Increased EZH2 Expression Research Articles

Epigenetic Silencing of miRNA-338-5p and miRNA-421 Drives SPINK1-Positive Prostate Cancer.

Serine peptidase inhibitor, Kazal type-1 (SPINK1) overexpression defines the second most recurrent and aggressive prostate cancer subtype. However, the underlying molecular mechanism and pathobiology of SPINK1 in prostate cancer remains largely unknown. miRNA prediction tools were employed to examine the SPINK1-3'UTR for miRNA binding. Luciferase reporter assays were performed to confirm the SPINK1-3'UTR binding of shortlisted miR-338-5p/miR-421. Furthermore, miR-338-5p/-421-overexpressing cancer cells (SPINK1-positive) were evaluated for oncogenic properties using cell-based functional assays and a mouse xenograft model. Global gene expression profiling was performed to unravel the biological pathways altered by miR-338-5p/-421. IHC and RNA in situ hybridization were carried out on prostate cancer patients' tissue microarray for SPINK1 and EZH2 expression, respectively. Chromatin immunoprecipitation assay was performed to examine EZH2 occupancy on the miR-338-5p/-421-regulatory regions. Bisulfite sequencing and methylated DNA immunoprecipitation were performed on prostate cancer cell lines and patients' specimens. We established a critical role of miRNA-338-5p/-421 in posttranscriptional regulation of SPINK1. Ectopic expression of miRNA-338-5p/-421 in SPINK1-positive cells abrogates oncogenic properties including cell-cycle progression, stemness, and drug resistance, and shows reduced tumor burden and distant metastases in a mouse model. Importantly, we show that patients with SPINK1-positive prostate cancer exhibit increased EZH2 expression, suggesting its role in epigenetic silencing of miRNA-338-5p/-421. Furthermore, presence of CpG dinucleotide DNA methylation marks on the regulatory regions of miR-338-5p/-421 in SPINK1-positive prostate cancer cells and patients' specimens confirms epigenetic silencing. Our findings revealed that miRNA-338-5p/-421 are epigenetically silenced in SPINK1-positive prostate cancer, although restoring the expression of these miRNAs using epigenetic drugs or synthetic mimics could abrogate SPINK1-mediated oncogenesis.See related commentary by Bjartell, p. 2679.

EZH2 promotes gastric cancer cells proliferation by repressing p21 expression

EZH2 is a core component of the polycomb repressive complex 2 (PRC2), which catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) and promotes carcinogenesis by epigenetically silencing many tumor suppressor genes. Increased EZH2 expression is a marker of advanced and metastatic in many cancers, including lung, prostate and breast cancer, and it has been considered as a potential novel therapeutic target. However, the clinical significance and molecular mechanisms of EZH2 controlling gastric cancer cell proliferation and invasion are not well documented. In this study, immunohistochemical analysis was conducted to investigate the EZH2 expression in gastric cancer. We found that EZH2 levels were increased in gastric cancer tissues compared with adjacent normal tissues. Moreover, patients with high levels of EZH2 expression had a relatively poor prognosis. Furthermore, knockdown of EZH2 expression by siRNA could impair cell proliferation and invasion both in vitro and vivo. Finally, we found that EZH2 influences gastric cancer cells proliferation partly through regulating p21 expression. Our findings present that EZH2 over-expression can be identified as a poor prognostic biomarker in gastric cancer.

SOX5 promotes breast cancer proliferation and invasion by transactivation of EZH2.

