantly suppressed lipid accumulation and inflammation in vitro, and NAFLD development in vivo. Therefore, boosting MEG3 level may benefit the treatment of NAFLD.

LncRNA UCA1 Antagonizes Arsenic-Induced Cell Cycle Arrest through Destabilizing EZH2 and Facilitating NFATc2 Expression

Arsenic (As) is a widespread metalloid contaminant, and its internal exposure is demonstrated to cause serious detrimental health problems. Albeit considerable studies are performed to interrogate the molecular mechanisms responsible for As-induced toxicities, the exact mechanisms are not fully understood yet, especially at the epigenetic regulation level. In the present study, it is identified that long non-coding RNA (lncRNA) urothelial cancer associated 1 (UCA1) alleviates As-induced G2/M phase arrest in human liver cells. Intensive mechanistic investigations illustrate that UCA1 interacts with enhancer of zeste homolog 2 (EZH2) and accelerates the latter's protein turnover rate under normal and As-exposure conditions. The phosphorylation of EZH2 at the Thr-487 site by cyclin dependent kinase 1 (CDK1) is responsible for As-induced EZH2 protein degradation, and UCA1 enhances this process through increasing the interaction between CDK1 and EZH2. As a consequence, the cell cycle regulator nuclear factor of activated T cells 2 (NFATc2), a downstream target of EZH2, is upregulated to resist As-blocked cell cycle progress and cytotoxicity. In conclusion, the findings decipher a novel prosurvival signaling pathway underlying As toxicity from the perspective of epigenetic regulation: UCA1 facilitates the ubiquitination of EZH2 to upregulate NFATc2 and further antagonizes As-induced cell cycle arrest.

EZH2 activates CHK1 signaling to promote ovarian cancer chemoresistance by maintaining the properties of cancer stem cells.

Background: Ovarian cancer is a fatal malignant gynecological tumor. Ovarian cancer stem cells (OCSCs) contribute to resistance to chemotherapy. The polycomb group protein enhancer of zeste homolog 2 (EZH2) plays a key role in maintaining CSCs. Here, we aimed to investigate the specific mechanism by which EZH2 regulates CSCs to result in chemoresistance and poor prognosis of ovarian cancer.Methods: We used a nude mouse model to obtain a cell line enriched for OCSCs, named SK-3rd cells. The CRISPR and Cas9 endonuclease system was used to establish an EZH2-knockout SK-3rd ovarian cancer cell line. High-throughput PCR array and bioinformatics methods were used to screen the EZH2 target involved in CSC stemness. A luciferase reporter assay and chromatin immunoprecipitation assay were performed to identify activation of CHK1 by EZH2. We evaluated associations between EZH2/CHK1 expression and the chemoresistance and prognosis of ovarian cancer patients.Results: EZH2 plays a critical role in maintaining ovarian CSC stemness and chemo-resistance. CHK1 is an EZH2 target involved in CSC stemness. Knockdown of EZH2 in ovarian CSCs decreased CHK1 expression, while CHK1 overexpression was sufficient to reverse the inhibitory effect on spheroid formation and chemoresistance caused by repression of EZH2. In addition, EZH2 was also shown to play a unique role in activating rather than repressing CHK1 signaling through binding to the CHK1 promoter in epithelial ovarian cancer cells. Finally, in clinical samples, ovarian cancer patients with high levels of EZH2 and CHK1 not only were more resistant to platinum but also had a poorer prognosis.Conclusions: Our data revealed a previously unidentified functional and mechanistic link between EZH2 levels, CHK1 signaling activation, and ovarian CSCs and provided strong evidence that EZH2 promotes ovarian cancer chemoresistance and recurrence.

Methylation of EZH2 by PRMT1 regulates its stability and promotes breast cancer metastasis.

Enhancer of zeste homolog 2 (EZH2), a key histone methyltransferase and EMT inducer, is overexpressed in diverse carcinomas, including breast cancer. However, the molecular mechanisms of EZH2 dysregulation in cancers are still largely unknown. Here, we discover that EZH2 is asymmetrically dimethylated at R342 (meR342-EZH2) by PRMT1. meR342-EZH2 was found to inhibit the CDK1-mediated phosphorylation of EZH2 at T345 and T487, thereby attenuating EZH2 ubiquitylation mediated by the E3 ligase TRAF6. We also demonstrate that meR342-EZH2 resulted in a decrease in EZH2 target gene expression, but an increase in breast cancer cell EMT, invasion and metastasis. Moreover, we confirm the positive correlations among PRMT1, meR342-EZH2 and EZH2 expression in the breast cancer tissues. Finally, we report that high expression levels of meR342-EZH2 predict a poor clinical outcome in breast cancer patients. Our findings may provide a novel diagnostic target and promising therapeutic target for breast cancer metastasis.

