Molecular and Cellular Pathobiology Snail Recruits Ring 1 B toMediate Transcriptional Repression and Cell Migration in Pancreatic Cancer Cells

Transcriptional repressor Snail is a master regulator of epithelial–mesenchymal transition (EMT), yet the epigenetic mechanism governing Snail to induce EMT is not well understood. Here, we report that in pancreatic ductal adenocarcinoma (PDAC), elevated levels of the ubiquitin E3 ligase Ring1B and Snail, along with elevated monoubiquitination of H2A at K119 (H2AK119Ub1), are highly correlated with poor survival. Mechanistic investigations identified Ring1B as a Snail-interacting protein and showed that the carboxyl zinc fingers of Snail recruit Ring1B and its paralog Ring1A to repress its target promoters. Simultaneous depletion of Ring1A and Ring1B in pancreatic cancer cells decreased Snail binding to the target chromatin, abolished H2AK119Ub1 modification, and thereby compromised Snail-mediated transcriptional repression and cell migration. We found that Ring1B and the SNAG-associated chromatin modifier EZH2 formed distinct protein complexes with Snail and that EZH2 was required for Snail-Ring1A/B recruitment to the target promoter. Collectively, our results unravel an epigenetic mechanism underlying transcriptional repression by Snail, suggest Ring1A/B as a candidate therapeutic target, and identify H2AK119Ub1 as a potential biomarker for PDAC diagnosis and prognosis. Cancer Res; 74(16); 1–11. 2014 AACR. Introduction Snail is a member of SNAG domain containing zinc finger proteins and a master regulator of epithelial–mesenchymal transition (EMT) and metastasis in various tumor types (1–3). Snail can directly bind to the E-boxes of E-cadherin gene promoter to repress its transcription and convert normal epithelial cells into mesenchymal cell phenotype (4, 5). Mechanistically, Snail recruitsmultiple repressive protein complexes involving histone deacetylation and methylation as well as DNAmethylation to its target promoters and exerts its repressive function (6–10). However, how these protein complexes are assembled at the target chromatin regions remains elusive. Ring1A and Ring1B belong to the RING domain containing ubiquitin E3 ligase family and are crucial components of the polycomb repressive complex 1 (PRC1) by catalyzing monoubiquitination of histone H2A at lysine 119 (H2AK119Ub1; ref. 11). H2AK119Ub1 status is associated with gene silencing (12), chromatin remodeling (13), and X chromosome inactivation (14). Genetic disruption of Ring1B in mice causes embryonic lethality due to gastrulation arrest anddefectivemesoderm formation (15), which are reminiscent of the Snail null mice. Snail-deficient mouse embryos also die early in gestation, displaying defects in gastrulation and mesoderm formation (16, 17). The striking genetic evidence strongly indicates that Snail very likely correlates with Ring1B in regulation of gastrulation and mesoderm formation during embryo development. A recent study found an elevated Ring1B expression in highgrade pancreatic ductal adenocarcinoma (PDAC; ref. 18); however, no biologic and mechanistic studies on Ring1B were performed. In this present study, we report that Ring1A and Ring1B interact with Snail and are important coregulators for Snail-mediated transcriptional repression and cell migration in pancreatic cancer cells. Snail enhances the binding of Ring1A and Ring1B to the promoter region of E-cadherin to increase H2AK119Ub1 at this locus, and an elevated Snail, Ring1B, and H2AK119Ub1 modification in PDAC is highly correlated with poor prognosis. Materials and Methods IHC and tissues microarray IHC staining was performed as described (18) with specific antibodies against Snail, Ring1B, or H2AK119Ub1. Tissue Department of Surgery, Ruijin Hospital, Department of Biochemistry and Molecular Cell Biology, Shanghai Jiaotong University School of Medicine, Shanghai, China. Department of Hepatobiliary Surgery, Union Hospital, Fujian Medical University, Fuzhou, China. Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai Jiaotong University School of Medicine, Shanghai, China. Institute of Health Sciences, Chinese Academy of Sciences, Shanghai, China. The Wistar Institute, Philadelphia, Pennsylvania. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). J. Chen and H. Xu contributed equally to this article. CorrespondingAuthors:ZhaoyuanHou,Shanghai JiaotongUniversity, 280 South Chongqing Road, Building #7, Room 110, Shanghai 200025, China. Phone: 15121148204; Fax: 86-021-64661525; E-mail: houzy@sjtu.edu.cn; and Chenghong Peng, No.197, Ruijin 2nd Road, Building #36, Floor 5, Shanghai 200025, China. E-mail: chhpeng@yearh.net doi: 10.1158/0008-5472.CAN-14-0181 2014 American Association for Cancer Research. Cancer Research www.aacrjournals.org OF1 Research. on July 21, 2017. © 2014 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst June 5, 2014; DOI: 10.1158/0008-5472.CAN-14-0181