Sex determining region Y-box protein 5 (SOX5) is a transcriptional factor and serves important roles in various cancer types; however, the pathological role of SOX5 in patients with breast cancer remains unclear. In the present study, the expression and potential role of SOX5 in patients with breast cancer and in breast cancer cells was investigated. The data indicated that SOX5 was highly expressed in breast cancer tissues compared with adjacent healthy tissues, and overexpression of SOX5 was associated with a reduced overall survival rate in patients with breast cancer. Gain and loss of function studies with MTT, colony formation, wound healing and Matrigel invasion assays demonstrated that SOX5 significantly promoted breast cancer cell proliferation and invasion. The chromatin immunoprecipitation (ChIP) assay sequence, quantitative ChIP and luciferase reporter assays were used to identify enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) as a downstream target gene of SOX5. Furthermore, it was determined that ectopic expression of SOX5 increased EZH2 expression at the mRNA and protein level, while the knockdown of SOX5 decreased EZH2 expression. Additionally, the biological effect of SOX5 was investigated, and it was determined to be dependent on the regulation of EZH2 expression. The present results may provide important insights into the biological significance of SOX5 serving as a candidate therapeutic target in breast cancer progression.

Modulation of EZH2 expression in T cells improves efficacy of anti-CTLA-4 therapy.

Enhancer of zeste homolog 2-mediated (EZH2-mediated) epigenetic regulation of T cell differentiation and Treg function has been described previously; however, the role of EZH2 in T cell-mediated antitumor immunity, especially in the context of immune checkpoint therapy, is not understood. Here, we showed that genetic depletion of EZH2 in Tregs (FoxP3creEZH2fl/fl mice) leads to robust antitumor immunity. In addition, pharmacological inhibition of EZH2 in human T cells using CPI-1205 elicited phenotypic and functional alterations of the Tregs and enhanced cytotoxic activity of Teffs. We observed that ipilimumab (anti-CTLA-4) increased EZH2 expression in peripheral T cells from treated patients. We hypothesized that inhibition of EZH2 expression in T cells would increase the effectiveness of anti-CTLA-4 therapy, which we tested in murine models. Collectively, our data demonstrated that modulating EZH2 expression in T cells can improve antitumor responses elicited by anti-CTLA-4 therapy, which provides a strong rationale for a combination trial of CPI-1205 plus ipilimumab.

Clinicopathological Significances of EZH2 & Twist-1 Combined Expressions in Renal Cell Carcinoma (RCC)

Abstract Background: Renal cell carcinoma (RCC) is considered the commonest and most fatal malignant neoplasm of the kidney. Distant metastases are found in 30% of RCC patients at diagnosis, which is considered an important sign of predic-tion of patients' prognosis. So trying to clarify molecular mechanisms of metastases in RCC could help to discover a recent therapeutic target to decrease and control distant metastases and subsequently might improve patients prognosis.Aim of the Study: Was to assess the EZH2 & Twist-1 combined expressions in RCC tissues with different histopatho-logical subtypes and to correlate their expression with clin-icopathological parameters.Material and Methods: We have assessed expression of EZH2 & Twist-1 in tissue by using immunohistochemistry in 50 paraffin blocks of RCC different histopathological subtypes, analyzed correlations between the levels of combined expres-sions and clinicopthological data. Results: Increased EZH2 expression was positively asso-ciated with advanced AJCC stage, Lymph Node (L.N) metas-tases (p=0.006), higher grade (p<0.001), and presence of distant metastases (p=0.045). Increased Twist-1 expression was positively associated with higher grade, L.N (p<0.001) & distant metastases (p=0.04) and AJCC stage (p=0.01). Both EZH& Twist-1 expressions were positively correlated to each other (p<0.001).Conclusion: EZH2 & Twist-1 are considered markers of poor prognosis in RCC and so they could be used as novel therapeutic targets in RCC patients.