Increased EZH2 Levels in Anterior Cingulate Cortex Microglia Aggravate Neuropathic Pain by Inhibiting Autophagy Following Brachial Plexus Avulsion in Rats.

After brachial plexus avulsion (BPA), microglia induce inflammation, initiating and maintaining neuropathic pain. EZH2 (enhancer of zeste homolog 2) has been implicated in inflammation and neuropathic pain, but the mechanisms by which it regulates neuropathic pain remain unclear. Here, we found that EZH2 levels were markedly upregulated during BPA-induced neuropathic pain in vivo and in vitro, stimulating pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) secretion in vivo. In rats with BPA-induced neuropathic pain, mechanical and cold hypersensitivities were induced by EZH2 upregulation and inhibited by EZH2 downregulation in the anterior cingulate cortex. Microglial autophagy was also significantly inhibited, with EZH2 inhibition activating autophagy and reducing neuroinflammation in vivo. However, this effect was impaired by inhibiting autophagy with 3-methyladenine, suggesting that the MTOR signaling pathway is a functional target of EZH2. These data suggest that EZH2 regulates neuroinflammation and neuropathic pain via a novel MTOR-mediated autophagy signaling pathway, providing a promising approach for managing neuropathic pain.

LncRNA UCA1 Antagonizes Arsenic-Induced Cell Cycle Arrest through Destabilizing EZH2 and Facilitating NFATc2 Expression.

Arsenic (As) is a widespread metalloid contaminant, and its internal exposure is demonstrated to cause serious detrimental health problems. Albeit considerable studies are performed to interrogate the molecular mechanisms responsible for As‐induced toxicities, the exact mechanisms are not fully understood yet, especially at the epigenetic regulation level. In the present study, it is identified that long non‐coding RNA (lncRNA) urothelial cancer associated 1 (UCA1) alleviates As‐induced G2/M phase arrest in human liver cells. Intensive mechanistic investigations illustrate that UCA1 interacts with enhancer of zeste homolog 2 (EZH2) and accelerates the latter's protein turnover rate under normal and As‐exposure conditions. The phosphorylation of EZH2 at the Thr‐487 site by cyclin dependent kinase 1 (CDK1) is responsible for As‐induced EZH2 protein degradation, and UCA1 enhances this process through increasing the interaction between CDK1 and EZH2. As a consequence, the cell cycle regulator nuclear factor of activated T cells 2 (NFATc2), a downstream target of EZH2, is upregulated to resist As‐blocked cell cycle progress and cytotoxicity. In conclusion, the findings decipher a novel prosurvival signaling pathway underlying As toxicity from the perspective of epigenetic regulation: UCA1 facilitates the ubiquitination of EZH2 to upregulate NFATc2 and further antagonizes As‐induced cell cycle arrest.

Abstract TMIM-066: EZH2 REGULATES THE EPIGENETIC REPROGRAMMING OF OVARIAN CANCER PROMOTING CARCINOMA-ASSOCIATED MESENCHYMAL STEM CELLS

