Suppression of TGFb-Induced Epithelial-Mesenchymal Transition Like Phenotype by a PIAS1 Regulated Sumoylation Pathway in NMuMG Epithelial Cells Stuart J. Netherton, Shirin Bonni* Department of Biochemistry and Molecular Biology, Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta, Canada Abstract Epithelial-mesenchymal-transition (EMT) is a fundamental cellular process that is critical for normal development and tumor metastasis. The transforming growth factor beta (TGFb) is a potent inducer of EMT like effects, but the mechanisms that regulate TGFb-induced EMT remain incompletely understood. Using the widely employed NMuMG mammary epithelial cells as a model to study TGFb-induced EMT, we report that TGFb downregulates the levels of the SUMO E3 ligase PIAS1 in cells undergoing EMT. Gain and loss of function analyses indicate that PIAS1 acts in a SUMO ligase dependent manner to suppress the ability of TGFb to induce EMT in these cells. We also find that TGFb inhibits sumoylation of the PIAS1 substrate SnoN, a transcriptional regulator that antagonizes TGFb-induced EMT. Accordingly, loss of function mutations of SnoN sumoylation impair the ability of SnoN to inhibit TGFb-induced EMT in NMuMG cells. Collectively, our findings suggest that PIAS1 is a novel negative regulator of EMT and reveal that inhibition of the PIAS1-SnoN sumoylation pathway represents a key mechanism by which TGFb induces EMT, with important implications in normal development and tumor metastasis. Citation: Netherton SJ, Bonni S (2010) Suppression of TGFb-Induced Epithelial-Mesenchymal Transition Like Phenotype by a PIAS1 Regulated Sumoylation Pathway in NMuMG Epithelial Cells. PLoS ONE 5(11): e13971. doi:10.1371/journal.pone.0013971 Editor: Marc Vooijs, University Medical Center Maastricht, Netherlands Received April 6, 2010; Accepted October 19, 2010; Published November 12, 2010 Copyright: 2010 Netherton, Bonni. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is supported by grants from the Alberta Cancer Board (ACB), and the Canadian Institutes of Health Research to SB. SJN is an ACB Postdoctoral Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sbonni@ucalgary.ca Introduction Epithelial-mesenchymal transition (EMT) is a fundamental biological process that is critical for proper tissue and organ morphogenesis in the developing organism [1]. EMT also is important for wound healing [1]. EMT is reactivated in pathological conditions including fibrotic and neoplastic diseases, and importantly EMT contributes to tumor metastasis [1,2,3,4]. Epithelial cells are characterized by apical-basal polarity that is maintained by specialized junctions including the apical tight junctions and basolateral adherens junctions as well as by an organized cytoskeletal architecture. Thus, EMT induces a morphological alteration in epithelial cells from an apical-basal polarized to a spindle-like non- polarized phenotype. EMT comprises the loss of epithelial cell polarity and cell-cell adhesion due to changes in cytoskeletal architecture and cell junctional proteins [2,3,5,6]. Consistent with these morphological alterations, cells undergoing EMT show a change in the actin cytoskeleton from cortical F-actin type to stress fiber-like. EMT also induces downregulation or mislocalization of the epithelial marker and adherens junctional protein E-cadherin [7]. E- cadherin exerts a central role in epithelial homeostasis and its loss at the cell-cell junctions leads to reduced expression and/or organiza- tion of other epithelial markers including zona occludins-1 [8]. Reduction in E-cadherin levels at the sites of cell-cell attachments is considered to be a hallmark of EMT [9,10,11]. In addition, downregulation of E-cadherin is a predictive marker of invasiveness and metastatic potential of many forms of cancer, including breast and gastric tumors [12,13,14,15]. The transforming growth factor beta (TGFb) family of proteins plays pleiotropic and essential roles in normal development and homeostasis [16,17]. A key function of TGFb is its ability to induce EMT [1,5,6,18]. TGFb-induced EMT plays critical roles in development and wound healing, and contributes to the ability of TGFb to promote tumor progression observed at later stages of many malignancies including mammary, prostate, and colorectal carcinomas [5,19,20,21,22]. TGFb initiates signaling in responsive cells by forming an activated heteromeric complex with specific transmembrane TGFb type I and II serine/threonine kinase receptors [23,24,25,26]. The type II receptor kinase phosphorylates and activates the type I receptor, which in turn induces the activation of the canonical intracellular Smad signaling pathway [27,28,29,30,31]. The Smad proteins, which are transcription factors, are required for the ability of TGFb to induce EMT. In particular, the Smad proteins induce the expression of other transcription factors, including Snail and Zeb1, that are thought to repress the expression of the E-cadherin gene [22]. Although the mechanisms that mediate TGFb induction of EMT are beginning to be elucidated, how the function of TGFb in EMT is regulated has remained unexplored. PIAS1 [protein inhibitor of activated STAT (signal transducer and activator of transcription) 1] was originally identified based on its ability to interact with and inhibit STAT1 binding to DNA [32,33]. Later studies showed that PIAS1 is a SUMO E3 ligase [34,35,36]. Sumoylation involves the covalent attachment of the protein SUMO (small ubiquitin like modifier) to e-amino group in PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e13971