Epigenetic regulation of EZH2 by ncRNAs: mechanisms and oncogenic implications.

EZH2, the moderator in the discussion between methyltransferases at histone H3?

EZH2 takes the stage when BRCA1 loses

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.

Oncogenic Role of Chromatin Modifier Polycomb Repressive Complex-2 in Mantle Cell Lymphoma

Biomarkers that predict prostate cancer (PCa) adaptation to hormone therapy are essential for developing novel therapeutic strategies to manage castration-resistant PCa (CRPC) progression. Enhancer of zeste homolog 2 (EZH2) methyltransferase, the enzymatic component of polycomb repressor complex 2 (PRC2), mediates histone 3 lysine 27 trimethylation (H3K27me3) to represses target genes. Elevated EZH2 levels and decreased PRC2 target gene expression predict poor prognosis in PCa. To test whether altered EZH2 expression and H3K27me3 mark intensity are linked to CRPC progression, we analyzed these parameters in matched primary PCa and lymph node metastasis (LN-mets) from 18 therapy naïve (TN) and neo-adjuvant hormone therapy (NHT) -treated patients. EZH2 expression was observed at varying intensities in all adenocarcinoma samples. Median EZH2 expression did not differ between TN and NHT-treated prostate tumors, but was enriched in discrete foci of varying densities in TN prostatectomy specimens while being uniformly expressed in NHT-treated specimens. In LN-mets from TN patients, EZH2 expression was uniform and reflected the predominant expression pattern observed in matched prostatectomy specimens. In the NHT-treated LN-met cohort, EZH2 expression was localized to discrete foci, however its median expression was reduced relative to the TN LN-met cohort and to both primary PCa cohorts. In contrast, H3K27me3 intensity was uniform in all specimens and on average was indistinguishable between primary PCa and LN-mets irrespective of treatment. Median EZH2 expression in patient-matched prostatectomy and LN-met specimens was directly correlated in both TN and NHT-treated cohorts. H3K27me3 intensity levels were not correlated in patient-matched prostatectomy and LN-met specimens from TN patients, but were correlated in the NHT-treated cohort. While no correlation was observed for EZH2 expression and H3K27me3 intensity within each specimen, the ratio of H3K27me3 intensity to EZH2 expression was increased in the NHT-treated LN-met cohort relative to the TN LN-met cohort and to either primary PCa cohort. This effect was driven by the decreased EZH2 expression in the LN-mets from NHT-treated patients. We conclude that EZH2 expression in LN-mets is predicted by its expression in foci of the patient-matched primary tumors from both TN and NHT-treated patients. This correlation is not observed for H3K27me3 intensity levels of TN primary tumors and LN-mets, but is strongly correlated in NHT-treated specimens. NHT treatment appears to suppress EZH2 expression in LN-mets without impacting H3K27me3 intensity levels. These results suggest that while EZH2 expression and PRC2 activity may drive phenotypic changes that promote oncogenic potential, persistence of this epigenetic signature is a better predictor of CRPC progression. Citation Format: Mitali Pandey, Ladan Fazli, Antonio Hurtado-Coll, Martin E. Gleave, Michael E. Cox. Predictive significance of EZH2 expression and H3K27me3 intensity in therapy-naïve and neoadjuvant therapy-treated metastatic prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5359. doi:10.1158/1538-7445.AM2013-5359

A Novel Non-Canonical Phosphodegron Regulates EZH2 Proteasomal Degradation and H3K27 Trimethylation Activity in Hematopoietic Malignancies