Abstract Ovarian tumor cells reside within a complex tumor microenvironment (TME) critical to the formation and function of ovarian cancer. Carcinoma-associated mesenchymal stem cells (CA-MSCs) are stromal progenitor cells within the TME which significantly enhance ovarian tumor cell growth and chemotherapy resistance. We demonstrated that CA-MSCs arise from ovarian tumor cell mediated reprograming of normal tissue MSCs. The resulting CA-MSC phenotype has a unique and durable expression profile without acquisition of genetic mutations, suggesting epigenetic mechanisms underpin the induction of a CA-MSC. In support of this, EPIC methylation array and ATACseq data demonstrate CA-MSCs have significant global hypermethylation and overall “closed” chromatin structure compared to normal MSCs. Further, promoter methylation alterations correlate with the gene expression profile which characterize CA-MSCs. We thus sought to investigate the mechanism underlying the cancer-mediated epigenetic reprograming of CA-MSCs. The histone-lysine N-methyltransferase Enhancer of Zeste homolog 2 (EZH2) is induced early in the conversion of a CA-MSC through tumor secreted factors. EZH2 promotes transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3) leading to a closed chromatin state. EZH2 also acts as a scaffold for DNA-methyltransferases (DNMTs) thus enhancing DNA methylation. Western blotting confirmed increases in EZH2 and the EZH2 target repressive marks H3K27me3 and H2AK119ub in CA-MSCs versus MSCs. We therefore hypothesize that ovarian cancer mediated induction of EZH2 in normal tissue MSCs is critical to the formation of a CA-MSC through epigenetic reprogramming. Normal tissue MSCs derived from benign human ovary or omentum and CA-MSCs derived from high grade serous ovarian cancer patient samples were isolated as previously described. To model the tumor-mediated induction of CA-MSCs, direct co-culture of normal tissue MSCs with high grade serous cell lines CAOV3, PEO1 or OVCAR3 were used. MSC/tumor cell co-cultures were treated with the EZH2 inhibitor Tazemetostat and/or the DNMT inhibitor 5-Azacitidine. A mathematical model, referred to as the CA-MSC classifier which uses the expression of 6 genes to accurately distinguish normal MSCs from CA-MSCs, was utilized to assess the impact of EZH2 and DNMT inhibition on the formation of a CA-MSC. In this model, values closest to 1 indicate a high likelihood of being a CA-MSC (a value of >0.8 meets the threshold for a CA-MSC and <0.2 meets the threshold for a normal MSC). Pharmacologic EZH2 inhibition significantly decreased the induction of a CA-MSC phenotype in tumor cell co-culture without impacting tumor cell or CA-MSC viability. DNMT inhibition alone yielded a modest decrease in CA-MSC induction. However, the combination of EZH2 inhibition and DNMT inhibition blocked the formation of a CA-MSC with a classifier score going from 0.9 (in untreated co-cultures) to 0.1 in dual-inhibition co-cultures. Collectively, our data suggests that ovarian tumor cell mediated EZH2 induction plays a critical role in the conversion of a MSC into a CA-MSC through epigenetic reprogramming. This reprogramming can be interrupted through targeting EZH2 and DNMT. This presents a novel target to block the formation of the tumor enhancing stromal niche thus offering a potentially powerful new approach to the treatment of ovarian cancer. Citation Format: Lenard G. Frisbie, Hui Shen, and Lan Coffman. EZH2 REGULATES THE EPIGENETIC REPROGRAMMING OF OVARIAN CANCER PROMOTING CARCINOMA-ASSOCIATED MESENCHYMAL STEM CELLS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-066.

HNF1B-mediated repression of SLUG is suppressed by EZH2 in aggressive prostate cancer

Prostate cancer is the most common malignancy in men in developed countries. Overexpression of enhancer of zeste homolog 2 (EZH2), the major histone H3 lysine 27 methyltransferase, has been connected to prostate cancer malignancy. However, its downstream genes and pathways have not been well established. Here, we show tumor suppressor Hepatocyte Nuclear Factor 1β (HNF1B) as a direct downstream target of EZH2. EZH2 binds HNF1B locus and suppresses HNF1B expression in prostate cancer cell lines, which is further supported by the reverse correlation between EZH2 and HNF1B expression in clinical samples. Consistently, restored HNF1B expression significantly suppresses EZH2-mediated overgrowth and EMT processes, including migration and invasion of prostate cancer cell lines. Mechanistically, we find that HNF1B primarily binds the promoters of thousands of target genes, and differentially regulates the expression of 876 genes. We also identify RBBP7/RbAP46 as a HNF1B interacting protein which is required for HNF1B-mediated repression of SLUG expression and EMT process. Importantly, we find that higher HNF1B expression strongly predicts better prognosis of prostate cancer, alone or together with lower EZH2 expression. Taken together, we have established a previously underappreciated axis of EZH2-HNF1B-SLUG in prostate cancer, and also provide evidence supporting HNF1B as a potential prognosis marker for metastatic prostate cancer.

The EZH2 inhibitor GSK343 suppresses cancer stem-like phenotypes and reverses mesenchymal transition in glioma cells.

