Role Of EZH2 Research Articles

Abstract 4500: An AR-regulated kinase CCRK activates a tumor-initiating circuitry through dual regulation of EZH2

Abstract Hepatocellular carcinoma (HCC) is one of the commonest and deadliest cancers worldwide with a striking gender prevalence observed in men. Androgen receptor (AR) is a ligand-activated nuclear receptor that regulates the development of male sexual phenotype. Aberrant AR signaling, however, has detrimental consequences in the development of male-predominant cancers. Using genome-wide location analysis, we have recently uncovered cell cycle-related kinase (CCRK) as an AR direct transcriptional target that drives aberrant hepatocellular proliferation and tumorigenicity through β-catenin/T cell factor signaling (Feng et al. 2011). We also found that the Polycomb protein EZH2 promotes β-catenin-dependent hepatocarcinogenesis through concordant repression of Wnt antagonists via histone H3 lysine 27 trimethylation (Cheng et al. 2011). Recent studies have revealed the Polycomb-independent role of EZH2 in gene activation via AKT-mediated phosphorylation, however, the regulation and signaling network of EZH2 in HCC are poorly-understood. Here we show that the AR-regulated kinase CCRK activates a tumor-initiating circuitry by dual regulation of EZH2. Activation of CCRK phosphorylates glycogen synthase kinase 3β (GSK-3β) and AKT leading to transcriptional up-regulation and phosphorylation of EZH2 at Ser21 (pEZH2Ser9), respectively. pEZH2Ser9 in turn physically interacts with AR and cooperatively activate CCRK transcription via direct binding to the CCRK promoter, thus forming a feedback circuitry. In a chemical-induced murine HCC model, activation of the AR/CCRK/EZH2 regulatory loop precedes HCC formation, which is significantly reduced (>80%) by lentiviral-mediated knockdown of CCRK. In human HCC tissues, these proteins are highly up-regulated, starting from the adjacent precancerous liver tissues. Most importantly, hyper-activation of this loop is significantly associated with shorter overall (p<0.0001) and disease-free survival rates (p<0.01) of the HCC patients. This study not only advances our fundamental understanding of the gender disparity in HCC, but also provides a new paradigm for nuclear receptor regulation of cancer epigenome. Elucidating the detailed signaling network of AR/CCRK/EZH2 will lead to the discovery and development of targeted therapies tailored to inhibit cancer-specific pathways. This study was supported by a CUHK Focused Investments Scheme - Scheme B (fis1112/0674/12yc), a National Natural Science Foundation of China (373492), a Health and Medical Research Fund (12110532) and a General Research Fund (462710).

EZH2 promotes angiogenesis through inhibition of miR-1/Endothelin-1 axis in nasopharyngeal carcinoma.

Emerging evidence clearly indicates that EZH2 plays a crucial role in tumor angiogenesis. However, the role of EZH2 in angiogenesis is still unknown in nasopharyngeal carcinoma (NPC). We here showed that the elevated EZH2 level was closely associated with an aggressive and poor prognostic phenotype, and was positively correlated with microvessel density (MVD) in NPC tissues. Functional studies showed that EZH2 upregulation promoted cell proliferation, migration and tubule formation of endothelial cells, and knockdown of EZH2 suppressed tumor growth, metastasis and angiogenesis in vivo. Mechanistic investigations revealed that EZH2 inhibited miR-1 transcription via promoter binding activity, leading to enhanced expression of Endothelin-1 (ET-1) which is suppressed by miR-1 targeting of ET-1 3'UTR. Furthermore, knockdown of EZH2 or overexpression of miR-1 exerted anti-angiogenic effect on NPC cells. More importantly, the neutralizing antibody against ET-1 significantly abrogated the pro-angiogenic effect of EZH2, and forced expression of ET-1 rescued the anti-angiogenic effect induced by EZH2 knockdown. In clinical specimens, ET-1 was widely overexpressed and associated with clinical stage and MVD. Taken together, our results identify a novel signaling pathway involved in NPC angiogenesis, and also suggest that EZH2-miR-1-ET-1 axis represents multiple potential therapeutic targets for NPC.

