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Background Four transcription factors, Oct4, Sox2, Klf4, and c-Myc (the Yamanka factors), can reprogram somatic cells to induced pluripotent stem cells (iPSCs). Many studies have provided a number of alternative combinations to the non-Yamanaka factors. However, it is clear that many additional transcription factors that can generate iPSCs remain to be discovered. Methods The chromatin accessibility and transcriptional level of human embryonic stem cells and human urine cells were compared by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to identify potential reprogramming factors. Selected transcription factors were employed to reprogram urine cells, and the reprogramming efficiency was measured. Urine-derived iPSCs were detected for pluripotency by Immunofluorescence, quantitative polymerase chain reaction, RNA sequencing and teratoma formation test. Finally, we assessed the differentiation potential of the new iPSCs to cardiomyocytes in vitro. Results ATAC-seq and RNA-seq datasets predicted TEAD2, TEAD4 and ZIC3 as potential factors involved in urine cell reprogramming. Transfection of TEAD2, TEAD4 and ZIC3 (in the presence of Yamanaka factors) significantly improved the reprogramming efficiency of urine cells. We confirmed that the newly generated iPSCs possessed pluripotency characteristics similar to normal H1 embryonic stem cells. We also confirmed that the new iPSCs could differentiate to functional cardiomyocytes. Conclusions In conclusion, TEAD2, TEAD4 and ZIC3 can increase the efficiency of reprogramming human urine cells into iPSCs, and provides a new stem cell sources for the clinical application and modeling of cardiovascular disease. Graphical abstract

Background Transcriptional enhanced associated domain (TEAD) transcription factors are nuclear effectors of several oncogenic signalling pathways including Hippo, WNT, TGF-s and EGFR pathways that interact with various cancer genes. The subcellular localization of TEAD regulates the functional output of these pathways affecting tumour progression and patient outcome. However, the impact of the TEAD family on pancreatic ductal adenocarcinoma (PDAC) and its clinical progression remain elusive. Methods A cohort of 81 PDAC patients who had undergone surgery was established. Cytoplasmic and nuclear localization of TEAD1, TEAD2, TEAD3 and TEAD4 was evaluated with the immunoreactive score (IRS) by immunohistochemistry (IHC) using paraffin-embedded tissue. Results were correlated with clinicopathological data, disease-free and overall survival. Results Nuclear staining of all four TEADs was increased in pancreatic cancer tissue. Patients suffering from metastatic disease at time of surgery showed a strong nuclear staining of TEAD2 and TEAD3 (p cytoplasmic ratio of TEAD2 and TEAD3 was associated with a shorter overall survival and TEAD2 emerged as an independent prognostic factor for disease-free survival. Conclusion Our study underlines the importance of TEAD transcription factors in PDAC as a nuclear localization was found to be associated with metastatic disease and an unfavourable prognosis after surgical resection.

Xinxin Ren,1 Xiang Wang,2 Bi Peng,3 Qiuju Liang,2 Yuan Cai,3 Kewa Gao,3 Yongbin Hu,3 Zhijie Xu,3,4 Yuanliang Yan2 1Center for Molecular Medicine, Xiangya Hospital, Central South University, Changsha, People’s Republic of China; 2Department of Pharmacy, Xiangya Hospital, Central South University, Changsha, People’s Republic of China; 3Department of Pathology, Xiangya Hospital, Central South University, Changsha, People’s Republic of China; 4National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, People’s Republic of ChinaCorrespondence: Yuanliang YanDepartment of Pharmacy, Xiangya Hospital, Central South University, Changsha, 410008, Hunan, People’s Republic of ChinaEmail yanyuanliang@csu.edu.cnPurpose: To explore the molecular profiles of transcriptional enhanced associate domain (TEAD) family in ovarian serous carcinoma (OSC).Methods: In this study, we use bioinformatics methods including GEPIA, GE-mini, Oncomine 3.0, Kaplan–Meier plotter, cBioPortal, WebGestalt, TIMER2.0 and DiseaseMeth2.0, and in vitro experimental RT-PCR to assess the expression profiles and prognostic significance of TEAD family in OSC.Results: According to the bioinformatics analysis, TEAD family was abnormally expressed in OSC. In terms of prognosis, Kaplan–Meier plotter indicated that OSC patients with high level of TEAD4 showed poor overall survival (OS), progression-free survival (PFS) and post progression survival (PPS). TEAD family also had significantly diagnostic values for OSC patients. Tumor Immune Estimation Resource (TIMER) algorithm indicated that TEAD family was significantly associated with different types of infiltrating immune cells, including B cells, macrophages, dendritic cells, neutrophils, CD8+ T cells and CD4+ T cells. Gene set enrichment analysis of TEAD family-associated coexpression genes was further explored. In in vitro experiments, the RT-PCR results showed the upregulated TEAD2/4 in OSC tissues and cells (A2780 and TOV112D). Moreover, decreased expression of TEAD2 could induce the ferroptosis through increasing the ROS accumulation.Conclusion: Thus, TEAD family correlated with the diagnosis, prognosis and immune infiltration in OSC. These results could provide comprehensive understanding of TEAD family in the diagnosis and prognosis of OSC patients.Keywords: TEAD family, Hippo pathway, ovarian serous carcinoma, expression profiles, prognosis, immune infiltration