Multi-institutional evaluation of the prognostic significance of EZH2 expression in high-grade upper tract urothelial carcinoma

PurposeEnhancer of zeste homolog 2 is a methyltransferase encoded by the EZH2 gene, whose role in upper tract urothelial carcinoma (UTUC) is poorly understood. We sought to evaluate the prognostic value of EZH2 expression in UTUC. MethodsWe reviewed a multi-institutional cohort of patients who underwent radical nephroureterectomy for high-grade UTUC from 1990 to 2008. Immunohistochemistry for EZH2 was performed on tissue microarrays. Percentage of staining was evaluated, and the discriminative value of EZH2 was tested, with EZH2 positivity defined as>20% staining present. Clinicopathologic characteristics and oncologic outcomes (recurrence-free (RFS), cancer-specific (CSS), and overall survival (OS)) were compared, stratified by EZH2 positivity. The prognostic role of EZH2 was assessed using Kaplan-Meier, univariate (UVA), and multivariate (MVA) Cox regression analyses. Significance was defined for P<0.05. ResultsA total of 376 patients were included for analysis, with median follow-up 36.0 months. Overall, 78 (20.7%) were EZH2-positive. EZH2 expression was more often associated with ureteral location, lymphovascular invasion, sessile architecture, necrosis, and concomitant carcinoma in situ. On UVA, increased EZH2 expression was a significant predictor for inferior RFS (HR 1.63, P = 0.033), CSS (HR 2.03, P = 0.003), and OS (HR 2.11, P<0.001). On MVA EZH2 remained a significant predictor of worse CSS (HR 1.99 [95% CI: 1.21–3.27], P = 0.007) and OS (HR 1.54 [95% CI: 1.06–2.24], P = 0.024), while significance was lost for RFS. ConclusionIncreased EZH2 expression is associated with adverse pathologic features and inferior oncologic outcomes in patients with high-grade UTUC. The role of EZH2 biology in UTUC pathogenesis remains to be further elucidated.

The Histone Methyltransferase Ezh2 Controls Mechanisms of Adaptive Resistance to Tumor Immunotherapy

Immunotherapy and particularly immune checkpointinhibitors have resulted in remarkable clinical responses in patients with immunogenic tumors, although most cancers develop resistance to immunotherapy. The molecular mechanisms of tumor resistance to immunotherapy remain poorly understood. We now show that induction of the histone methyltransferase Ezh2 controls several tumor cell-intrinsic and extrinsic resistance mechanisms. Notably, Tcell infiltration selectively correlated with high EZH2-PRC2 complex activity in human skin cutaneous melanoma. During anti-CTLA-4 or IL-2 immunotherapy in mice, intratumoral tumor necrosis factor-α (TNF-α) production and Tcell accumulation resulted in increased Ezh2 expression in melanoma cells, which in turn silenced their own immunogenicity and antigen presentation. Ezh2 inactivation reversed this resistance and synergized with anti-CTLA-4 and IL-2 immunotherapy to suppress melanoma growth. These anti-tumor effects depended on intratumorally accumulating interferon-γ (IFN-γ)-producing PD-1low CD8+ Tcells and PD-L1 downregulation on melanoma cells. Hence, Ezh2 serves as a molecular switch controlling melanoma escape during Tcell-targeting immunotherapies.

Loss of TET1 facilitates DLD1 colon cancer cell migration via H3K27me3-mediated down-regulation of E-cadherin.

Epigenetic modifications such as histone modifications and cytosine hydroxymethylation are linked to tumorigenesis. Loss of 5-hydroxymethylcytosine (5 hmC) by ten-eleven translocation 1 (TET1) down-regulation facilitates tumor initiation and development. However, the mechanisms by which loss of TET1 knockdown promotes malignancy development remains unclear. Here, we report that TET1 knockdown induced epithelial-mesenchymal transition (EMT) and increased cancer cell growth, migration, and invasion in DLD1 cells. Loss of TET1 increased EZH2 expression and reduced UTX-1 expression, thus increasing histone H3K27 tri-methylation causing repression of the target gene E-cadherin. Ectopic expression of the H3K27 demethylase UTX-1 or EZH2 depletion both impeded EZH2 binding caused a loss of H3K27 methylation at epithelial gene E-cadherin promoter, thereby suppressing EMT and tumor invasion in shTET1 cells. Conversely, UTX-1 depletion and ectopic expression of EZH2 enhanced EMT and tumor metastasis in DLD1 cells. These findings provide insight into the regulation of TET1 and E-cadherin and identify EZH2 as a critical mediator of E-cadherin repression and tumor progression.