Background: Enhancer of zeste homolog 2 (EZH2) is a catalytic subunit of the polycomb repressive complex 2 (PRC2). When present in PRC2, EZH2 catalyzes trimethylation on lysine 27 residue of histone H3(H3K27Me3), resulting in epigenetic silencing of gene expression and cancer progression. Here, we investigated the expression and the function of EZH2 in intrahepatic and extrahepatic cholangiocarcinoma (ICC and ECC). Methods: The influence of EZH2 on cell growth and apoptosis was assessed by knockdown experiments using siRNA in cholangiocarcinoma cell lines, RBE and TFK-1. Target genes of EZH2 in cholangiocarcinoma cell lines were searched by real time PCR. The clinical significance of EZH2 in 84 cholangiocarcinoma patients (ICC and ECC in 45 and 41, respectively) who underwent a curative surgery was examined by immunohistochemistry. Results: In vitro analysis, a knockdown of EZH2 reduced cell growth and induced G1 arrest, and induced apoptosis confirmed by Annexin V staining and increasing sub-G1 population of two cholangiocarcinoma cell lines. Moreover, a knockdown of EZH2 increased the expression of p16INK4A and p27KIP1, in real time PCR. In immunohistochemical study, The upregulation of EZH2 was correlated with tumor diameter (P = 0.0103) in ICC, lymph node metastasis (P=0.0292) in ECC, and Ki67 index in both ICC (P=0.0364) and ECC (P=0.0017). In addition, EZH2 expression was correlated with the poor prognosis in both ICC (P=0.0447) and ECC (P=0.0227). Conclusion: The current study demonstrates that the high expression of EZH2 results in acceleration of cell cycle and anti-apoptosis in vitro analysis, and is related to poor prognosis in patients with ICC or ECC by IHC study. These results suggest that EZH2 is a potential target for cholangiocarcinoma therapeutics. Citation Format: Shigeki Nakagawa, Hirohisa Okabe, Yasuo Sakamoto, Hiromitsu Hayashi, Daisuke Hashimoto, Hideyuki Kuroki, Katsunori Imai, Hidetoshi Nitta, Akira Chikamoto, Masayuki Watanabe, Toru Beppu, Hideo Baba. Enhancer of zeste homolog 2 (EZH2) promotes progression of cholangiocarcinoma by regulating cell cycle and apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5350. doi:10.1158/1538-7445.AM2013-5350

Background: Breast cancer accounts for more than 40,000 deaths yearly in the United States, the majority of which are due to metastatic disease. The histone methyltransferase EZH2 (Enhancer of Zeste Homolog 2) is a member of the polycomb repressive complex 2 (PRC2) responsible for transcriptional repression by trimethylation of histone H3 at lysine 27. EZH2 is an independent predictor of recurrence and mortality that is overexpressed in 55% of invasive breast carcinomas. We previously identified a novel link between EZH2 and the p38 mitogen-activated protein kinase, an important mediator of breast cancer progression and metastasis. EZH2 binds to p38, and overexpression of EZH2 leads to phosphorylation and activation of p38. Based on in vitro evidence and recent studies implicating EZH2 in the methylation of non-histone targets, we hypothesized that EZH2 may methylate p38 in breast cancer cells. We further hypothesized that this methylation event may be important for p38 activation or stability. Methods: In order to test this hypothesis, we used stable shRNA knockdown of EZH2 in MDA-MB-231 and SUM149 breast cancer cell lines. We performed co-immunoprecipitation using p38 and pan-methyl lysine antibodies to assess changes in methylated p38 by immunoblot. We are currently performing LC MS/MS analysis to identify the specific residues that are methylated by PRC2 both in vitro and in vivo. Additionally, in order to localize this interaction, we performed co-immunoprecipitation experiments on fractionated MDA-MB-231 and SUM149 lysates. Results: Stable knockdown of EZH2 resulted in reduced methylated p38 compared to vector controls, suggesting that EZH2 methylates p38 in vivo. We found that EZH2 and p38 interact in nuclear and, surprisingly, cytoplasmic fractions. Conclusions. We provide evidence that in addition to being a nuclear protein, EZH2 can be found in the cytoplasm of TNBCs, where it is able to complex with p38. Taken together, our data suggest that p38 methylation may represent an additional mechanism by which EZH2 mediates breast cancer progression in TNBC. Citation Format: Talha Anwar, Heather Moore, Maria E. Gonzalez, Celina Kleer. Identification of a novel interaction between EZH2 and p38 in triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4975. doi:10.1158/1538-7445.AM2014-4975