Dysregulated alternative splicing (AS) plays critical roles in driving cancer progression, and the underlying mechanisms remain largely unknown. Here, we demonstrated that PHF5A, a component of U2 small nuclear ribonucleoproteins, was frequently upregulated in colorectal cancer (CRC) samples and associated with poor prognosis. PHF5A promoted proliferation and metastasis of CRC cells in vitro and in vivo. Transcriptomic analysis identified PHF5A-regulated AS targets and pathways. Particularly, PHF5A induced TEAD2 exon 2 inclusion to activate YAP signaling, and interference of TEAD2-L partially reversed the PHF5A-mediated tumor progression. Pharmacological inhibition of PHF5A using pladienolide B had potent antitumor activity. Collectively, these data revealed the oncogenic role of PHF5A in CRC through regulating AS and established PHF5A as potential therapeutic target., Graphical abstract, Dysregulated alternative splicing is closely associated with oncological processes. Our study revealed that splicing factor PHF5A was associated with poor prognosis of CRC patients and promoted CRC progression by inducing aberrant splicing of TEAD2 to activate YAP signaling. Splicing modulators targeting PHF5A had potent antitumor activity.

Transcriptional enhanced associate domain (TEAD) is a family of transcription factors that plays a significant role during embryonic developmental processes, and its dysregulation is responsible for tumour progression. TEAD is considered as druggable targets in various diseases, namely cancer, cardiovascular diseases and neurodegenerative disorders. Previous structural studies revealed the importance of the central hydrophobic pocket of TEAD as a potential target for small-molecule inhibitors and demonstrated flufenamic acid (FLU) (a COX-2 enzyme inhibitor) to bind and inhibit TEAD2 functions. However, to date, no drug candidates that bind specifically to TEAD2 with high selectivity and efficacy have been developed or proposed. Within this framework, we present here a case study where we have identified potential TEAD2 inhibitor candidates by integrating multiple computational approaches. Among the candidates, the top two ranked compounds ZINC95969481 (LG1) which is a fused pyrazole derivative and ZINC05203789 (LG2), a fluorene derivative resulted in much favourable binding energy scores than the reference ligand, FLU. The drug likeliness of the best compounds was also evaluated in silico to ensure the bioavailability of these compounds particularly LG1 as compared to FLU thus providing a strong rationale for their development as leads against TEAD. Molecular dynamics simulations results highlighted the role of key residues contributing to favourable interactions in TEAD2-LG1 complex with much favourable interaction and binding free energy values with respect to the reference compound. Altogether, this study provides a starting platform to be more exploited by future experimental research towards the development of inhibitors against TEAD, a persuasive strategy for therapeutic intervention in cancer treatment.

Trastuzumab is the first-line targeted therapeutic drug for HER2-positive breast cancer, leading to improved overall survival. However, acquired resistance inevitably occurs. We aimed to identify, quantify, and assess the mechanisms of acquired resistance to trastuzumab. We established an acquired trastuzumab-resistant model in vitro from BT-474, a trastuzumab-sensitive, HER2-amplified breast-cancer cell line. A multi-omic strategy was implemented to obtain gene, proteome, and phosphoproteome signatures associated with acquired resistance to trastuzumab in HER2-positive breast cancer, followed by validation in human clinical samples. YAP1 dephosphorylation and TEAD2 overexpression were detected as significant alterations in the Hippo pathway in trastuzumab-resistant breast cancer. Because of the emerging role of these proteins as mediators of normal growth and tumorigenesis, we assessed the exogenous modulation of their activity, either by in vitro gene silencing or by pharmacological inhibition of the YAP1/TEAD complexes, both in vitro and in vivo. Moreover, we identified increased signaling through the Hippo pathway in human samples after progression following trastuzumab treatment. Finally, YAP1/TAZ nuclear accumulation in malignant cells in HER2 breast tumor was significantly associated with worse progression-free and overall survival in metastatic HER2-positive breast-cancer patients. Our results suggest the involvement of Hippo signaling in acquired trastuzumab resistance in breast cancer. Additionally, we provide novel evidence for a potential breast-cancer treatment strategy based on dual targeting of HER2 and Hippo pathway effectors, which may improve the antitumor activity of trastuzumab and help overcome resistance., The present work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) with European Regional Development Fund (ERDF) funding through the Institute of Health Carlos III (AES Program, grants PI15/00934, PI18/00382 and PI18/00006; CIBERONC, Biomedical Research Networking Centre for Cancer; Biobanks Platform, PT13/0010/0012; ProteoRed, PRB2-ISCIII, PT13/0001); and the Community of Madrid (S2010/BMD-2344). P.G.-A. was supported by a Fundación Conchita Rábago de Jiménez Díaz grant. P.M. was supported by the ISCIII Miguel Servet Program (CP16/00116).

Neural stem cells (NSCs) generate neurons of the cerebral cortex with distinct morphologies and functions. How specific neuron production, differentiation and migration are orchestrated is unclear. Hippo signaling regulates gene expression through Tead transcription factors (TFs). We show that Hippo transcriptional coactivators Yap1/Taz and the Teads have distinct functions during cortical development. Yap1/Taz promote NSC maintenance and Satb2+ neuron production at the expense of Tbr1+ neuron generation. However, Teads have moderate effects on NSC maintenance and do not affect Satb2+ neuron differentiation. Conversely, whereas Tead2 blocks Tbr1+ neuron formation, Tead1 and Tead3 promote this early fate. In addition, we found that Hippo effectors regulate neuronal migration to the cortical plate (CP) in a reciprocal fashion, that ApoE, Dab2 and Cyr61 are Tead targets, and these contribute to neuronal fate determination and migration. Our results indicate that multifaceted Hippo signaling is pivotal in different aspects of cortical development., Scientific Reports, 10 (1), ISSN:2045-2322

TEA Domain Transcription Factors (TEADs) are important in development and serve essential roles in tumorigenesis; however, the role of TEAD2 expression in hepatocellular carcinoma (HCC) has not been widely examined. The present study was conducted to investigate the expression status of TEAD2 in HCC and to evaluate whether the expression of TEAD2 is associated with the prognosis of patients with HCC. mRNA expression data was retrieved for Hippo pathway genes of 50 normal control and 377 HCC samples from The Cancer Genome Atlas data portal. Gene set enrichment, GeneNeighbors, ClassNeighbors and survival analyses were then performed based on the gene expression levels. The mRNA expression of TEAD2 and VGLL4 was significantly higher in HCC compared with the normal control samples, and the mRNA expression of TEAD2 was higher in advanced stages than in early stages. Specifically, survival analysis revealed that higher mRNA expression of TEAD2 was significantly associated with a less favorable overall survival rate (P=0.0067) and there was a trend towards significance between higher mRNA expression of VGLL4 and poor overall survival rate (P=0.051). According to the gene set enrichment analysis, patients with higher mRNA expression of TEAD2 and VGLL4 had strongly enhanced epithelial‑mesenchymal transition and angiogenesis, which are associated with tumor progression. In conclusion, increased mRNA expression of TEAD2 is associated with a poor prognosis in patients with HCC. TEAD2 may serve as a prognostic factor for HCC and a novel therapeutic target.

The Hippo pathway is an evolutionarily conserved pathway involved in developmental biology. The biological functions of Hippo pathway are executed by the downstream transcriptional coactivator YAP/TAZ, which shuttle between the cytoplasm and the nucleus and have to interact with TEADs in nucleus for transcription activation. YAP/TAZ are master transcriptional factors widely activated in human cancers to promote cancer initiation, progression, metastasis and therapy resistance. It has been shown that aberrant YAP/TAZ activation is also associated with the adapted tumor microenvironment (TME) for tumor growth. Dysregulation in Hippo pathway and YAP/TAZ-TEADs transcriptional activity is pervasively associated with various types of cancers, including mesothelioma, squamous cell cancers, liver cancer and lung cancer, making it an attractive target for cancer therapy. Therefore, inhibition of YAP/TAZ oncogenic activity by blocking YAP/TAZ-TEADs interaction is an effective approach for cancer treatment. In this study, we aim to discover small molecule inhibitors that bind to TEADs and disrupt YAP/TAZ-TEADs interaction for cancer therapy. With superposition of all the available crystal structures of four TEAD subtypes in the public database, we found the YAP Ω loop binding site of TEADs is relatively rigid (of backbone atoms), but the side chain flexibility of K289, K265 and V406 in TEAD1 affects the shape of the binding site significantly. Notably, in two TEAD2 structures, e.g. PDB ID: 5dqe and 5dq8, K301 (K274 of TEAD1) adopts an open conformation and accommodates a flufenamic acid to bind in an induced pocket. Using 5dq8 as the protein model, we virtually screened a fragment library, and identified a hit compound that is suitable to combine with flufenamic acid. Interaction between the hit compound and TEAD1 was confirmed by 1D-NMR (STD). Based on the docking poses, we combined the hit and flufenamic acid, designed and synthesized several compounds. Interaction with TEAD1 of the compounds were confirmed by 1D-NMR, SPR and HTRF. Crystal structures of representative compounds confirmed the predicted binding mode in TEAD1. With further medicinal chemistry efforts, we have discovered a series of novel YAP/TAZ-TEAD PPI inhibitors bound at the Ω loop site of TEAD1. Citation Format: Lin Du, Jing Liu, Qinglong Zeng, Ming Xu, Jiting Lu, Qiangang Zheng, Jidong Zhu. Structural dynamics-based hit generation to disrupt YAP/TAZ-TEAD protein-protein interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 326.

Alternative splicing (AS) is a key process to expand the diversity of mRNA and protein from the genome and it is crucial for fate determination of embryonic stem cells (ESCs) by encoding isoforms with different functions to regulate the balance between pluripotency maintenance and differentiation. Since the role of the Hippo pathway in ESCs is controversial, there may be novel isoforms of Taz, a key effector of the Hippo pathway, previously unknown to us. Here, we identified three variants of Taz in mESCs. Apart from the canonical Taz1185, there were also two novel variants, Taz402 and Taz1086. We found their structure and subcellular localization to be different, while they could all interact with TEAD2 with similar binding affinities and activate transcription. Under the LIFlow condition, overexpression of them all induced apoptosis and differentiation of mESCs, among which the phenotype of Taz1086 was the most dramatic. Taken together, we discovered novel variants of Taz and compared their structure and functional differences in mESC pluripotency maintenance. These findings will help us to understand the Taz gene and clarify its role in mESC.

TEAD proteins constitute a family of highly conserved transcription factors, characterized by a DNA-binding domain called the TEA domain and a protein-binding domain that permits association with transcriptional co-activators. TEAD proteins are unable to induce transcription on their own. They have to interact with transcriptional cofactors to do so. Once TEADs bind their co-activators, the different complexes formed are known to regulate the expression of genes that are crucial for embryonic development, important for organ formation (heart, muscles), and involved in cell death and proliferation. In the first part of this review we describe what is known of the structure of TEAD proteins. We then focus on two members of the family: TEAD1 and TEAD2. First the different transcriptional cofactors are described. These proteins can be classified in three categories: i), cofactors regulating chromatin conformation, ii), cofactors able to bind DNA, and iii), transcriptional cofactors without DNA binding domain. Finally we discuss the recent findings that identified TEAD1 and 2 and its coactivators involved in cancer progression.