Targeting histone methyltransferase EZH2 as cancer treatment.

It is widely accepted that epigenetic alterations are associated with different stages of tumour formation and progression in many cancers. Therefore, epigenetic abnormalities in cancers are emerging as important biomarkers and may have therapeutic potential. The polycomb repressive complex 2 (PRC2) is a key epigenetic regulator that catalyses trimethylation of lysine 27 on histone H3 (H3K27me3) via the histone methyltransferase, EZH2, which confers stemness and regulates differentiation during embryonic development. Given these roles of EZH2 and H3K27me3, plastic and dynamic features of cancer cells, especially cancer stem cells (CSCs), may be closely associated with this epigenetic mechanism. In addition, recent sequencing technology revealed that there are many recurrent mutations in polycomb-related genes, including EZH2, in different types of cancers. Therefore, researchers focused on targeting EZH2 as a novel cancer treatment and identified small compounds that inhibit EZH2 activity. Some of them are now under clinical trial in B-cell lymphoma. However, the underlying mechanisms by which PRC2 precisely regulate epigenetic alterations at certain genomic loci under different cellular conditions remain unclear. In this review, I focus on the recent advancements in EZH2 research, especially its dynamic regulation of epigenetic alterations in tumour cells, including the CSC population, and discuss perspectives and challenges for cancer treatment in the near future.

Taming the young and restless—epigenetic gene regulation in pancreas and beta‐cell precursors

The in vivo assessment of epigenetic changes during mouse pancreatic beta‐cell differentiation reveals surprising differences to directed, in vitro differentiation of human embryonic stem cells. New findings reported in this issue of The EMBO Journal further identify Ezh2 as a critical determinant of endocrine progenitor number and could instruct improved protocols for stem cell-based therapies.

Dynamics of genomic H3K27me3 domains and role of EZH2 during pancreatic endocrine specification.

Endoderm cells undergo sequential fate choices to generate insulin-secreting beta cells. Ezh2 of the PRC2 complex, which generates H3K27me3, modulates the transition from endoderm to pancreas progenitors, but the role of Ezh2 and H3K27me3 in the next transition to endocrine progenitors is unknown. We isolated endoderm cells, pancreas progenitors, and endocrine progenitors from different staged mouse embryos and analyzed H3K27me3 genome-wide. Unlike the decline in H3K27me3 domains reported during embryonic stem cell differentiation in vitro, we find that H3K27me3 domains increase in number during endocrine progenitor development in vivo. Genes that lose the H3K27me3 mark typically encode transcriptional regulators, including those for pro-endocrine fates, whereas genes that acquire the mark typically are involved in cell biology and morphogenesis. Deletion of Ezh2 at the pancreas progenitor stage enhanced the production of endocrine progenitors and beta cells. Inhibition of EZH2 in embryonic pancreas explants and in human embryonic stem cell cultures increased endocrine progenitors in vitro. Our studies reveal distinct dynamics in H3K27me3 targets in vivo and a means to modulate beta cell development from stem cells.

Polycomb protein EZH2 expression in diffuse large B-cell lymphoma is associated with better prognosis in patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone

Polycomb group (PcG) proteins are evolutionarily conserved regulators of gene expression that contribute to normal lymphocyte development, and are involved in malignant transformation of these cells. Recently, BMI1 and EZH2 have been shown to be involved in lymphomagenesis and oncogenesis. We tried to elucidate the role of EZH2 as a prognostic factor for diffuse large B-cell lymphoma (DLBCL). High-level expression of EZH2 (EZH2 ≥ 70%) was associated with superior overall survival (OS) of 85.8% compared to low expression (EZH2 < 70%), with OS of 44.5% (p = 0.005). Subgroup analysis showed that the activated B-cell (ABC) subtype with high EZH2 expression had the highest overall survival (p = 0.011). In analysis of EZH2 expression within low International Prognostic Index (IPI) score, high EZH2 expression had a significant statistical correlation with longer OS (p = 0.034). With high IPI score, high EZH2 expression tended to be associated with longer OS (p = 0.130). Our results showed that EZH2 expression had a high prognostic relevance to survival outcomes. We demonstrated that DLBCL was associated with increased expression of the EZH2 PcG protein and Ki67. The distribution of EZH2 expression was wider than that of Ki67. In summary, increased EZH2 expression of tumor cells was associated with improvements in OS.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 5217: Integrative sequencing reveals novel alterations in untreated and castration resistant prostate cancer.

Abstract Prostate cancer is the third most common source of male cancer deaths in developed countries. The standard of care for aggressive prostate cancer is androgen ablation, which prolongs survival until the tumor acquires a castration resistant phenotype. The molecular pathology underlying prostate cancer progression is not yet fully understood. We used integrative high throughput sequencing to study cancer-associated alterations in 53 prostate cancer related neoplasia at the DNA, RNA and epigenetic levels. The cohort included both hormone-naive and castration resistant prostate cancers, along with two neuroendocrine prostate cancers. We identified two new fusion genes, one of which associated with neuronal differentiation and castration resistance. We also identified a number of novel prostate cancer associated transcripts, including transcripts specific to castration resistant tumors. Based on ChIP-seq data from prostate cancer cell lines, many of the novel transcripts were regulated by known oncogenes such as ERG and AR. Methylation sequencing revealed a near-identical pattern of promoter hypermethylation in both hormone-naive and castration resistant tumors. Enrichment of hypermethylation was observed at EZH2 binding sites, supporting the role of EZH2 in the recruitment of DNA methyltransferase in prostate cancer. Promoter hypermethylation suppressed the expression of hundreds of genes, but a subset of genes characterized by promoter H3K27 trimethylation responded to hypermethylation with increased expression. Citation Format: Matti J. Annala, Kati K. Waltering, Antti Ylipää, Kimmo Kartasalo, Kirsi Tuppurainen, Serdar Karakurt, Leena Latonen, Outi Saramäki, Simo-Pekka Leppänen, Janne Seppälä, Hanna E. Rauhala, Teuvo LJ Tammela, Olli Yli-Harja, Wei Zhang, Tapio Visakorpi, Matti Nykter. Integrative sequencing reveals novel alterations in untreated and castration resistant 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 5217. doi:10.1158/1538-7445.AM2013-5217

Abstract 4643: EZH2 promote a malignant phenotype in lung adenocarcinoma and VEGF/VEGFR2 pathways mediates regulation of EZH2 through E2F3/HIF-1α and downregulation of miR-101.