Background: Triple negative (estrogen receptor, progesterone receptor, HER2-neu negative) breast cancers (TNBC) account for 15% of all breast cancers but are responsible for a disproportionate number of deaths. Overexpression of the histone methyltransferase EZH2 (Enhancer of Zeste Homolog 2) is an independent prognostic biomarker significantly associated with poorly-differentiated TNBCs and poor patient outcome. As a member of the polycomb repressive complex 2 (PRC2), the canonical function of EZH2 is to mediate transcriptional repression of target genes via trimethylation of histone H3 at lysine 27 (H3K27me3). We previously identified a novel interaction between EZH2 and the p38 mitogen-activated protein kinase, an important mediator of progression and metastasis of TNBC. We found that EZH2 can bind to p38 and that overexpression of EZH2 leads to phosphorylation and activation of p38. Based on these data and preliminary in vitro studies, we hypothesized that EZH2 may have functions independent of its H3K27me3 activity in TNBC, where it can methylate p38 and other non-histone proteins. We further hypothesized that these methylation events may be important for p38 activation or stability. Methods: In order to test this hypothesis, we performed an in vitro methyltransferase assay with recombinant PRC2 complex, using p38α as substrate. We then employed LC MS/MS to determine the lysine residues methylated on p38α by PRC2. In order to determine the functional consequences of this modification, we assessed for changes in p38 isoform protein stability in a cycloheximide pulse-chase assay with genetic or pharmacologic inhibition of EZH2 in MDA-MB-231 and SUM149 triple-negative breast cancer cell lines. As a complementary approach, we used site-directed mutagenesis to block methylation at these sites and to determine resulting changes in protein stability. Results: EZH2 methylates p38α in vitro at two lysine residues. Enzymatic inhibition of EZH2 results in reduced levels of methylated p38α, as well as decreased stability in vivo in an isoform-specific manner. Consistent with these results, lysine-to-alanine mutagenesis of these residues results in significantly reduced protein stability. We are currently investigating the possibility that EZH2 methylation may block other modifications, such as ubiquitination, at these sites. Conclusions: We provide evidence that EZH2 directly methylates oncoprotein p38α at two novel sites and that these modifications confer stability. Taken together, our data suggest that p38 methylation may represent an additional mechanism by which EZH2 contributes to TNBC progression. Citation Format: Talha Anwar, Heather Moore, Sarah Bergholtz, Celina Kleer. Noncanonical functions of EZH2 in triple-negative breast cancers. [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 4790. doi:10.1158/1538-7445.AM2015-4790

The histone methyltransferase Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2). Its overexpression in triple-negative breast cancer (TNBC) correlates with aggressive, poorly differentiated phenotypes and poor prognosis. EZH2 methylates histone 3 at lysine 9/27 and controls the transcription of many genes, such as cyclin D1, Cyclin E and MMP9, which are involved in tumor survival, proliferation, migration, invasion, and angiogenesis. EZH2 also promotes breast tumor-initiating cell expansion (BITC) through impairment of the DNA repair process and the favoring of oncogenes. Small-molecule EZH2 inhibitors, including Tazmetostat (TAZVERIK), mainly inhibit the SET domain methylase activity which may not completely abrogate the actions of EZH2. Recently, TAZVERIK was FDA approved for follicular lymphoma and is in clinical trials for other solid tumors including the breast. The PI3K-AKT pathway in TNBC plays a pivotal role in metastasis and tumorigenesis, and it associates and interacts with other proteins and pathways. Post translational modifications of EZH2 lead to its role in cancer progression, and importantly, AKT phosphorylates EZH2 and promotes oncogenesis independent of PRC2-mediated gene transcriptional silencing. Docking studies using PyRx 0.8 software revealed AKT inhibitor MK2206 interact with EZH2. We hypothesize that enhancing EZH2 inhibition by combining Tazmetostat with an Akt inhibitor will be highly effective in suppressing progression and metastasis in TNBC. The in-silico analysis revealed MK2206 actively interacts with the CXC (Cystein rich region) an integral part of the catalytic site of EZH2. The binding affinity of MK2206 was observed to be -6.9 kcal/mol, which is -1.3 kcal/mol less than the affinity of Tazmetostat (-8.2Kcal/mol). Tazmetostat binds to the SET domain, inhibiting methyltransferase activity, while MK2206 binds towards the CXC region. Tazmetostat by itself effectively inhibit cell growth (IC50: 20.3 μM) but combining with MK2206 caused a synergistic inhibition of cell viability. The combination of 8µM Tazmetostat and 5μM MK2206 significantly suppressed EZH2 activity and migration. Additionally, we investigated the Protein-Protein interaction STRING between AKT and EZH2 in humans, which revealed interactions with proteins involved in regulation of cell cycle progression (CDK2), RING -type E3 Ubiquitin Transferase (TTC3), and the triggering of histone H3-k27 methylation (EED). The combination of Tazmetostat and MK2206 significantly inhibits TNBC proliferation, invasion and migration by disrupting the catalytic activity by preventing EZH2-DNA interactions, abrogating methylation and inhibiting the EZH2-Akt interaction in the TNBC phenotype. (Work supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: Gowripriya Thirumugam, Gautham Sarathy, Shiv Verma, Shivani Dhekne, Balamurugan Krishnaswamy, Sanjay Gupta, Bhuvaneswari Ramaswamy, Eswar Shankar. Computational drug repositioning identifies Akt inhibitor MK2206 binding to EZH2 as enhancer of Tazmetostat efficacy in inhibition of triple negative breast cancer proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2561.