Interferon tau (IFNT) is the pregnancy recognition protein in all ruminants, and its expression is restricted to trophoblast cells. Interferon tau production increases as the conceptus elongates; however, its expression is downregulated soon after the initiation of conceptus attachment to the uterine epithelium. Our previous study identified that among 8 bovine IFNT genes, only 2 forms of IFNTs, IFNT2 and IFN-tau-c1, were expressed by the conceptuses during the periattachment period. To characterize whether Hippo signaling including a transcription cofactor yes-associated protein (YAP) was involved in the IFNT regulation, we examined the expression and effects of YAP and/or TEAD in human choriocarcinoma JEG3 and bovine trophoblast CT-1 cells, and in bovine conceptuses obtained from day 17, 20 or 22 pregnant animals (pregnant day 19.5 = day of conceptus attachment to the endometrium). YAP was expressed in bovine conceptuses and transfection of YAP or TEAD4, a transcription factor partner of YAP, expression plasmid increased the luciferase activity of IFNT2 and IFN-tau-c1 reporter plasmids in JEG3 cells. In the presence of YAP expression plasmid, TEAD2 or TEAD4 expression plasmid further upregulated transcriptional activity of IFNT2 or IFN-tau-c1 constructs, which were substantially reduced in the absence of the TEAD-binding site on IFNT2 or IFN-tau-c1 promoter region in JEG3 cells. In CT-1 cells, treatment with TEAD2, TEAD4, or YAP small-interfering RNA downregulated endogenous IFNT expression. It should be noted that TEAD2 and TEAD4 were predominantly localized in the nuclei of trophectoderm of Day 17 conceptuses, but nuclear localization appeared to be lower in those cells of conceptuses on days 20 and 22 of pregnancy. Moreover, the binding of TEAD4 to the TEAD-binding site of the IFN-tau-c1 promoter region in day 17 conceptuses was less in day 20 and 22 conceptuses. Furthermore, the level of YAP phosphorylation increased in day 20 and 22 conceptuses. These results indicated that although YAP/TEAD had the ability to up-regulate IFNT gene transcription on day 17, IFNT2 or IFN-tau-c1 was down-regulated following changes in the localization of TEAD2 and TEAD4 from the nucleus to the cytoplasm and increases in phosphorylation and degradation of YAP. These data suggest that TEAD relocation and/or YAP degradation following its phosphorylation down-regulates IFNT gene transcription after conceptus attachment to the uterine endometrium.

Transcriptional enhanced associate domain (TEAD) proteins are the downstream effectors of the Hippo signaling pathway that regulate cell proliferation and stem cell functions. The expression of TEAD family is upregulated in many cancer types including pancreatic, colorectal, breast, and prostate cancers, which is correlated with poor survival in patients. Pharmacologic modulators of TEADs could therefore find application in cancer treatment and regenerative medicine. In this presentation, we report our exploration of the TEAD2 Yap-binding domain conformational ensemble and its binding interaction characteristics with peptides and small molecules, using both molecular dynamics simulation and experimental binding studies with FP probes. We carried a series of conventional, cosolvent, and Gaussian-biased MD studies and discovered that the binding subsites II and III possess considerable flexibility as the free energy calculations indicate that multiple low-energy states exist with only about 1.4 Kcal/mol barriers among them. Together with the binding results, our study points to additional “hot spots”/pharmacophores for fresh design of novel small-molecule inhibitors disrupting YAP/TEAD protein-protein interactions. Note: This abstract was not presented at the conference. Citation Format: Yiping Li, Wei Zhou, Chenglong Li. Computational insights on the druggability of TEAD YAP-binding domain [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B11.

TEAD transcription factors bind to the transcription co-activators YAP/TAZ and control the transcriptional output of the Hippo pathway. Deregulation of Hippo-YAP signaling is implicated in diverse human cancers; however, it remains difficult to directly target TEAD-YAP complex with small molecules. We previously reported that TEADs possess intrinsic “enzyme-like” activities and undergo autopalmitoylation. Palmitate binds into a deep hydrophobic pocket and is essential for TEAD protein stability and transcriptional activation. Here we report MGH-CP1 as a small-molecule inhibitor of TEAD autopalmitoylation. We determined the crystal structure of inhibitor-bound TEAD2 and showed that MGH-CP1 binds to the conserved lipid-binding pocket in TEAD2 and displaces palmitate binding. In cells, MGH-CP1 maintains TEAD stability and TEAD-Vgll4 repressor complex and allosterically inhibits TEAD-YAP association and YAP-induced transcriptional activation. Through a large-scale profiling of cancer cell line growth inhibition, followed by intersecting with the Cancer Dependence Map analysis, we demonstrate that the sensitivity to MGH-CP1 treatment correlates significantly with YAP-dependency in a broad range of human cancer cells. Furthermore, we show that MGH-CP1 blocks YAP-dependent liver overgrowth induced by liver-specific Lats1/2 deletion in mice and inhibits xenograft tumor growth of human uveal melanoma cells in vivo. Together, our work suggests pharmacologic inhibition of TEAD palmitoylation as a promising therapeutic strategy for diseases associated with deregulated Hippo signaling, and opens a door to target “autopalmitoylation” of proteins. Citation Format: Xu Wu. Targeting autopalmitoylation of TEAD transcription factors [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA12.