Targeting EZH2 in acral lentiginous melanoma (ALM).

9534 Background: Efforts to identify targeted therapies that can improve treatment outcome in metastatic ALM have been unsuccessful. In a previous genomic screening, we identified copy number amplification of the histone methyltransferase EZH2 in 47% of ALM cases, a higher frequency than previously reported in cutaneous melanomas (CM) (5%). Here, we tested the hypothesis that increased EZH2 expression contributes to ALM progression and may confer selective sensitivity to EZH2 inhibition. Methods: EZH2 expression was examined by immunohistochemistry (IHC) in 51 primary (21 stage I, 13 Stage II and 17 Stage III) and 23 metastatic (11 in transit, 8 nodal and 4 visceral) ALM cases with extensive clinicopathological data and protocol-driven follow up. Colony formation and cell proliferation was assessed following treatment of ALM and CM cell lines with three EZH2 inhibitors, including GSK126, currently in clinical trials. The effect of GSK126 on H3K27me3 and downstream EZH2 targets was analyzed by western blotting. Results: EZH2 is commonly overexpressed in both primary (30/51; 65%) and metastatic (20/23; 87%) ALM cases, with a significant increase in mean IHC score between primary and metastatic tumors (1.9 vs. 2.7, respectively, p = 0.047). EZH2 expression increased in 6/10 metastatic ALM tumors compared to their matched primary tumors. ALM tumors with EZH2 gene amplification showed increased EZH2 protein expression; however more cases showed overexpression with no amplification suggesting a potential epigenetic component of EZH2 regulation. GSK126 significantly suppressed ALM colony formation at lower doses compared to CM (1 µM vs. 5 µM, respectively). EZH2 inhibition also increased expression of the downstream tumor suppressor E-cadherin in ALM but not in CM cell lines. Finally, ALM cell lines had significantly lower basal H3K27me3 levels than CM cell lines, suggesting an additional, histone methyltransferase-independent function of EZH2 in ALM. Conclusions: Our data demonstrate thatEZH2 upregulation is common in ALM, and suggest that it may play a role in ALM’s metastatic progression that requires further investigation. Selective sensitivity of ALM cell lines to EZH2 inhibitors supports the therapeutic potential of EZH2-targeted therapy in ALM.

EZH2 protein expression in normal breast epithelium and risk of breast cancer: results from the Nurses\u2019 Health Studies

BackgroundEnhancer of zeste homolog 2 (EZH2) is a polycomb-group protein that is involved in stem cell renewal and carcinogenesis. In breast cancer, increased EZH2 expression is associated with aggressiveness and has been suggested to identify normal breast epithelium at increased risk of breast cancer development. However, the association between EZH2 expression in benign breast tissue and breast cancer risk has not previously been evaluated in a large prospective cohort.MethodsWe examined the association between EZH2 protein expression and subsequent breast cancer risk using logistic regression in a nested case-control study of benign breast disease (BBD) and breast cancer within the Nurses’ Health Studies. EZH2 immunohistochemical expression in normal breast epithelium and stroma was evaluated by computational image analysis and its association with breast cancer risk was analyzed after adjusting for matching factors between cases and controls, the concomitant BBD diagnosis, and the Ki67 proliferation index.ResultsWomen with a breast biopsy in which more than 20% of normal epithelial cells expressed EZH2 had a significantly increased risk of developing breast cancer (odds ratio (OR) 2.95, 95% confidence interval (CI) 1.11–7.84) compared to women with less than 10% EZH2 epithelial expression. The risk of developing breast cancer increased for each 5% increase in EZH2 expression (OR 1.22, 95% CI 1.02–1.46, p value 0.026). Additionally, women with high EZH2 expression and low estrogen receptor (ER) expression had a 4-fold higher risk of breast cancer compared to women with low EZH2 and low ER expression (OR 4.02, 95% CI 1.29–12.59).ConclusionsThese results provide further evidence that EZH2 expression in the normal breast epithelium is independently associated with breast cancer risk and might be used to assist in risk stratification for women with benign breast biopsies.