Ovarian cancer is a lethal gynecological tumor, typically diagnosed at an advanced stage and associated with limited treatment options. The Enhancer of Zeste Homolog 2 (EZH2) is a crucial methyltransferase that serves as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), alongside other core subunits (EED, SUZ12, and RbAp46/48). EZH2 contributes to ovarian cancer progression through both its catalytic and non-catalytic functions, making it a promising therapeutic target. Data from the TCGA database and the Human Protein Atlas indicate that EZH2 mRNA and protein levels are elevated in ovarian cancer tissues compared to normal ovary tissues, correlating with poor prognosis in patients. This provides a rationale for targeting EZH2 in ovarian cancer treatment. However, traditional EZH2 enzyme inhibitors only block its catalytic function and are ineffective against ovarian cancer driven by its non-catalytic functions. EZH2 degraders can effectively inhibit both functions. To identify phytochemicals that promote EZH2 degradation, we screened and identified 20 candidates that can directly or indirectly modulate EZH2 expression. Western blot analysis revealed that Triptolide (TPL, 1 µM) significantly degrades EZH2 protein. TPL is a diterpene compound extracted from the roots, leaves, flowers, and fruits of Tripterygium wilfordii, known for its anti-inflammatory and anti-tumor properties. However, its role in regulating EZH2 in ovarian cancer has not been previously reported. Our western blot results showed that TPL significantly degrades EZH2 and other core PRC2 subunits (EED and SUZ12) in a concentration-dependent manner, with a half-degradation concentration (DC50) as low as 25 nM in the ovarian cancer cell line A2780. The degradation effect of TPL on EZH2 and PRC2 increased with longer exposure times. Further experiments indicated that TPL induces degradation of EZH2 or the PRC2 complex via the ubiquitin-proteasome pathway. A biotin-labeled TPL probe, Biotin-TPL, used in a pull-down assay, confirmed the interaction between TPL and EZH2. Additionally, TPL effectively inhibits the proliferation of ovarian cancer cells in a time- and concentration-dependent manner, with IC50 values in low nanomolar range. Colony formation and 3D cell spheroid formation assays demonstrated that TPL (3.7-33.3 nM) significantly reduces colony formation in A2780 and ES-2 ovarian cancer cells, as well as the formation of ovarian cancer 3D cell spheroids. RNA-seq data and subsequent validation experiments showed that TPL can inhibit signaling pathways regulated by EZH2's non-catalytic functions. Collectively, these findings suggest that TPL may inhibit ovarian cancer growth by degrading the EZH2/PRC2 complex and modulating the signaling pathways associated with EZH2’s non-catalytic functions. Acknowledgements: This work was supported by National Natural Science Foundation of China (82304454). Citation Format: Yongxia Zhu, Qing Tang, Hongtao Xiao, Ningyu Wang. Triptolide inhibits ovarian cancer growth by enhancing EZH2 degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 609.

