ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1523–1536 doi:10.1242/jcs.139865 RESEARCH ARTICLE Tead2 expression levels control the subcellular distribution of Yap and Taz, zyxin expression and epithelial–mesenchymal transition Maren Diepenbruck1,*, Lorenz Waldmeier1,*, Robert Ivanek1, Philipp Berninger2, Phil Arnold2, Erik van Nimwegen2 and Gerhard Christofori1,` ABSTRACT tumor, but also results in the acquisition of stem-cell-like traits, which has implications for cancer therapy and might also be The cellular changes during an epithelial–mesenchymal transition important for colonization at distant organs (Chaffer and Weinberg, (EMT) largely rely on global changes in gene expression 2011; Magee et al., 2012; Polyak and Weinberg, 2009; Scheel and orchestrated by transcription factors. Tead transcription factors Weinberg, 2012). Among the many genes and signaling pathways and their transcriptional co-activators Yap and Taz have been active during EMT, transcription factors are the master coordinators previously implicated in promoting an EMT; however, their direct of the EMT program (Acloque et al., 2009; Moreno-Bueno et al., transcriptional target genes and their functional role during EMT 2008; Nieto, 2011). have remained elusive. We have uncovered a previously The Hippo tumor suppressor signaling pathway plays a critical unanticipated role of the transcription factor Tead2 during EMT. role in restricting organ size by antagonizing the oncogenic During EMT in mammary gland epithelial cells and breast cancer transcriptional co-activators Yap and Taz (Hong and Guan, 2012; cells, levels of Tead2 increase in the nucleus of cells, thereby Zhao et al., 2011). A complex network of cell adhesion and signaling directing a predominant nuclear localization of its co-factors molecules, including the tumor suppressor neurofibromin-2/ Yap and Taz via the formation of Tead2–Yap–Taz complexes. Merlin, regulates the Hippo kinase cascade, leading from the Genome-wide chromatin immunoprecipitation and next generation protein kinases Mst1 and Mst2 via the protein kinases Lats1 and sequencing in combination with gene expression profiling revealed Lats2 to the transcriptional co-factors Yap and Taz. When the the transcriptional targets of Tead2 during EMT. Among these, zyxin Hippo pathway is active, Yap and Taz are phosphorylated by contributes to the migratory and invasive phenotype evoked by Lats1 and Lats2, and phosphorylated Yap and Taz are retained in Tead2. The results demonstrate that Tead transcription factors are the cytoplasm. In the absence of activated Hippo signaling, crucial regulators of the cellular distribution of Yap and Taz, and unphosphorylated Yap and Taz are imported into the nucleus together they control the expression of genes critical for EMT and where they, together with Tead DNA-binding transcription metastasis. factors, activate the expression of proliferative and anti- apoptotic genes. KEY WORDS: Breast cancer, EMT, metastasis, Taz, Tead, Yap, Zyxin In mammals, Tead transcription factors comprise a family of four members (Tead1–Tead4). They are ubiquitously expressed (Jacquemin et al., 1996; Kaneko and DePamphilis, 1998) and INTRODUCTION exert partially redundant roles in regulating the development Epithelial–mesenchymal transition (EMT) is a cell-biological of various embryonic tissues, including neural crest, whose program that is required at various stages of embryonic formation depends on EMT (Thiery et al., 2009), notochord and development. Activation of EMT in epithelial cells induces trophectoderm (Milewski et al., 2004; Sawada et al., 2008; a loss of cell–cell adhesions and apical-basal polarity, and Sawada et al., 2005; Yagi et al., 2007). Transcriptional activity promotes trans-differentiation into a mesenchymal state, which is of the DNA-binding Teads requires their physical association characterized by a migratory and invasive phenotype (Kalluri and with the transcriptional co-activators Yap or Taz (Mahoney Weinberg, 2009; Nieto, 2011). During solid tumor progression, a et al., 2005; Vassilev et al., 2001). Upon Yap- or Taz-mediated reactivation of some of these features in epithelial tumor cells activation, Teads can exert multiple functions. For example, they (oncogenic EMT) is regarded as one of the mechanisms that can control proliferation in epithelial cells and fibroblasts (Ota and facilitate metastatic spread (Chaffer and Weinberg, 2011; Thiery Sasaki, 2008; Zhang et al., 2009; Zhao et al., 2008b). Moreover, et al., 2009). Oncogenic EMT not only provides tumor cells with Yap and Taz are sufficient to induce EMT of MCF10A human invasive properties that permit dissemination from the primary