Opposing roles of PIK3CA gene alterations to EZH2 signaling in non-muscle invasive bladder cancer.

The high rates of tumor recurrence and progression represent a major clinical problem in non-muscle invasive bladder cancer. Previous data showed that EZH2-dependent signaling mediates these processes, whereas the frequent alterations of PIK3CA gene (copy gains and mutations) are predictive of reduced recurrence. Here we show, using clinical samples and bladder cancer cell lines, a functional interaction between EZH2- and PIK3CA-dependent signaling pathways. PIK3CA alterations mediated, on the one hand, the increased expression of two miRNAs, miR-101 and miR-138, which posttranscriptionally downregulate EZH2 expression. On the other hand, PIK3CA alterations facilitate the activation of Akt which phosphorylates EZH2 on Ser21, precluding the trimethylation of histone H3 in K27. Remarkably the increased expression of miR101 or miR138 and the expression of Ser21-phosphorylated EZH2 are good prognostic factors regarding non-muscle invasive bladder cancer recurrence and progression. Collectively, this study provides molecular evidences indicating that the gene expression rewiring occurring in primary bladder tumors, associated with increased EZH2 expression and activity and mediating the increased recurrence and progression risk, are prevented by PIK3CA-dependent signaling. This molecular process may have deep implications in the management of bladder cancer patients and in the design of novel molecularly targeted therapeutic approaches.

Global Transcriptome Analysis Reveals That Poly(ADP-Ribose) Polymerase 1 Regulates Gene Expression through EZH2.

Posttranslational modifications, such as poly(ADP-ribosyl)ation (PARylation), regulate chromatin-modifying enzymes, ultimately affecting gene expression. This study explores the role of poly(ADP-ribose) polymerase (PARP) on global gene expression in a lymphoblastoid B cell line. We found that inhibition of PARP catalytic activity with olaparib resulted in global gene deregulation, affecting approximately 11% of the genes expressed. Gene ontology analysis revealed that PARP could exert these effects through transcription factors and chromatin-remodeling enzymes, including the polycomb repressive complex 2 (PRC2) member EZH2. EZH2 mediates the trimethylation of histone H3 at lysine 27 (H3K27me3), a modification associated with chromatin compaction and gene silencing. Both pharmacological inhibition of PARP and knockdown of PARP1 induced the expression of EZH2, which resulted in increased global H3K27me3. Chromatin immunoprecipitation confirmed that PARP1 inhibition led to H3K27me3 deposition at EZH2 target genes, which resulted in gene silencing. Moreover, increased EZH2 expression is attributed to the loss of the occupancy of the transcription repressor E2F4 at the EZH2 promoter following PARP inhibition. Together, these data show that PARP plays an important role in global gene regulation and identifies for the first time a direct role of PARP1 in regulating the expression and function of EZH2.

Abstract 1448: Dynamic chromatin modification by tumor secreted Hsp90 modulates EMT and tumor invasion