Background: EZH2 component of the polycomb repressive complex 2 (PRC2) is a histone methyltransferase whose function is to methylate lysine 27 of histone 3. EZH2 is overexpressed in 49% of breast cancers and is associated with worse outcomes especially in TNBC. EZH2 is overrepresented in African American and Hispanic women, suggesting that clinical targeting of this protein may particularly help improve the disproportionately poor outcomes in these populations. In addition to its role in metastasis, EZH2 regulates tumor immune microenvironment (TIME) by inhibiting T cell activation via suppression of MHC-1 antigen presentation pathway, upregulating PD-L1 expression causing suppression of an antitumor immune response. Current EZH2 inhibitors only targets the catalytic activity of EZH2, leaving its function as a gene activator unaffected. Despite the advancement in the discovery of inhibitors for EZH2 that attenuate its catalytic activity, resistance to these small molecules limits their use in solid tumors. The neurotransmitter dopamine via its D1 receptor activation in TNBC cell lines induces apoptosis and autophagy, as well as inhibits the invasion and regress in mammary tumors in vivo. In addition, dopamine D1 receptor signaling has been reported to attenuate the immunosuppressive effects of myeloid-derived suppressor cells (MDSCs) on T cell proliferation and IFN-g production. We hypothesize that combined treatment of dopamine D1 receptor agonist (A77636) and EZH2 inhibitors (GSK126) inhibits tumor growth and metastasis of TNBC cells both in vitro and in vivo. Methods: To test the efficacy of the combination inhibiting metastasis we employed an in vivo model system to confirm the results we had obtained from our invitro 3D culture system and 3D organ-on-chip-based microphysiological (MPS) platform (SynTumor). 4-6 weeks old female NSG mice were injected with MDA-MB-231 cells on the breast fat pads. When the tumors became palpable, they were randomized into four groups, Vehicle, GSK126 (2mg/kg BW), A77637 (50mg/kg BW) and the combination. The drugs individually or in combination were administered intraperitoneally five days for 4 weeks. Tumor measurements were also done during the time and at the end of 4 weeks the animals were euthanized. The tissues, tumor, bone, blood, and spleen were harvested and processed for flow cytometer analysis. Tumor weight and volume were also calculated. Results: The combination of GSK126 and A77636 demonstrated a synergistic effect inhibiting the tumor weight and tumor volume when compared to the individual treatments or the vehicle treated animals. These results matched with our in-vitro data, where the combination synergistically inhibited the growth of spheroids. in the microfluidic SynTumor model, the combination reduced circulating tumor cell numbers by half. The combination significantly decreased the monocyte population in the blood and tumor. Also, the EZH2 expression in the monocytes and neutrophils were significantly decreased by the combination. Conclusion: Our data indicate that the combinatorial effect of DRD1 agonist and EZH2 inhibitor efficiently attenuates the EZH2-mediated tumor growth and innate immune environment in TNBC (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Dedicated in memory of Dr. Bhuvaneswari Ramaswamy. Citation Format: Eswar Shankar, Rajni Kant Shukla, Gautam Sarathy, Kate Ormiston, Xilal Rima, Chunyu Hu, Divya S Patel, Radha vaddavalli, Deborah Ramsey, Gwen Fewell, Bhuvaneswari Ramaswamy, Eduardo Reátegui. Enhancer of zeste homologue 2 (EZH2) inhibition in Triple Negative Breast Cancer (TNBC) attenuates tumor growth in vitro and in vivo altering the tumor immune microenvironment [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-09-20.