Abstract Background. EZH2 overexpression has been described in a wide variety of tumors, including lung cancer. EZH2 has been implicated in neoplastic transformation, tumor progression and activation of angiogenesis. The mechanisms associated with the regulation of EZH2 expression in lung cancer cells are mostly unknown. In this study, we investigated the role of EZH2 in cell proliferation/migration and response to chemotherapy in lung adenocarcinoma (LADCa) cell lines. Moreover, we studied the mechanisms of EZH2 regulation associated to the VEGF/VEGFR2 pathway expression. Methods. LADCa cell lines were inhibited pharmacologically with different doses of DZNep (0, 2.5 and 5μM) and by knockdown of EZH2 by siRNA. EZH2, H3K27me3 and PARP-C expressions were determined by Western-blots (WB). Cisplatin and carboplatin sensitivity (IC50) and proliferation were determined by MTS assay. Cell migration was measured using Boyden chamber assay. After stimulation of cell lines with VEGF-A, EZH2, VEGFR-2, E2F3 and HIF-1α expression were determined using WBs, and EZH2 and miR-101 expressions were determined by quantitative PCR. To study the role of VEGFR-2 and HIF-1α in the regulation of EZH2, we examined the effect of knockdown VEGFR-2 and HIF-1α by siRNA. Results. We found that inhibition with DZNep and knockdown by siRNA decreased the expression of EZH2, reduced H3K27me3 level, and increased PARP-C levels in LADCa cell lines. Treatment with DZNep and knockdown of EZH2 significantly increased sensitivity to cisplatin and carboplatin (p&amp;lt;0.05) by MTS assay of LADCa cell lines. Knockdown of EZH2 reduced significantly cell proliferation and migration of LADCa cell lines (p&amp;lt;0.05). VEGF stimulation induced expression of EZH2 and H3K27me3 levels. Concomitantly, we found that stimulation with VEGF induced expression of E2F3 and HIF-1α, and downregulation of miR-101. This phenomenon was observed only in LADCa cell lines expressing VEGFR-2, and not in cell lines with low or lack of expression of VEGFR-2. The increased expression of EZH2, E2F3 and HIF-1α, and the down-regulation of miR-101 in response to VEGF, were reduced by knocking down VEGFR-2 and to a lesser degree by knocking down HIF-1α in LADCa. Conclusion. Our in vitro findings suggest that EZH2 overexpression may promote a more malignant phenotype of lung adenocarcinoma. The pharmacologic inhibition with DZNep and knockdown of EZH2 by siRNA increased sensitivity of LADCa cell lines to cisplatin and carboplatin, and reduced cell migration capabilities. Our results suggest that VEGF/VEGFR-2 axis plays a role in the regulation of EZH2 through E2F3/HIF-1α and miR-101 regulations. EZH2 is a potential therapeutic target in lung cancer that regulates the epigenome to overcome drug resistance. (Supported in part by grants DoD PROSPECT W81XWH-07-1-0306, NCI/UT Lung SPORE 5P50CA70907-11, and Becas Chile) Citation Format: Erick M. Riquelme, Carmen Berhens, Milind Suraokar, Ignacio I. Wistuba. EZH2 promote a malignant phenotype in lung adenocarcinoma and VEGF/VEGFR2 pathways mediates regulation of EZH2 through E2F3/HIF-1α and downregulation of miR-101. [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 4643. doi:10.1158/1538-7445.AM2013-4643

Abstract 5356: EZH2-mediated H3K27me3 is involved in epigenetic repression of DLC1 (Deleted in liver cancer 1) in human cancers.

Abstract Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive Complex 2 catalyzing histone H3 lysine 27 tri-methylation (H3K27me3), is frequently up-regulated in human cancers, including hepatocellular carcinoma (HCC). In this study, we identified the tumor suppressor Deleted in liver cancer 1 (DLC1) as a target of repression by EZH2-mediated H3K27me3. DLC1 is a GTPase-activating protein for Rho family proteins. Inactivation of DLC1 results in hyper-activated Rho/ROCK signaling and is implicated in actin cytoskeleton reorganization to promote cancer metastasis. By chromatin immunoprecipitation assay, we demonstrated that H3K27me3 was significantly enriched at the DLC1 promoter region of a DLC1-nonexpressing HCC cell line, MHCC97L. Depletion of EZH2 in MHCC97L by short hairpin RNA reduced H3K27me3 level at DLC1 promoter and induced DLC1 gene re-expression. Conversely, transient overexpression of GFP-EZH2 in DLC1-expressing Huh7 cells reduced DLC1 mRNA level with a concomitant enrichment of EZH2 on DLC1 promoter. An inverse relation in the expression pattern between EZH2 and DLC1 was observed in the liver, lung, breast, prostate, and ovarian cancer tissues. Treating cancer cells with the EZH2 small molecular inhibitor, 3-Deazaneplanocin A (DZNep), restored DLC1 expression in different cancer cell lines, indicating that EZH2-mediated H3K27me3 epigenetic regulation of DLC1 was a common mechanism in human cancers. Importantly, we found that DZNep treatment inhibited HCC cell migration through disrupting actin cytoskeleton network, suggesting the therapeutic potential of DZNep in targeting cancer metastasis. Taken together, our study has shed mechanistic insight into EZH2-H3K27me3 epigenetic repression of DLC1 and advocated the significant pro-metastatic role of EZH2 via repressing tumor and metastasis suppressors. Citation Format: Leung Kuen Au, Carmen Chak-Lui Wong, Joyce Man-Fong Lee, Chun-Ming Wong, Irene Oi-Lin Ng. EZH2-mediated H3K27me3 is involved in epigenetic repression of DLC1 (Deleted in liver cancer 1) in human cancers. [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 5356. doi:10.1158/1538-7445.AM2013-5356