Enhancer of zeste homolog 2 (EZH2) is the catalytic unit of polycomb repressive complex 2 (PRC2) which epigenetically silences many genes involved in tumor-suppressive mechanisms via the trimethylation of lysine 27 of histone H3 (H3K27me3). We recently found that overexpression of EZH2 was associated with poor outcome of glioblastoma (GBM). In this study, we examined the antitumor effects of the EZH2 inhibitor GSK343 on glioma cells in vitro and in vivo. The proliferation and cell cycle of glioma cells was measured. Wound healing assay and transwell invasion assay were performed to evaluate the capacity of migration and invasion of glioma cells. Western blot, qPCR, immunoprecipitation and fluorescent staining were used to test the levels of EZH2 and associated proteins. Spheroid formation assay and clonogenic assays were conducted to assess the stemness of glioma stem cells. Finally, the effect of GSK343 was measured through a nude mice model with intracranially xenotransplanted glioma. We found that GSK343 reduced proliferation, attenuated cell motility and reversed epithelial-mesenchymal transition in U87 and LN229 glioma cells. GSK343 also suppressed the stemness of cell lines and patient derived glioma stem cells. Further, GSK343 inhibited histone H3K27 methylation and upregulated the expression of EZH2 target genes thereby regulating the levels of markers involved in epithelial-mesenchymal transition and stemness. Taken together, our results indicate that GSK343 could be a potential drug against glioblastoma.

High expression of ABCG2 induced by EZH2 disruption has pivotal roles in MDS pathogenesis.

Both proto-oncogenic and tumor-suppressive functions have been reported for enhancer of zeste homolog 2 (EZH2). To investigate the effects of its inactivation, a mutant EZH2 lacking its catalytic domain was prepared (EZH2-dSET). In a mouse bone marrow transplant model, EZH2-dSET expression in bone marrow cells induced a myelodysplastic syndrome (MDS)-like disease in transplanted mice. Analysis of these mice identified Abcg2 as a direct target of EZH2. Intriguingly, Abcg2 expression alone induced the same disease in the transplanted mice, where stemness genes were enriched. Interestingly, ABCG2 expression is specifically high in MDS patients. The present results indicate that ABCG2 de-repression induced by EZH2 mutations have crucial roles in MDS pathogenesis.

Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2H)-ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors

A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound 18. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound 31. Compound 31 displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound 18. Inhibitor 31 also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes.

Abstract 2304: The role of methyltransferase, enhancer of zeste homolog 2 (EZH2) in mouse hepatocyte and human hepatocellular carcinoma

Abstract To investigate the role of EZH1 and EZH2 in liver homeostasis, mice were generated that carried Ezh1-/- and EZH2fl/fl alleles and an Alb-Cre transgene. Only combined loss of EZH1 and EZH2 in mouse hepatocytes caused a depletion of global H3K27me3 marks and the specific loss over ∼1900 genes at 3 months of age. Ezh1-/-,Ezh2fl/flAlb-Cre mice exhibited progressive liver abnormalities manifested by development of regenerative nodules and concomitant periportal fibrosis, inflammatory infiltration and activation of A6-positive hepatic progenitor cells at 8 months of age. In response to chronic treatment with CCl4, all experimental mice but none of the controls (n = 27 each) showed increased hepatic degeneration associated with liver dysfunction and reduced ability to proliferate. After 2/3ds partial hepatectomy, mutant mice (n = 5) displayed increased liver injury and a blunted regenerative response. Genome-wide analyses at 3-months of age identified 51 genes that had lost H3K27me3 marks and their expression was significantly increased. These genes were involved in regulation of cell survival, fibrosis, and proliferation. H3K27me3 levels and liver physiology were unaffected in mice lacking either EZH1 globally or EZH2 specifically in hepatocytes. This work demonstrates a critical redundancy of EZH1 and EZH2 in maintaining hepatic homeostasis and regeneration. Next, we checked the expression EZH2 and H3K27me3 status in 67 human hepatocellular carcinoma (HCC) samples. We also checked the gene expression by the next-generation sequencing based on EZH2 expression. This work demonstrates candidate EZH2 target genes in human HCC. Citation Format: Woo Kyun Bae, Hyun Jeong Shim, Sang Hee Cho, Ik-Joo Chung, In-Kyu Park, Lothar Hennighausen. The role of methyltransferase, enhancer of zeste homolog 2 (EZH2) in mouse hepatocyte and human hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2304. doi:10.1158/1538-7445.AM2015-2304