Abstract Tumor metastasis is the main cause of prostate cancer lethality. Pathological reactivation of the developmental genetic program epithelial to mesenchymal (EMT) is associated with increased tumorigenesis and invasion, and is considered a primary culprit for tumor dissemination. Hallmarks of EMT include upregulation of the core transcription factors Zeb and Snail, concomitant with suppression of the junctional protein E-cadherin. Overexpression of the epigenetic repressor EZH2, a histone methyltransferase, is also implicated in EMT activation. Although these molecular events are well known to trend with cancer progression, a paucity of knowledge exists regarding identification of the clinically-relevant upstream triggers capable of setting these events into motion. We recently demonstrated that tumor secreted extracellular heat shock protein 90 (eHsp90) initiates EMT events in prostate cancer cells. Moreover, we reported the presence of surface Hsp90 in human prostatectomy specimens, an event highly correlated with elevated expression of a subset of EMT transcripts. The present study reveals novel mechanistic aspects of eHsp90 action. We define an epigenetic function for eHsp90 as a significant regulator of EZH2 expression and activity. This eHsp90-EZH2 axis was found to govern a majority of eHsp90's pro-EMT activity in vitro, and was essential for E-cadherin suppression and tumor invasion in vivo. Upon further investigation, it was revealed that eHsp90 sustained ERK activation. This eHsp90-ERK pathway was required for the increased expression of EZH2, as well as for its directed recruitment to the E-cadherin promoter. Moreover, eHsp90-ERK signaling augmented the epigenetic landscape of Zeb and Snail towards a profile permissive for transcriptional activation. Hence, our findings indicate that eHsp90-ERK signaling has profound effects upon key drivers of cancer progression via the concerted action of epigenetic modulation of EMT effectors and increased EZH2 expression. Collectively, these data support a model wherein tumor eHsp90 functions as an upstream rheostat for EZH2 expression and activity to orchestrate mesenchymal properties and coincident aggressive behavior. Importantly, our findings suggest that eHsp90 may be a clinically relevant instigator in the transition from localized to invasive disease. Citation Format: Krystal D. Nolan, Michael W. Hance, Omar E. Franco, Simon W. Hayward, Jennifer S. Isaacs. Dynamic chromatin modification by tumor secreted Hsp90 modulates EMT and tumor invasion. [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 1448. doi:10.1158/1538-7445.AM2015-1448

EZH2 is a negative prognostic factor and exhibits pro-oncogenic activity in glioblastoma

The identification of single or less genes based on mRNA expression as clinical diagnostic markers for glioblastoma (GBM) remains a challenge. Recent data have shown the potential oncogenic role and prognostic significance of EZH2 in several human cancers. However, the clinical signature and further mechanisms of EZH2 function in gliomagenesis are still poorly understood. In this study, we found that increased EZH2 expression was associated with tumor grade. High expression of EZH2 in GBM was determined to be a strong and independent predictor of short overall survival. Further, we screened EZH2 targets and associated genes in GBM. Repression of EZH2 induced cell cycle arrest and inhibited tumor growth in vivo. This event represents a positive feedback loop with β-catenin/TCF4 and STAT3 signaling. Taken together, EZH2 could be an independent prognostic factor and potential therapeutic target for GBM.

Expression and clinicopathological significance of EED, SUZ12 and EZH2 mRNA in colorectal cancer.

Enhancer of zeste 2 (EZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 homolog (SUZ12), the key component of polycomb repressive complex 2, are of great importance in human cancer pathogenesis. This study was designed to investigate the clinical and prognostic significances of EZH2, EED and SUZ12 in colorectal cancer (CRC) patients. The expression of EZH2, EED and SUZ12 mRNA was evaluated in 82 primary CRC and paired non-cancerous mucosa samples by qRT-PCR. We found that overall EZH2, EED and SUZ12 mRNA expression in the CRC tissues was significantly increased than in the non-cancerous tissue (p < 0.05). Increased EZH2, EED and SUZ12 mRNA expression was directly correlated with primary tumor size, regional lymph node metastases, distant metastasis and AJCC stage. Furthermore, CRC patients with higher level of EED, SUZ12 or EZH2 showed a worse disease-free survival (DFS) (p < 0.01). In multivariate analysis, the increased EZH2 expression may be a risk factor for the patients' 3-year DFS (HR 2.517; 95% CI 1.104, 5.736; p = 0.028). Furthermore, the k-means cluster analysis showed that high mRNA expression of EED, SUZ12 and EZH2 was significantly correlated with the aggressive clinical behavior and poor prognosis. High expression of EED, SUZ12 and EZH2 might contribute to the CRC development/progression.