Update of research on the role of EZH2 in cancer progression

Accumulating evidence shows that enhancer of zeste homolog 2 (E2H2) is upregulated in a broad range of cancer types, such as breast cancer, prostate cancer, ovarian cancer, and colon cancer. Therefore, inhibiting EZH2 expression may be a promising strategy for anticancer therapy. This review focuses on the current understanding of the mechanisms underlying EZH2 regulation that are involved in cancer progression. Also, it introduces two EZH2 inhibitors that target EZH2 and could be potentially applied in the treatment of cancer in the future.

Role of EZH2 in Epithelial Ovarian Cancer: From Biological Insights to Therapeutic Target

EZH2 is the catalytic subunit of polycomb repressive complex 2 (PRC2), which generates a methylation epigenetic mark at lysine 27 residue of histone H3 (H3K27me3) to silence gene expression. EZH2 target genes are involved in a variety of biological processes such as stem cell pluripotency, cell proliferation, and oncogenic transformation. EZH2 is often over-expressed in epithelial ovarian cancer (EOC) cells and in ovarian cancer-associated stromal endothelial cells. Notably, EZH2 promotes cell proliferation, inhibits apoptosis and enhances angiogenesis in EOCs. In contrast to genetic alterations, which are typically non-reversible, epigenetic alterations are reversible. Thus, inhibiting EZH2/PRC2 activity represents an attractive strategy for developing ovarian cancer therapeutics by targeting both ovarian cancer cells and ovarian tumor microenvironment. Here we discuss the progress recently obtained in understanding how EZH2/PRC2 promotes malignant phenotypes of EOC. In addition, we focus on strategies for targeting EZH2/PRC2 to develop novel EOC epigenetic therapeutics.

Alternative Role for EZH2

Alternative Role for EZH2

EZH2 promotes a bi-lineage identity in basal-like breast cancer cells

The mechanisms regulating breast cancer differentiation state are poorly understood. Of particular interest are molecular regulators controlling the highly aggressive and poorly differentiated traits of basal-like breast carcinomas. Here we show that the Polycomb factor EZH2 maintains the differentiation state of basal-like breast cancer cells, and promotes the expression of progenitor associated and basal-lineage genes. Specifically, EZH2 regulates the composition of basal-like breast cancer cell populations by promoting a ‘bi-lineage’ differentiation state, in which cells co-express basal- and luminal-lineage markers. We show that human basal-like breast cancers contain a subpopulation of bi-lineage cells, and that EZH2-deficient cells give rise to tumors with a decreased proportion of such cells. Bi-lineage cells express genes that are active in normal luminal progenitors, and possess increased colony-formation capacity, consistent with a primitive differentiation state. We found that GATA3, a driver of luminal differentiation, performs a function opposite to EZH2, acting to suppress bi-lineage identity and luminal-progenitor gene expression. GATA3 levels increase upon EZH2 silencing, mediating a decrease in bi-lineage cell numbers. Our findings reveal a novel role for EZH2 in controlling basal-like breast cancer differentiation state and intra-tumoral cell composition.

Downregulation of Ezh2 expression by RNA interference induces cell cycle arrest in the G0/G1 phase and apoptosis in U87 human glioma cells

The Ezh2 gene is an important member of the polycomb-group (PcG) family. As a newly identified oncogene, the expression of Ezh2 has been shown to be significantly increased in prostate cancer, breast cancer, renal cell carcinoma and hepatic cancer; however, a role for Ezh2 in the occurrence of glioma has not yet been reported. In this study, we found that the Ezh2 gene is highly expressed in U87 human glioma cells. Using RNA interference, we demonstrated that the downregulation of Ezh2 expression in U87 human glioma cells resulted in apoptosis and a cell cycle arrest in the G0/G1 phase. In addition, we found that silencing of the Ezh2 gene altered the mitochondrial membrane potential and promoted the release of cytochromec from the mitochondria. Furthermore, the reduced expression of Ezh2 altered the Bax and Bcl-2 protein levels and led to the activation of caspase 9 and 3. These results indicate that the apoptosis induced in U87 human glioma cells by the silencing of the Ezh2 gene is related to the mitochondrial pathway.