The histone methyltransferase enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex (PRC2) and is involved in chromatin remodeling and gene silencing through its methylation of histone H3 on lysine 27 (H3K27). Specifically, trimethylation of H3K27 is associated with gene silencing and plays critical role in cancer initiation and progression. Therefore, blocking EZH2 catalytic activity may present a valid preventive and/or therapeutic strategy for the treatment of cancers with EZH2 overexpression including prostate cancer. Our multistep approach of molecular modeling and direct binding has led to the identification of plant flavone luteolin (3’, 4’, 5, 7-tetrahydroxyflavone) as a specific inhibitor of EZH2 with preferential blocking of its catalytic site. Here we report luteolin, in micro molar range, inhibits EZH2 catalytic activity, demonstrates anti-proliferative and anti-invasive activities in functional cell based assays. Treatment of human prostate cancer DU145 and 22Rv1 cells, which possess high constitutive EZH2 expression, with 5-20 μM luteolin significantly inhibits EZH2 and SUZ12 protein expression in dose and time dependent manner, although luteolin treatment did not affect protein expression of EED and RbAp46/48 protein, other members of PRC2 complex. Treatment of both cell lines with luteolin also reduced H3K27me3 and H3K27me2 protein and its enzymatic activity in dose and time dependent manner without affecting total H3 protein. In addition, luteolin was also effective in suppressing in vitro methylation performed using recombinant PCR2 complex. These events led to increase in the expression of downstream tumor suppressor genes including E-cadherin, SLIT2 and TIMP3, respectively. Interestingly, treatment of cells with proteasome inhibitor, MG132 together with luteolin did not prevent EZH2 degradation indicating that proteasomal degradation might not contribute to EZH2 inhibition. Taken together, our study suggest that luteolin acts on the catalytic binding site of EZH2 to exhibit downregulation of histone H3 methylation and could be developed as a potential promising agent for the prevention and/or treatment of various human cancers including prostate cancer with EZH2 overexpression. Citation Format: Rajnee Kanwal, Xiaoping Yang, Eswar Shankar, Sanjay Gupta. Luteolin selectively inhibits EZH2 and blocks H3K27 methylation in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2230. doi:10.1158/1538-7445.AM2017-2230

Purpose: Colorectal cancer (CRC) is the second most commonly diagnosed cancer and the third leading cause of cancer mortality in Japan. Almost 15% and 10% of patients with CRC are diagnosed with synchronous and metachronous liver metastasis, respectively. In the last decade, the therapeutic strategies including chemotherapy and molecular targeted therapy have improved. However, the prognosis of patients with CRC remains poor because of their acquirement of chemoresistance. Previous studies have shown that enhancer of zeste homologue 2 (EZH2) is associated with proliferation of various cancers. EZH2 is the catalytic subunit of Polycomb repressive complex2 (PRC2) which catalyzes the methylation of lysine 27 on histone H3 (H3K27), leading to repress the transcription of tumor-suppressor genes. In addition, high EZH2 expression is associated with the resistance to cisplatin in non-small cell lung carcinoma. However, the association between the resistance to oxaliplatin and the expression of EZH2 is unrevealed. The aim of this study is to investigate the correlation between the expression of EZH2 and response to oxaliplatin-based chemotherapy as well as survival of patients with colorectal liver metastasis (CRLM). Experimental Design: Sixty-one patients who underwent first-line oxaliplatin-based chemotherapy before resection of CRLM were enrolled in this study. Immunohistochemistry of EZH2 was performed, and then the correlation between response to chemotherapy and survival was evaluated. Results: Immunohistochemistry revealed 38 patients (62.3%) with high EZH2 expression, and 23 patients (37.7%) with low EZH2 expression. Of the 38 patients with EZH2 high expression, 15 patients (39.5%) responded to oxaliplatin-based chemotherapy with either complete response or partial response. Of the 23 patients with low EZH2 expression, 14 patients (60.9%) exhibited a response to chemotherapy. There was significant difference in response to oxaliplatin-based chemotherapy between high and low EZH2 expression patients (p = 0.04). Univariate survival analysis indicated that patients with high EZH2 expression had a lower overall survival than those with low EZH2 expression. Moreover, multivariate Cox regression analysis revealed that high EZH2 expression was an independent prognostic factor for overall survival. Kaplan-Meier survival curves also confirmed that high EZH2 expression correlates with poor prognosis in patients who underwent hepatectomy for CRLM. Conclusions: Our results indicated that prognosis of patients with high EZH2 expression who received preoperative oxaliplatin-based chemotherapy and underwent hepatectomy for CRLM was worse than those with low EZH2 expression. EZH2 may be a predictive factor for oxaliplatin-based chemotherapy response and overall survival in patients with CRLM. Citation Format: Mayuko Ohuchi, Yasuo Sakamoto, Ryuma Tokunaga, Kenichi Nakamura, Yuki Kiyozumi, Daisuke Izumi, Keisuke Kosumi, Kazuto Harada, Junji Kurashige, Yukiharu Hiyoshi, Shiro Iwagami, Yoshifumi Baba, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Prognostic impact of enhancer of zeste homologue 2 (EZH2) in patients underwent hepatectomy for colorectal liver metastases who received preoperative oxaliplatin-based chemotherapy. [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 2277. doi:10.1158/1538-7445.AM2015-2277