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.

Abstract 5353: EZH2-mediated, but not BMI-1, epigenetic alteration may be associated with the accelerated cell proliferation and malignant step in IPMN.

Abstract Backgrounds. Histologic examination of IPMNs shows the co-existence of benign and malignant lesions within a single specimen of the pancreas represented by multifocality. Thus, IPMNs of pancreas are characterized by multifocal and malignant potential represented by an adenomacarcinoma sequence, whereas its molecular mechanism of oncogenesis remains largely unknown. Recently, epigenetic gene silencing in cancer is now increasingly recognized as a novel therapeutic target. The histone methyltransferase Enhancer of Zeste 2 (EZH2) is associated with transcriptional repression. B-lymphoma moloney murine leukemia virus insertion-1 (BMI-1). is a proto-oncogene that belongs to polycom group gene family. The role of epigenetic alteration mediated by EZH2 and BMI-1 in IPMNs progression is unclear. Method. From 2002 to 2011, 54 patients with IPMN underwent curative pancreatic resection. The total 133 IPMN lesions and 48 normal duct epithelial lesions within IPMN lesion were analyzed in this study. In the present study, a total of 133 IPMN lesions were classified as either benign (adenoma and borderline tumors)or malignant (carcinoma in situ or invasive cancer). Result. Fifty-four cases of IPMNs consists of 23 IPMC (91 lesions) and 31 IPMA (90 lesions). We assessed to a proliferative activity in IPMNs by Ki-67 immunostainig. The number of Ki-67 positive cell was significantly increased during the progression of IPMNs. In malignant IPMN, Ki-67 positive cell was significantly increased compared with that in benign IPMN. Thus, the increased proliferative activity plays an important role in an adenoma-carcinoma sequence of IPMNs. The expression of cell-cycle related suppressor protein, p27Kip1 expression was inversely down-regulated during the progression of IPMNs. To further assess the molecular mechanism of the downregulated p27KIP1 expression during the progression of IPMNs, we focused the epigenetic epigenetic gene silencing during the tumor progression. The EZH2 expression was increased step by step during the progression of the IPMNs. In benign IPMN (IPMA), EZH2-negative cells expressed p27KIP1 protein. In contrast, in malignant IPMN (CIS lesion), EZH2-positive cells unexpressed p27KIP1 protein, suggesting that the increased EZH2 expression during the progression of IPMNs suppress the p27KIP1 protein expression. Furthermore, EZH2 expression level in IPMNs showed the significantly positive correlation with the Ki-67 positive nuclear ratio (p&amp;lt;0.0001). However, there was no significant association between BMI-1 expression and Ki-67 positive nuclear ratio. Conclusion. Here, we show EZH2-mediated, but not BMI-1, epigenetic alteration as a novel oncogenetic mechanism in IPMNs. EZH2-mediated epigenetic alteration may be associated with the accelerated cell proliferation and malignant step in IPMN via a downregulation of p27KIP1. Citation Format: Hideyuki Kuroki. EZH2-mediated, but not BMI-1, epigenetic alteration may be associated with the accelerated cell proliferation and malignant step in IPMN. [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 5353. doi:10.1158/1538-7445.AM2013-5353