Ezh2 augments leukemogenicity by reinforcing differentiation blockage in acute myeloid leukemia

AbstractEZH2, a catalytic component of the polycomb repressive complex 2, trimethylates histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Although EZH2 is overexpressed in various cancers, including some hematologic malignancies, the role of EZH2 in acute myeloid leukemia (AML) has yet to be examined in vivo. In the present study, we transformed granulocyte macrophage progenitors from Cre-ERT;Ezh2flox/flox mice with the MLL-AF9 leukemic fusion gene to analyze the function of Ezh2 in AML. Deletion of Ezh2 in transformed granulocyte macrophage progenitors compromised growth severely in vitro and attenuated the progression of AML significantly in vivo. Ezh2-deficient leukemic cells developed into a chronic myelomonocytic leukemia–like disease with a lower frequency of leukemia-initiating cells compared with the control. Chromatin immunoprecipitation followed by sequencing revealed a significant reduction in the levels of trimethylation at H3K27 in Ezh2-deficient leukemic cells, not only at Cdkn2a, a known major target of Ezh2, but also at a cohort of genes relevant to the developmental and differentiation processes. Overexpression of Egr1, one of the derepressed genes in Ezh2-deficient leukemic cells, promoted the differentiation of AML cells profoundly. Our findings suggest that Ezh2 inhibits differentiation programs in leukemic stem cells, thereby augmenting their leukemogenic activity.

Abstract 2483: Inhibition of the polycomb group gene EZH2 suppresses growth and radiosensitizes ATRT cells by promoting senescence and inhibiting the CycinD-E2F1 axis

Abstract Overexpression of the polycomb group gene Enhancer of Zeste 2 (EZH2) occurs in many tumors including prostate cancer, breast cancer and brain tumors such as Glioblastoma multiform and Medulloblastoma. Recent evidence suggests it may also have a role in rhabdoid tumors. Atypical teratoid rhabdoid tumor (ATRT) is a rare high-grade brain tumor with very poor survival that occurs most commonly in children. AT/RT tumors are characterized by deletion of the chromatin associated protein SMARCB1. Given the role of EZH2 in regulating epigenetic changes we chose to investigate the role of EZH2 in AT/RT. Here we show that targeted disruption of EZH2 by RNAi or pharmacologic means strongly impairs ATRT cell growth, induces apoptosis and potently sensitizes these cells to radiation. Using functional analysis of transcription factor activity we found the Cyclin D-E2F1 axis to be repressed upon EZH2 depletion in ATRT cells. Taken together our observations provide evidence that EZH2 regulates cell cycle progression by targeting the G1-S transition machinery and may be an important new therapeutic target particularly in combination with radiation in ATRT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2483. doi:1538-7445.AM2012-2483

Abstract 5208: Overexpression of EZH2 increases multipotent mammary stem cell self-renewal and differentiation

Abstract BACKGROUND: A role for EZH2 in stem cell maintenance was recently reported. Our lab has previously demonstrated that overexpression of EZH2 accelerates the initiation of ErbB2-induced mammary tumors. To understand underlying mechanism, we investigated the role of EZH2 in regulation of stem cells and luminal progenitor cells utilizing xenotransplantation studies of preneoplastic mammary glands of EZH2+; neu and EZH2 wt; neu transgenic mice. METHODS: Generation of crossing EZH2+; neu and EZH2 wt; neu transgenic mice was described previously. Mouse handling and experimental procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the IACUC of the UM. Stem cells (Lin-/CD49f+/ESA-) and luminal progenitor cells (Lin-/CD49f+/ESA+) were isolated from the hyperplastic mammary glands of 8 weeks old EZH2+; neu and EZH2 wt; neu mice by flow cytometry (n=15 per group). Stem cells and luminal progenitor cells were injected into cleared fat pads of 3 weeks old FVB mice. After 5 weeks the mammary glands were subjected to flow cytometry and histology. RESULTS: Histologic study showed that only the stem cells, but not the luminal progenitor cells, extracted from EZH2+; neu and EZH2 wt; neu transgenic mice were able to form mammary outgrowths. Outgrowths of EZH2+;neu mice had significantly increased epithelial proliferation and increased numbers of terminal end buds (p&amp;lt;0.05) compared to EZH2 wt;neu outgrowths. EZH2 +;neu outgrowths had significant upregulation of Ki67 and pSTAT5 proteins. Flow cytometry of the outgrowths revealed that while all cell populations were increased, EZH2 induced a more pronounced expansion of the stem cell and luminal progenitor cell populations. CONCLUSIONS: Our data show that EZH2 overexpression in ErbB2 mice induces an expansion of the multipotent mammary stem cell population with the ability to regenerate the cellular heterogeneity of the mammary gland and develop hyperplastic outgrowths. We found that EZH2 promotes hyperproliferation and upregulation of STAT5 phosphorylation in the mammary epithelium in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5208. doi:1538-7445.AM2012-5208

Abstract LB-148: The role of EZH2 in the regulation of the activity of matrix metalloproteinases in prostate cancer cells

Abstract Degradation of the extracellular matrix (ECM), a critical step in cancer metastasis, is determined by the balance between MMPs (matrix metalloproteinases) and their inhibitors TIMPs (tissue inhibitors of metalloproteinases). In cancer cells, this balance is shifted towards MMPs, promoting ECM degradation. Here, we show that EZH2 plays an active role in this process by repressing the expression of TIMP2 and TIMP3 in prostate cancer cells. The TIMP genes are derepressed by knockdown of EZH2 expression in human prostate cancer cells but repressed by overexpression of EZH2 in benign human prostate epithelial cells. EZH2 catalyzes H3K27 trimethylation and subsequent DNA methylation of the TIMP gene promoters. Overexpression of EZH2 confers an invasive phenotype on benign prostate epithelial cells; however, this phenotype is suppressed by cooverexpression of TIMP3. EZH2 knockdown markedly reduces the proteolytic activity of MMP-9, thereby decreasing the invasive activity of prostate cancer cells. These results suggest that the transcriptional repression of the TIMP genes by EZH2 may be a major mechanism to shift the MMPs/TIMPs balance in favor of MMP activity and thus to promote ECM degradation and subsequent invasion of prostate cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-148. doi:1538-7445.AM2012-LB-148

Conditional Ablation of Ezh2 in Murine Hearts Reveals Its Essential Roles in Endocardial Cushion Formation, Cardiomyocyte Proliferation and Survival

Ezh2 is a histone trimethyltransferase that silences genes mainly via catalyzing trimethylation of histone 3 lysine 27 (H3K27Me3). The role of Ezh2 as a regulator of gene silencing and cell proliferation in cancer development has been extensively investigated; however, its function in heart development during embryonic cardiogenesis has not been well studied. In the present study, we used a genetically modified mouse system in which Ezh2 was specifically ablated in the mouse heart. We identified a wide spectrum of cardiovascular malformations in the Ezh2 mutant mice, which collectively led to perinatal death. In the Ezh2 mutant heart, the endocardial cushions (ECs) were hypoplastic and the endothelial-to-mesenchymal transition (EMT) process was impaired. The hearts of Ezh2 mutant mice also exhibited decreased cardiomyocyte proliferation and increased apoptosis. We further identified that the Hey2 gene, which is important for cardiomyocyte proliferation and cardiac morphogenesis, is a downstream target of Ezh2. The regulation of Hey2 expression by Ezh2 may be independent of Notch signaling activity. Our work defines an indispensible role of the chromatin remodeling factor Ezh2 in normal cardiovascular development.