Hepatoblastoma (HB) is the most common type of pediatric liver cancer. Activation of yes-associated protein (YAP) has been implicated in HB molecular pathogenesis. The transcriptional co-activator Yap regulates downstream gene expression through interaction with the TEA domain (TEAD) proteins. Nonetheless, YAP also displays functions that are independent of its transcriptional activity. The underlying molecular mechanisms by which Yap promotes HB development remain elusive. In the current study, we demonstrated that blocking TEAD function via the dominant-negative form of TEAD2 abolishes Yap-driven HB formation in mice and restrains human HB growth invitro. When TEAD2 DNA-binding domain was fused with virus protein 16 transcriptional activation domain, it synergized with activated β-catenin to promote HB formation invivo. Among TEAD genes, silencing of TEAD4 consistently inhibited tumor growth and Yap target gene expression in HB cell lines. Furthermore, TEAD4 mRNA expression was significantly higher in human HB lesions when compared with corresponding nontumorous liver tissues. Human HB specimens also exhibited strong nuclear immunoreactivity for TEAD4. Altogether, data demonstrate that TEAD-mediated transcriptional activity is both sufficient and necessary for Yap-driven HB development. TEAD4 is the major TEAD isoform and Yap partner in human HB. Targeting TEAD4 may represent an effective treatment option for human HB.

SummaryThe Hippo signaling pathway is responsible for regulating the function of TEAD family transcription factors in metazoans. TEADs, with their co-activators YAP/TAZ, are critical for controlling cell differentiation and organ size through their transcriptional activation of genes involved in cell growth and proliferation. Dysregulation of the Hippo pathway has been implicated in multiple forms of cancer. Here, we identify a novel form of regulation of TEAD family proteins. We show that human TEADs are palmitoylated at a universally conserved cysteine, and report the crystal structures of the human TEAD2 and TEAD3 YAP-binding domains in their palmitoylated forms. These structures show a palmitate bound within a highly conserved hydrophobic cavity at each protein's core. Our findings also demonstrate that this modification is required for proper TEAD folding and stability, indicating a potential new avenue for pharmacologically regulating the Hippo pathway through the modulation of TEAD palmitoylation.

Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)-dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development.

The TEAD family of transcription factors (TEAD1-4) bind the MCAT element in the regulatory elements of both growth promoting and myogenic differentiation genes. Defining TEAD transcription factor function in myogenesis has proved elusive due to overlapping expression of family members and their functional redundancy. We show that silencing of either Tead1, Tead2 or Tead4 did not effect primary myoblast (PM) differentiation, but that their simultaneous knockdown strongly impaired differentiation. In contrast, Tead1 or Tead4 silencing impaired C2C12 differentiation showing their different contributions in PMs and C2C12 cells. Chromatin immunoprecipitation identified enhancers associated with myogenic genes bound by combinations of Tead4, Myod1 or Myog. Tead4 regulated distinct gene sets in C2C12 cells and PMs involving both activation of the myogenic program and repression of growth and signaling pathways. ChIP-seq from mature mouse muscle fibres in vivo identified a set of highly transcribed muscle cell-identity genes and sites bound by Tead1 and Tead4. Although inactivation of Tead4 in mature muscle fibres caused no obvious phenotype under normal conditions, notexin-induced muscle regeneration was delayed in Tead4 mutants suggesting an important role in myogenic differentiation in vivo. By combining knockdown in cell models in vitro with Tead4 inactivation in muscle in vivo, we provide the first comprehensive description of the specific and redundant roles of Tead factors in myogenic differentiation., Author summary Aspects of muscle differentiation can be reproduced using the C2C12 cell line or primary myoblasts both of which can be differentiated to form myotubes in vitro. While the functions of recognised myogenic proteins such as Myogenin, Myod1 and MEF-family transcription factors in this process have been extensively studied, the role of the Tead factors has received only limited attention. Tead factors have well defined roles as mediators of Hippo signalling in promoting cell growth and oncogenic transformation, but are also involved in myogenic differentiation involving cell cycle arrest and activation of the myogenic gene expression program. Using integrative genomics and knockdowns in cell based models, we show that Tead factors are essential for differentiation of C2C12 cells and primary myoblasts, but make different contributions activating a distinct set of myogenesis genes in each cell type. We also developped effective chromatin immunoprecipitation from mature mouse muscle fibres allowing identification of highly transcribed muscle identify genes and identification of Tead1 and Tead4 occupied sites. Somatic inactivation in vivo revealed an important role for Tead4 in muscle fibre regeneration. The integration of genomics and loss of function in cell models in vitro and muscle in vivo provide the first comprehensive description Tead factors in myogenic differentiation.

