Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press αE-catenin inhibits a Src–YAP1 oncogenic module that couples tyrosine kinases and the effector of Hippo signaling pathway Peng Li,1,4 Mark R. Silvis,1,4 Yuchi Honaker,1,4 Wen-Hui Lien,1,3 Sarah T. Arron,2 and Valeri Vasioukhin1 1Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA; 2Department of Dermatology, University of California at San Fricisco, San Francisco, California, 94143, USA Cell–cell adhesion protein αE-catenin inhibits skin squamous cell carcinoma (SCC) development; however, the mechanisms responsible for this function are not completely understood. We report here that αE-catenin inhibits β4 integrin-mediated activation of SRC tyrosine kinase. SRC is the first discovered oncogene, but the protein substrate critical for SRC-mediated transformation has not been identified. We found that YAP1, the pivotal effector of the Hippo signaling pathway, is a direct SRC phosphorylation target, and YAP1 phosphorylation at three sites in its transcription activation domain is necessary for SRC–YAP1-mediated transformation. We uncovered a marked in- crease in this YAP1 phosphorylation in human and mouse SCC tumors with low/negative expression of αE-catenin. We demonstrate that the tumor suppressor function of αE-catenin involves negative regulation of the β4 integrin– SRC signaling pathway and that SRC-mediated phosphorylation and activation of YAP1 are an alternative to the canonical Hippo signaling pathway that directly connect oncogenic tyrosine kinase signaling with YAP1. [Keywords: αE-catenin; SRC; YAP1; SCC; keratinocytes; skin] Supplemental material is available for this article. Received November 23, 2015; revised version accepted February 29, 2016. Adherens junctions (AJs) are the cadherin–catenin protein growth (Ewing et al. 1995; Bullions et al. 1997; Benjamin complexes that are essential for proper intercellular adhe- and Nelson 2008; Vasioukhin 2012). Genetic experiments sion. In addition to their structural function, these pro- in mice demonstrated the tumor suppressor function of teins have an important role in regulating intracellular αE-catenin in skin squamous cell carcinoma (SCC) (Kobie- signaling pathways, which help to maintain normal tissue lak and Fuchs 2006; Silvis et al. 2011). The analysis of homeostasis and protect from abnormal cellular prolifera- mechanisms responsible for this function identified tion and cancer (Klezovitch and Vasioukhin 2015). AJs a functional connection between αE-catenin and YAP1, play a critical role in the regulation of contact inhibition a pivotal transcriptional coactivator of the Hippo signal- of cellular proliferation. They can sense an increase in ing pathway (Schlegelmilch et al. 2011; Silvis et al. 2011). local cell density and respond by signaling to halt prolifer- YAP1 binds numerous transcription factors and potent- ation (Stepniak et al. 2009; Vasioukhin 2012). The molec- ly stimulates transcription (Yagi et al. 1999; Basu et al. ular mechanisms responsible for the signaling function of 2003; Komuro et al. 2003; Zaidi et al. 2004; Zhao et al. the AJ proteins are not fully understood. Epithelial α-cate- 2008; Rosenbluh et al. 2012). YAP1 is a critical down- nin (encoded by Ctnna1) is an essential AJ protein that stream target of the Hippo signaling pathway, which is links cadherin–catenin protein complexes at the mem- necessary for the proper regulation of contact inhibition brane to the actin cytoskeleton (Buckley et al. 2014). αE- and tumor suppression in mammalian organisms (Barry catenin also has AJ-independent functions in the regula- and Camargo 2013; Yu and Guan 2013). At the core of tion of intracellular trafficking events (Lien et al. 2008a). the Hippo pathway is a kinase cascade consisting of It is frequently missing or down-regulated in various hu- the serine/threonine kinases MST1/2, which activate man epithelial cancers, and re-expression of αE-catenin LATS1/2, which in turn phosphorylate and inactivate in cancer cells has a prominent negative impact on tumor YAP1 (Enderle and McNeill 2013; Yu and Guan 2013). 3Present address: de Duve Institute, Université catholique de Louvain, © 2016 Li et al. This article is distributed exclusively by Cold Spring Brussels B-1200, Belgium. Harbor Laboratory Press for the first six months after the full-issue 4These authors contributed equally to this work. publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). Corresponding author: [email protected] After six months, it is available under a Creative Commons License Article published online ahead of print. Article and publication date are (Attribution-NonCommercial 4.0 International), as described at http:// online at http://www.genesdev.org/cgi/doi/10.1101/gad.274951.115. creativecommons.org/licenses/by-nc/4.0/. 