Background: Neuroendocrine prostate cancer (NEPC) is the most aggressive malignant variant of prostate cancer (PCa). Since there is no effective treatment available for NEPC to date, devising a therapeutic intervention requires identifying the mechanisms of how prostate cancer cells gain neuroendocrine phenotype. Evidence suggest that neuroendocrine differentiation is associated with elevated expression of Enhancer of Zeste Homolog 2 (EZH2). EZH2 is the catalytic subunit of Polycomb repressive complex 2 (PRC2) that functions to regulate histone H3 methylation and causes transcriptional repression of target genes. We hypothesize that the elevated expression of EZH2 in NEPC may render the tumors cells an undifferentiated state which enables the lineage switch and trans-differentiation into neuroendocrine malignancies. Method and Results: First, we extracted microarray data to analyze the expression of polycomb genes during PCa progression, using Gene Expression Omnibus (GEO) database from NCBI. The expressions of Polycomb genes (EZH2, EED, SUZ2, CBX1) were up-regulated in metastatic castrate-resistant prostate cancer and the increased EZH2 expression was in concert with elevated expressions of NEPC marker (CHGA, FOXA2, SOX2) and decreased expression of FOXA1, a prostate differentiation marker. Additionally, we conducted IHC staining in sections derived from benign prostate hyperplasia, low- and high- grade prostate adenocarcinoma, patient-derived NEPC xenograft tumors (LuCaP), and mouse models of NEPC (12T10 LADY and TRAMP). EZH2 and FOXA2 were overexpressed in all NEPC tumors while FOXA1 was abundant in prostate adenocarcinoma but decreased in NEPC. Also, we cultured prostate adenocarcinoma, LNCaP cells in androgen-depleted media and analyzed the gene expression of major polycomb genes and NEPC markers by qPCR. The expressions of Polycomb genes (BMI1, RING1a, CBX1) were induced by androgen depletion, concurrent with the induction of NEPC gene, CD56. Furthermore, we stably knocked down EZH2 in DU145 and PC3 cell lines (both have NEPC features) using shRNA and measured both the RNA and protein level of EZH2, PCa and NEPC markers using qPCR and western blot. We found that the levels of AR, FOXA1 and E-cadherin are upregulated in both DU145 and PC3 cells when EZH2 was knocked down. Conclusion: Our data support that EZH2 is functionally involved in the development of NEPC. Future direction: We will overexpress EZH2 in LNCaP cells to study if elevated EZH2 expression promotes NEPC. We will also perform RNA-seq and ChIP-seq to study how the altered expression of EZH2 affects the global transcriptome and identify the downstream target genes of EZH2 in PCa. Additionally, we will conduct in vivo animal experiments to determine EZH2’s role in NEPC using NEPC PDX models and transgenic mouse models. Findings from this research would provide detailed insights into developing therapeutics against EZH2 mediated NEPC. (Funding source: R03 and FWCC). Citation Format: Md Imtiaz Khalil, Shu Yang, Anthony Blankenship, Zachary Connelly, Xiuping Yu. Functional role of EZH2 in neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5191.