Abstract SY02-02: Inhibition of methyltransferase EZH2

Abstract Enhance of Zeste Homolog 2 (EZH2) is the biochemical activity of the Polycomb Repressive Complex 2 (PRC2) that transfers a methyl group from the cofactor S-adenosyl methionine (SAM) to histone H3 on lysine 27 (H3K27), resulting in epigenetic silencing of target gene expression. Activity of the multi-component PRC2 complex is crucial for normal development and differentiation. However, dysregulation of H3K27 methylation is implicated in tumorigenesis and occurs through multiple mechanisms. Elevated levels of EZH2 correlate with poor prognosis in a number of solid tumors including prostate, breast, kidney and lung. The increased EZH2 expression observed in many solid tumors has been linked to loss of one or more miRNAs including mir-101, mir-26A, and mir-214; aberrant E2F activity and chromosomal amplification. Inactivating mutations in UTX, an H3K27 demethylase which acts in opposition to EZH2, have been described in several tumor types including transitional cell bladder carcinoma, esophageal squamous cell carcinoma, renal cell carcinoma and multiple myeloma. Recently, somatic activating mutations in EZH2 have been identified in follicular lymphoma (FL) and GCB diffuse large B cell lymphoma (DLBCL). The frequency of the most prevalent mutation, Y641, is reported to be 7-12% in FL and as high as 22% in DLBCL. In view of the above, biochemical inhibitors of EZH2 may have therapeutic utility and herein we report on the identification and characterization of potent and selective small molecule EZH2 inhibitors with in vitro and in vivo antiproliferative activity in cancer cell lines and mouse xenografts. The studies provide a compelling rationale for the clinical evaluation of EZH2 inhibitors in EZH2 driven human cancers. 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 SY02-02. doi:1538-7445.AM2012-SY02-02

High miR-26a and low CDC2 levels associate with decreased EZH2 expression and with favorable outcome on tamoxifen in metastatic breast cancer

For patients with metastatic breast cancer, we previously described that increased EZH2 expression levels were associated with an adverse outcome to tamoxifen therapy. Main objective of the present study is to investigate miR-26a and miR-101 levels, which both target EZH2, for their association with molecular pathways and with efficacy of tamoxifen as first-line monotherapy for metastatic breast cancer. Expression levels were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens of 235 estrogen receptor-α (ER)-positive patients. Pathway analysis was performed on microarray data available for 65 of these tumors. Logistic regression and Cox uni- and multivariate analysis were performed to relate expression levels with clinical benefit and time to progression (TTP). Increasing levels of miR-26a were significantly (P < 0.005) associated with both clinical benefit and prolonged TTP, whereas miR-101 was not. Cell cycle regulation and CCNE1 and CDC2 were the only significant overlapping pathway and genes differentially expressed between tumors with high and low levels of miR-26a and EZH2, respectively. In addition, increasing mRNA levels of CCNE1 (P < 0.05) and CDC2 (P < 0.001) were related to poor outcome. Multivariate analysis revealed miR-26a and CDC2 as an optimal set of markers associated with outcome on tamoxifen therapy, independently of traditional predictive factors. To summarize, only miR-26a levels are related with treatment outcome. Cell cycle regulation is the only overlapping pathway linked to miR-26a and EZH2 levels. Low mRNA levels of EZH2, CCNE1, and CDC2, and high levels of miR-26a are associated with favorable outcome on tamoxifen.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-011-1877-4) contains supplementary material, which is available to authorized users.

EZH2 Promotes Expansion of Breast Tumor Initiating Cells through Activation of RAF1-β-Catenin Signaling

It has been proposed that an aggressive secondary cancer stem cell population arises from a primary cancer stem cell population through acquisition of additional genetic mutations and drives cancer progression. Overexpression of Polycomb protein EZH2, essential in stem cell self-renewal, has been linked to breast cancer progression. However, critical mechanism linking increased EZH2 expression to BTIC (breast tumor initiating cell) regulation and cancer progression remains unclear. Here, we identify a mechanism in which EZH2 expression-mediated downregulation of DNA damage repair leads to accumulation of recurrent RAF1 gene amplification in BTICs, which activates p-ERK-β-catenin signaling to promote BTIC expansion. We further reveal that AZD6244, a clinical trial drug that inhibits RAF1-ERK signaling, could prevent breast cancer progression by eliminating BTICs.