A role of the sphingosine-1-phosphate (S1P)–S1P receptor 2 pathway in epithelial defense against cancer (EDAC)

At the initial step of carcinogenesis, transformation occurs in single cells within epithelia, where the newly emerging transformed cells are surrounded by normal epithelial cells. A recent study revealed that normal epithelial cells have an ability to sense and actively eliminate the neighboring transformed cells, a process named epithelial defense against cancer (EDAC). However, the molecular mechanism of this tumor-suppressive activity is largely unknown. In this study, we investigated a role for the sphingosine-1-phosphate (S1P)–S1P receptor 2 (S1PR2) pathway in EDAC. First, we show that addition of the S1PR2 inhibitor significantly suppresses apical extrusion of RasV12-transformed cells that are surrounded by normal cells. In addition, knockdown of S1PR2 in normal cells induces the same effect, indicating that S1PR2 in the surrounding normal cells plays a positive role in the apical elimination of the transformed cells. Of importance, not endogenous S1P but exogenous S1P is involved in this process. By using FRET analyses, we demonstrate that S1PR2 mediates Rho activation in normal cells neighboring RasV12-transformed cells, thereby promoting accumulation of filamin, a crucial regulator of EDAC. Collectively these data indicate that S1P is a key extrinsic factor that affects the outcome of cell competition between normal and transformed epithelial cells. INTRODUCTION At the initial stage of carcinogenesis, it is generally believed that oncogenic transformation occurs in single cells within epithelia. However, it is not clearly understood what happens at the interface between normal epithelial cells and newly emerging transformed cells. In previous studies, we demonstrated that RasV12or Src-transformed cells are apically extruded when they are surrounded by normal epithelial cells. When transformed cells alone are present, apical extrusion does not occur, indicating that the presence of neighboring normal cells profoundly influences the behavior of the transformed cells (Hogan et al., 2009; Kajita et al., 2010). In addition, it has become clear that normal epithelial cells have an ability to actively eliminate transformed cells from the epithelium (Kajita et al., 2014). When normal and Rasor Src-transformed cells are copresent within the epithelial monolayer, filamin and vimentin are accumulated in normal cells at the interface with the neighboring transformed cells. Knockdown of filamin or vimentin suppresses apical extrusion of transformed cells, indicating an active role of these proteins in this process. Filamin acts upstream of vimentin and regulates its dynamic accumulation, which produces physical forces for the apical extrusion. Furthermore, we show that the Rho–Rho kinase pathway regulates the accumulation of filamin. Collectively these results indicate that normal epithelial cells have antitumor activity that does not involve immune systems. This Monitoring Editor Kozo Kaibuchi Nagoya University Received: Mar 20, 2015 Revised: Nov 20, 2015 Accepted: Nov 24, 2015 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E15-03-0161) December 2, 2015. *These authors equally contributed to this work. Address correspondence to: Yasuyuki Fujita (yasu@igm.hokudai.ac.jp). © 2016 Yamamoto, Yako, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by -nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. Abbreviations used: EDAC, epithelial defense against cancer; MDCK, Madin– Darby canine kidney; shRNA, short hairpin RNA; SphKI, sphingosine kinase inhibitor; S1P, sphingosine-1-phosphate; S1PR, sphingosine-1-phosphate receptor. Sayaka Yamamotoa,*, Yuta Yakoa,*, Yoichiro Fujiokab, Mihoko Kajitaa, Takeshi Kameyamac, Shunsuke Kona, Susumu Ishikawaa, Yusuke Ohbab, Yusuke Ohnod, Akio Kiharad, and Yasuyuki Fujitaa aDivision of Molecular Oncology and cSignaling in Cancer and Immunology, Institute for Genetic Medicine, Hokkaido University Graduate School of Chemical Sciences and Engineering, Sapporo 060-0815, Japan; bDepartment of Cell Physiology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan; dFaculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan http://www.molbiolcell.org/content/suppl/2015/11/29/mbc.E15-03-0161v1.DC1.html Supplemental Material can be found at: