The putative Tumor Suppressor VILIP-1 Counteracts Epidermal Growth Factor-Induced Epidermal- Mesenchymal Transition in Squamous Carcinoma Cells Katharina Scho ¨ nrath 1 , Andres J. Klein-Szanto 2 , Karl H. Braunewell 1,3,4 * 1 Signal Transduction Research Group, Institute of Neurophysiology, Charite ´ University Medicine Berlin, Berlin, Germany, 2 Department of Pathology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America, 3 Institute of Neurophysiology, Ruhr-University Bochum, Bochum, Germany, 4 Molecular and Cellular Neurosciences Laboratory, Department Biochemistry and Molecular Biology, Southern Research Institute, Birmingham, Alabama, United States of America Abstract Epithelial-mesenchymal transition (EMT) is a crucial step for the acquisition of invasive properties of carcinoma cells during tumor progression. Epidermal growth factor (EGF)-treatment of squamous cell carcinoma (SCC) cells provokes changes in the expression of lineage markers, morphological changes, and a higher invasive and metastatic potential. Here we show that chronic stimulation with EGF induces EMT in skin-derived SCC cell lines along with the down-regulation of the epithelial marker E-cadherin, and of the putative tumor suppressor VILIP-1 (visinin-like protein 1). In esophageal squamous cell carcinoma and non-small cell lung carcinoma the loss of VILIP-1 correlates with clinicopathological features related to enhanced invasiveness. VILIP-1 has previously been shown to suppress tumor cell invasion via enhancing cAMP-signaling in a murine SCC model. In mouse skin SCC cell lines the VILIP-1-negative tumor cells have low cAMP levels, whereas VILIP-1- positive SCCs possess high cAMP levels, but low invasive properties. We show that in VILIP-1-negative SCCs, Snail1, a transcriptional repressor involved in EMT, is up-regulated. Snail1 expression is reduced by ectopic VILIP-1-expression in VILIP-1-negative SCC cells, and application of the general adenylyl cyclase inhibitor 29,39-dideoxyadenosine attenuated this effect. Conversely, EGF-stimulation of VILIP-1-positive SCC cells leads to the down-regulation of VILIP-1 and the induction of Snail1 expression. The induction of Snail is inhibited by elevated cAMP levels. The role of cAMP in EMT was further highlighted by its suppressive effect on the EGF-induced enhancement of migration in VILIP-1-positive SCC cells. These findings indicate that VILIP-1 is involved in EMT of SCC by regulating the transcription factor Snail1 in a cAMP-dependent manner. Citation: Scho ¨ nrath K, Klein-Szanto AJ, Braunewell KH (2012) The putative Tumor Suppressor VILIP-1 Counteracts Epidermal Growth Factor-Induced Epidermal- Mesenchymal Transition in Squamous Carcinoma Cells. PLoS ONE 7(3): e33116. doi:10.1371/journal.pone.0033116 Editor: H. Peter Soyer, The University of Queensland, Australia Received November 23, 2011; Accepted February 9, 2012; Published March 30, 2012 Copyright: ß 2012 Scho ¨ nrath et al. 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 was funded by a grant from Deutsche Krebshilfe to KHB. AJK-S was supported from the National Institutes of Health CA107257, CA06927 and by an appropriation from the Commonwealth of Pennsylvania. 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: braunewell@sri.org Introduction Cell motility is a prerequisite for tumor progression and for invasive migration of carcinoma cells into surrounding tissue. In order to acquire a motile phenotype carcinoma cells undergo a dramatic morphological alteration, termed epithelial–mesenchy- mal transition (EMT), wherein they lose their epithelial charac- teristics and acquire the motility of mesenchymal cells [1]. In the case of many carcinomas, EMT-inducing signals, such as HGF, EGF, PDGF, and TGF-b, emanate from the tumor-associated stroma and activate a series of EMT-inducing transcription factors, including Snail, Slug, zinc finger E-box binding homeobox 1 (ZEB1), Twist, Goosecoid, and FOXC2. These transcription factors pleiotropically orchestrate the complex EMT program [2]. The loss of cell–cell contacts mediated by E-cadherin, an epithelial marker, is a typical hallmark of EMT [3]. The down-regulation of E-cadherin is common in squamous cell carcinomas (SCC) and is associated with an enhanced ability of invasion and/or metastasis and with a poor prognosis [4–6], reflective of its critical role in tumor progression. It is widely believed that the down-regulation of E-cadherin occurs through the transcriptional repression mediated by binding of transcriptional repressors, such as Snail1 (SNAI1) [7,8], to E-box sequences in the proximal E-cadherin promoter [9]. The EMT program and the activation of Snail1 depends on a series of intracellular signaling networks and feedback loops involving ERK, MAPK, PI3K, and Akt signaling pathways [10]. In contrast, little is known about the involvement of cyclic nucleotide-mediated signaling pathways in EMT. These pathways are implicated in many biological processes that cooperate in organ development and differentiation of epithelial cells. The effects of cyclic adenosine monophosphate (cAMP) via protein kinase A (PKA) on changes in cell motility and via exchange protein activated by cAMP (EPAC) on cell migration [11] and integrin-mediated cell adhesion [12] are particularly important for tumor invasion. Intracellular cAMP concentrations are regulated by adenyl cyclases (AC), which use ATP to produce cAMP, and by phosphodiesterases (PDEs), which catalyze the degradation of cAMP to AMP [13]. Visinin-like protein 1 (VILIP-1, gene name VSNL1), a member of the family of neuronal calcium sensor proteins [14], modulates PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e33116