798 GENES & DEVELOPMENT 30:798–811 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/16; www.genesdev.org Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press α-Catenin inhibits ITGB4–SRC–YAP oncogenic pathway We found previously that YAP1 is constitutively nucle- In addition to Src, Itgb4 (encoding β4 integrin) was neces- ar and is necessary for the hyperproliferative phenotype in sary for hyperproliferation of αE-catenin−/− cells (Fig. 1A). αE-catenin−/− keratinocytes (Schlegelmilch et al. 2011; There is significant coordination between cadherin- Silvis et al. 2011). Furthermore, analysis of the Hippo sig- based and integrin-based adhesion structures within the naling pathway demonstrated that the activity of LATS1/ cell (Collins and Nelson 2015). Moreover, β4 integrin is 2 and phosphorylation of YAP1 at S127 (LATS1/2 phos- known as a potent activator of SRC (Bertotti et al. 2006). phorylation site) were not affected by αE-catenin, suggest- Therefore, we analyzed whether β4 integrin is required ing that a novel regulatory pathway may be responsible for hyperactivation of SRC in αE-catenin−/− cells. Knock- for constitutive nuclear localization of YAP1 in αE-cate- down of Itgb4 eliminated the increased activity of SRC nin−/− keratinocytes (Silvis et al. 2011). We report here in αE-catenin−/− keratinocytes (Fig. 1E). Re-expression of that αE-catenin regulates a Hippo-independent signaling siRNA-resistant human β4 integrin rescued this pheno- pathway responsible for YAP1 activation. We found that type (Fig. 1F). Moreover, re-expression of the triple tyrosine αE-catenin attenuates β4 integrin-mediated activation of to phenylalanine mutant and therefore signaling-in- SRC. SRC is hyperactive in αE-catenin−/− keratinocytes; competent β4 integrin (Bertotti et al. 2006) failed to rescue it phosphorylates YAP1 at three sites in its transcriptional hyperactivation of SRC in αE-catenin−/− cells (Fig. 1F). activation domain, and this phosphorylation not only is Coimmunoprecipitation experiments revealed a promi- required for transformation of αE-catenin−/− keratino- nent increase in the interaction between β4 integrin and cytes but is also necessary for general SRC-mediated on- SRC in αE-catenin−/− keratinocytes (Fig. 1G). These data cogenic transformation. demonstrate that β4 integrin interacts with SRC and is necessary for SRC hyperactivation in αE-catenin−/− keratinocytes. Results β4 integrin is involved in the formation of adhesive hemidesmosomes in wild-type keratinocytes. To deter- αE-catenin negatively regulates a β4 integrin–SRC mine whether αE-catenin regulates localization of β4 signaling pathway, which is required for integrin, we performed immunofluorescent stainings of hyperproliferation of αE-catenin−/− keratinocytes keratinocytes with anti-β4 integrin antibodies. As expect- Our siRNA screen previously identified Yap1 as a gene ed, β4 integrin was present at cell–substratum adhesion necessary for the hyperproliferative and contact inhibi- structures in wild-type cells; however, it was localized to tion-defective phenotype of αE-catenin−/− mouse kerati- E-cadherin-positive cell–cell adhesion structures in αE- nocytes, and subsequent analysis demonstrated that catenin−/− cells (Fig. 2A,B). In addition, while SRC dis- YAP1 is constitutively active in these cells (Silvis et al. played diffuse cytoplasmic localization in wild-type cells, 2011). Since we did not observe changes in LATS1/2-me- it was localized to cell–cell junctions in αE-catenin−/− ker- diated phosphorylation of YAP1 that could account for atinocytes (Fig. 2B). its constitutive activation, we extended our siRNA screen Immunofluorescent stainings of skin and tumor sec- to examine genes necessary for hyperproliferation of tions from control and GFAP-Cre/αE-cateninfl/fl mice αE-catenin−/− keratinocytes. We found that, in addition revealed mislocalization of β4 integrin and nuclear locali- to Yap1, Src and Itgb4 were necessary for this phenotype zation of YAP1 in αE-catenin−/− keratinocytes in vivo (Fig. 1A,A′; Supplemental Fig. S1A,B). Moreover, αE-cate- (Fig. 2C). Overall, we conclude that αE-catenin prevents nin−/− cells were also hypersensitive to PP2, a specific Src junctional localization of β4 integrin and SRC and that family kinase (SFK) inhibitor (Supplemental Fig. S1C). β4 integrin targeted to cell–cell junctions activates − − Thus, Src expression and SFK activity are essential for SRC, which drives hyperproliferation of αE-catenin / hyperproliferation of αE-catenin−/− keratinocytes. We keratinocytes. next explored whether loss of αE-catenin expression