Abstract B15: Combination chemotherapy in melanoma using EZH2 inhibitor

Abstract Melanoma is the deadliest variety of skin cancer, which according to the American Cancer Society (ACS) will account for around 9710 deaths and diagnosis of around 76,100 new melanoma cases in the current year. According to the ACS, melanoma is also the fastest growing cancer, and hence it is vital to investigate the pathogenesis involved in the advanced stage of melanoma, and design effective therapeutics for its treatment. Aberrant activity of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) has been reported to be associated with different types of cancer. The EZH2 catalyzes the deposition of the repressive mark histone H3 lysine 27 trimethylation (H3K27me3) on the promoter of the target genes using the catalytic SET domain, thereby epigenetically silencing the expression of the target genes. This is because the H3K27me3 mark results in chromatin compaction which reduces the accessibility of various transcription factors to the gene, and hence adversely affects transcription. Several of such EZH2 target genes have been found to be tumor suppressor genes like RUNX3, DAB2IP, and E-cadherin, and silencing of such tumor suppressor genes is a chief mechanism by which EZH2 leads to cancer progression. Hence derepression of the tumor suppressor genes by inhibition of EZH2 could be an effective treatment strategy in different types of cancer. There has been no study till date investigating the role of EZH2 in melanoma. Our laboratory is investigating the role of EZH2 in the pathogenesis involved in the advanced stage of melanoma, to enable the design of effective therapeutic strategies for the treatment of melanoma. Drugs targeting epigenetic modifiers are becoming increasingly popular because of the fact that most epigenetic modifications and their effects can be reversed using these drugs. Our lab has strong evidence that metastatic (or advanced) melanoma is associated with upregulation of EZH2 and H3K27me3. We have also observed that EZH2 inhibition inhibits proliferation and invasion, and also induces apoptosis in metastatic melanoma cells. Recently our lab is performing combination chemotherapeutic studies in vitro, where EZH2 inhibitor GSK126 (S-adenosyl-methionine competitive inhibitor) is being used in combination with conventional chemotherapeutics like cisplatin and Bcl-2 inhibitors. Our results strongly suggest that when EZH2 inhibitor is used in combination with cisplatin or Bcl-2 inhibitor in metastatic melanoma cell lines, the inhibition is synergistic. This synergistic effect of GSK126 and cisplatin or Bcl-2 inhibitor has been found to be caspase-mediated. Citation Format: Deepanwita Sengupta, Nathan L. Avaritt, Alan J. Tackett. Combination chemotherapy in melanoma using EZH2 inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B15.

EZH2: Not EZHY (Easy) to Deal

Seminal discoveries have established that epigenetic modifications are important for driving tumor progression. Polycomb group (PcG) proteins are highly conserved epigenetic effectors that maintain, by posttranslational modification of histones, the silenced state of genes involved in critical biologic processes, including cellular development, stem cell plasticity, and tumor progression. PcG proteins are found in two multimeric protein complexes called Polycomb repressive complexes: PRC1 and PRC2. Enhancer of zeste homolog 2 (EZH2), catalytic core subunit of PRC2, epigenetically silences several tumor-suppressor genes by catalyzing the trimethylation of histone H3 at lysine 27, which serves as a docking site for DNA methyltransferases and histone deacetylases. Evidence suggests that overexpression of EZH2 is strongly associated with cancer progression and poor outcome in disparate cancers, including hematologic and epithelial malignancies. The regulatory circuit and molecular cues causing EZH2 deregulation vary in different cancer types. Therefore, this review provides a comprehensive overview on the oncogenic role of EZH2 during tumorigenesis and highlights the multifaceted role of EZH2, as either a transcriptional activator or repressor depending on the cellular context. Additional insight is provided on the recent understanding of the causes and consequences of EZH2 overexpression in specific cancer types. Finally, evidence is discussed on how EZH2 has emerged as a promising target in anticancer therapy and the prospects for targeting EZH2 without affecting global methylation status. Thus, a better understanding of the complex epigenetic regulatory network controlling EZH2 expression and target genes facilitates the design of novel therapeutic interventions.

De novo peptide design and experimental validation of histone methyltransferase inhibitors.

Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2) maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 mM, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2. These inhibitors should prove useful for further chromatin biology investigations.

De Novo Peptide Design and Experimental Validation of Histone Methyltransferase Inhibitors

Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2) maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 M, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2. These inhibitors should prove useful for further chromatin biology investigations.

Abstract A30: Epigenetic regulation of Wnt/β-catenin signaling in vivo by PRC2 protein EZH2

Abstract Introduction: Colon cancer results from the accumulation of both acquired genetic and epigenetic changes that transform normal glandular epithelium into invasive adenocarcinomas. The Enhancer of Zeste Homolog 2 (EZH2) protein is a core component of the Polycomb Repressive Complex 2 (PRC2) that represses gene transcription through histone H3 lysine 27 trimethylation (H3K27me3). EZH2 is overexpressed in several malignancies including colon cancer; EZH2's role in tumor initiation however, is less clear. Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) that utilizes a type-three secretion system (T3SS) to activate Wnt/β-catenin signaling, represents the earliest molecular and functional changes associated with colon carcinogenesis; the increases in β-catenin recorded during TMCH however, are neither due to mutations in Adenomatous polyposis coli (Apc) or β-catenin (CTNNB1) gene. Aim: To investigate if EZH2 is critical in epigenetically regulating Wnt/β-catenin signaling in response to bacterial infection. Experimental Procedures: TMCH or tumorigenesis were induced by CR infection (108cfu) in wild type and ApcMin/+ mice, respectively. AOM (@10mg/Kg body wt.)/DSS (2.5%; 3 cycles) model of colitis-associated cancer and de-identified human adenoma and adenocarcinoma samples were used to further examine evidence of epigenetically-regulated Wnt/β-catenin signaling. Results: Following CR infection, EZH2 protein increased significantly in the crypt at day 6 and particularly at day 12 (peak hyperplasia) and coincided with expression of EZH2 target protein H3K27me3. The changes accrued were specific to CR as T3SS mutant escV failed to elicit alterations in either EZH2, H3K27me3 or crypt hyperplasia. Mechanistically, siRNA-mediated knockdown of EZH2 led to: i) attenuated cell migration, ii) reduced colonosphere formation and, iii) decreased β-catenin/TCF4-dependent TOPflash reporter activity due to loss of β-catenin. Chromatin immunoprecipitation revealed EZH2's occupancy on WIF1 (Wnt Inhibitory Factor 1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown either via siRNA or EZH2-inhibitor DZNep (5μM), relative levels of H3K27me3 decreased which led to the transcriptional upregulation of WIF1 as was evidenced by WIF1 promoter activity. Besides EZH2, we also discovered increases in miR-203 expression in the crypts at days-6 and 12 post-infection that correlated with reduced levels of its target WIF1, at these time points; overexpression of miR-203 in Young Adult Mouse Colon primary cells resulted in decreased WIF1 transcription. Tumors in the distal colons of ApcMin/+ mice following CR infection expressed high levels of EZH2 and β-catenin and a concomitant decrease in WIF1. In response to AOM/DSS, colonic tumors at 24 weeks expressed high levels of EZH2 and β-catenin and a concomitant decrease in WIF1. Pharmacological intervention with honokiol (HNK; @5mg/Kg body wt. p.o.) a week prior to and during AOM/DSS-induced colon carcinogenesis reduced both tumor burden and tumor size. Immunohistochemical analysis of HNK-treated tissues revealed significant reduction in EZH2 and β-catenin staining while WIF1 expression was dramatically upregulated. In human adenomas/adenocarcinomas, simultaneous increases in EZH2 and β-catenin staining coincided with a corresponding decrease in WIF1 positivity. Conclusions: 1. CR infection induces EZH2 overexpression that correlates with aberrant Wnt/β-catenin signaling in the colonic crypts.2. EZH2 downregulates WIF1 expression which may correlate with earliest stages of colon tumorigenesis. 3. Honokiol-induced upregulation of WIF1 to block aberrant Wnt/β-catenin signaling may be a novel strategy to target epigenetic abnormalities for colon cancer prevention and therapeutics. Citation Format: Badal C. Roy, Dharmalingam Subramaniam, Ishfaq Ahmed, Shrikant Anant, Shahid Umar. Epigenetic regulation of Wnt/β-catenin signaling in vivo by PRC2 protein EZH2. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A30.

Expression of polycomb targets predicts breast cancer prognosis.

Global changes in the epigenome are increasingly being appreciated as key events in cancer progression. The pathogenic role of enhancer of zeste homolog 2 (EZH2) has been connected to its histone 3 lysine 27 (H3K27) methyltransferase activity and gene repression; however, little is known about relationship of changes in expression of EZH2 target genes to cancer characteristics and patient prognosis. Here we show that through expression analysis of genomic regions with H3K27 trimethylation (H3K27me3) and EZH2 binding, breast cancer patients can be stratified into good and poor prognostic groups independent of known cancer gene signatures. The EZH2-bound regions were downregulated in tumors characterized by aggressive behavior, high expression of cell cycle genes, and low expression of developmental and cell adhesion genes. Depletion of EZH2 in breast cancer cells significantly increased expression of the top altered genes, decreased proliferation, and improved cell adhesion, indicating a critical role played by EZH2 in determining the cancer phenotype.