Regulation of organ size is important for development and tissue homeostasis. In Drosophila, Hippo signaling controls organ size by regulating the activity of a TEAD transcription factor, Scalloped, through modulation of its co-activator protein Yki. Here, we show that mouse Tead proteins regulate cell proliferation by mediating Hippo signaling. In NIH3T3 cells, cell density and Hippo signaling regulated the activity of endogenous Tead proteins by modulating nuclear localization of a Yki homolog, Yap1, and the resulting change in Tead activity altered cell proliferation. Tead2-VP16 mimicked Yap1 overexpression, including increased cell proliferation, reduced cell death, promotion of EMT, lack of cell contact inhibition and promotion of tumor formation. Growth-promoting activities of various Yap1 mutants correlated with their Tead-co-activator activities. Tead2-VP16 and Yap1 regulated largely overlapping sets of genes. However, only a few of the Tead/Yap1-regulated genes in NIH3T3 cells were affected in Tead1-/-;Tead2-/- or Yap1-/- embryos. Most of the previously identified Yap1-regulated genes were not affected in NIH3T3 cells or mutant mice. In embryos, levels of nuclear Yap1 and Tead1 varied depending on cell type. Strong nuclear accumulation of Yap1 and Tead1 were seen in myocardium,correlating with requirements of Tead1 for proliferation. However,their distribution did not always correlate with proliferation. Taken together, mammalian Tead proteins regulate cell proliferation and contact inhibition as a transcriptional mediator of Hippo signaling, but the mechanisms by which Tead/Yap1 regulate cell proliferation differ depending on the cell type, and Tead, Yap1 and Hippo signaling may play multiple roles in mouse embryos.

The YAP transcription coactivator has been implicated as an oncogene and is amplified in human cancers. Recent studies have established that YAP is phosphorylated and inhibited by the Hippo tumor suppressor pathway. Here we demonstrate that the TEAD family transcription factors are essential in mediating YAP-dependent gene expression. TEAD is also required for YAP-induced cell growth, oncogenic transformation, and epithelial–mesenchymal transition. CTGF is identified as a direct YAP target gene important for cell growth. Moreover, the functional relationship between YAP and TEAD is conserved in Drosophila Yki (the YAP homolog) and Scalloped (the TEAD homolog). Our study reveals TEAD as a new component in the Hippo pathway playing essential roles in mediating biological functions of YAP.

Four members of the TEAD/TEF family of transcription factors are expressed widely in mouse embryos and adult tissues. Although in vitro studies have suggested various roles for TEAD proteins, their in vivo functions remain poorly understood. Here we examined the role of Tead genes by generating mouse mutants for Tead1 and Tead2. Tead2(-/-) mice appeared normal, but Tead1(-/-); Tead2(-/-) embryos died at embryonic day 9.5 (E9.5) with severe growth defects and morphological abnormalities. At E8.5, Tead1(-/-); Tead2(-/-) embryos were already small and lacked characteristic structures such as a closed neural tube, a notochord, and somites. Despite these overt abnormalities, differentiation and patterning of the neural plate and endoderm were relatively normal. In contrast, the paraxial mesoderm and lateral plate mesoderm were displaced laterally, and a differentiated notochord was not maintained. These abnormalities and defects in yolk sac vasculature organization resemble those of mutants for Yap, which encodes a coactivator of TEAD proteins. Moreover, we demonstrated genetic interactions between Tead1 and Tead2 and Yap. Finally, Tead1(-/-); Tead2(-/-) embryos showed reduced cell proliferation and increased apoptosis. These results suggest that Tead1 and Tead2 are functionally redundant, use YAP as a major coactivator, and support notochord maintenance as well as cell proliferation and survival in mouse development.