breast epithelial cells in a Tead-dependent manner (Lei et al., 2008; Zhang et al., 2009; Zhao et al., 2008b), and the nuclear 1Department of Biomedicine, University of Basel, 4058 Basel, Switzerland. accumulation of Yap and Taz in EpH4 murine mammary 2 Biozentrum, University of Basel, and Swiss Institute of Bioinformatics, epithelial cells is required for these cells to undergo TGFb- 4056 Basel, Switzerland. *These authors contributed equally to this work induced EMT (Varelas et al., 2010b). Finally, elevated levels of ` Yap trigger increased tumor growth and a pro-metastatic Author for correspondence ([email protected]) phenotype through binding to Tead in breast cancer and This is an Open Access article distributed under the terms of the Creative Commons Attribution melanoma cells (Lamar et al., 2012). Even though these studies License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. clearly demonstrate a crucial role for Teads, Yap and Taz in mediating EMT induction and cancer progression, the Received 6 August 2013; Accepted 18 January 2014 mechanisms involved in the regulation of Tead transcriptional Journal of Cell Science 1523 RESEARCH ARTICLE Journal of Cell Science (2014) 127, 1523–1536 doi:10.1242/jcs.139865 activity and the direct target genes during an EMT remain to be Py2T cells stimulated with TGFb, whereas other growth factors like identified. EGF, FGF, HGF, IGF, PDGF or IL-6 were not able to induce Tead2 To delineate the mechanisms underlying the transcriptional expression (supplementary material Fig. S1F). Furthermore, small activities of Teads and to identify their transcriptional target interfering RNA (siRNA)-mediated ablation of Smad4 expression genes during EMT, we have utilized cellular model systems of during TGFb-induced EMT in NMuMG cells did not affect Tead2 EMT in non-transformed murine mammary gland epithelial cells expression levels, suggesting that TGFb-induced Tead2 expression and in murine breast cancer cells. We report that the expression of is independent of canonical TGFb signaling (supplementary Tead family members is upregulated during EMT, concomitant material Fig. S1G). Notably, siRNA-mediated ablation of Sox4 with an overall increase in Tead transcriptional activity. We expression, a transcription factor known to be a crucial regulator demonstrate that elevated levels of Tead2 lead to increased of EMT (Tiwari et al., 2013), revealed that Tead2 expression is nuclear localization of Yap and Taz, where they form a complex strictly dependent on Sox4 activity during TGFb-induced EMT in with Tead2. As a result, increased Tead2 transcriptional activity NMuMG and Py2T cells (supplementary material Fig. S1H). provokes the induction of EMT and a malignant tumor phenotype. Immunofluorescence analysis showed that low levels of nuclear Conversely, knockdown of Teads in cells undergoing an EMT Tead2 were present in epithelial cells, yet a much stronger nuclear prevents efficient subcellular redistribution of Yap and Taz and staining could be observed in cells that were in the process of blocks EMT. Genome-wide chromatin immunoprecipitation and undergoing EMT (Fig. 1C, NMuMG and Py2T) or had undergone next generation sequencing (ChIP-Seq) in combination with gene EMT (Fig. 1C, MTDEcad). Immunofluorescence staining of the expression profiling identified several EMT-relevant genes as Tead transcriptional co-activators Yap and Taz revealed that they direct transcriptional targets of Tead2 during EMT. Among these, were distributed throughout the cytoplasm and in the nuclei of the gene encoding zyxin, a component of focal adhesions and an epithelial cells, as expected in sparsely growing, proliferating cells actin cytoskeleton remodeling protein, is required for EMT-related (Zhao et al., 2010) (Fig. 1C). Conversely, NMuMG and Py2T migration and invasion. cells undergoing TGFb-induced EMT or stably mesenchymal MTDEcad cells displayed predominantly a nuclear localization of RESULTS Yap and Taz, highly similar to that of Tead2 (Fig. 1C). Formation of a nuclear Tead2–Yap–Taz complex and its We next investigated whether Tead2 binding to Yap and/or Taz transcriptional activity during EMT was subject to change during an EMT. Co-immunoprecipitation To identify crucial genes underlying the multiple stages of an EMT, experiments in total cell lysates (Fig. 1D, Py2T, NMuMG) or in we induced EMT in the untransformed normal murine mammary cytoplasmic and nuclear cell extracts (supplementary material gland cell line NMuMG (Maeda et al., 2005) by treatment with Fig. S1I) revealed that, indeed, the binding of Yap and Taz to TGFb for 0, 1, 4, 7, 10 and 20 days (data not shown). During this Tead2 increased exclusively in