SummaryThe human TEAD family of transcription factors (TEAD1–4) is required for YAP-mediated transcription in the Hippo pathway. Hyperactivation of TEAD's co-activator YAP contributes to tissue overgrowth and human cancers, suggesting that pharmacological interference of TEAD-YAP activity may be an effective strategy for anticancer therapy. Here we report the discovery of a central pocket in the YAP-binding domain (YBD) of TEAD that is targetable by small-molecule inhibitors. Our X-ray crystallography studies reveal that flufenamic acid, a non-steroidal anti-inflammatory drug (NSAID), binds to the central pocket of TEAD2 YBD. Our biochemical and functional analyses further demonstrate that binding of NSAIDs to TEAD inhibits TEAD-YAP-dependent transcription, cell migration, and proliferation, indicating that the central pocket is important for TEAD function. Therefore, our studies discover a novel way of targeting TEAD transcription factors and set the stage for therapeutic development of specific TEAD-YAP inhibitors against human cancers.

Fgfr4 has been shown to be important for appropriate muscle development in chick limb buds; however, Fgfr4 null mice show no phenotype. Here, we show that staged induction of muscle regeneration in Fgfr4 null mice becomes highly abnormal at the time point when Fgfr4 is normally expressed. By 7 days of regeneration, differentiation of myotubes became poorly coordinated and delayed by both histology and embryonic myosin heavy chain staining. By 14 days much of the muscle was replaced by fat and calcifications. To begin to dissect the molecular pathways involving Fgfr4, we queried the promoter sequences for transcriptional factor binding sites and tested candidate regulators in a 27-time point regeneration series. The Fgfr4 promoter region contained a Tead protein binding site (M-CAT 5′-CATTCCT-3′), and Tead2 showed induction during regeneration commensurate with Fgfr4 regulation. Co-transfection of Tead2 and Fgfr4 promoter reporter constructs into C2C12 myotubes showed Tead2 to activate Fgfr4, and mutation of the M-CAT motif in the Fgfr4 promoter abolished these effects. Immunostaining for Tead2 showed timed expression in myotube nuclei consistent with the mRNA data. Query of the expression timing and genomic sequences of Tead2 suggested direct regulation by MyoD, and consistent with this, MyoD directly bound to two strong E-boxes in the first intron of Tead2 by chromatin immunoprecipitation assay. Moreover, co-transfection of MyoD and Tead2 intron reporter constructs into 10T1/2 cells activated reporter activity in a dose-dependent manner. This activation was greatly reduced when the two E-boxes were mutated. Our data suggest a novel MyoD-Tead2-Fgfr4 pathway important for effective muscle regeneration.

We have previously demonstrated that the Src family kinase Yes, the Yes-associated protein (YAP) and TEA domain TEAD2 transcription factor pathway are activated by leukemia inhibitory factor (LIF) and contribute to mouse embryonic stem (mES) cell maintenance of pluripotency and self-renewal. In addition, we have shown that fetal bovine serum (FBS) induces Yes auto-phosphorylation and activation. In the present study we confirm that serum also activates TEAD-dependent transcription in a time- and dose-dependent manner and we identify Inter-α-inhibitor (IαI) as a component in serum capable of activating the Yes/YAP/TEAD pathway by inducing Yes auto-phosphorylation, YAP nuclear localization and TEAD-dependent transcription. The cleaved heavy chain 2 (HC2) sub-component of IαI, is demonstrated to be responsible for this effect. Moreover, IαI is also shown to efficiently increase expression of TEAD-downstream target genes including well-known stem cell factors Nanog and Oct 3/4. IαI is not produced by the ES cells per se but is added to the cells via the cell culture medium containing serum or serum-derived components such as bovine serum albumin (BSA). In conclusion, we describe a novel function of IαI in activating key pluripotency pathways associated with ES cell maintenance and self-renewal.

We have identified and characterized by transient transfection assays the cell-specific 117-bp enhancer sequence in the first intron of the mouse ETF ( E mbryonic T EA domain-containing f actor)/Tead2 gene required for transcriptional activation in ETF/Tead2 gene-expressing cells, such as P19 cells. The 117-bp enhancer contains one GC-rich sequence (5′-GGGGCGGGG-3′), termed the GC box, and two tandemly repeated GA-rich sequences (5′-GGGGGAGGGG-3′), termed the proximal and distal GA elements. Further analyses, including transfection studies and electrophoretic mobility shift assays using a series of deletion and mutation constructs, indicated that Sp1, a putative activator, may be required to predominate over its competition with another unknown putative repressor, termed the GA element-binding factor, for binding to both the GC box, which overlapped with the proximal GA element, and the distal GA element in the 117-bp sequence in order to achieve a full enhancer activity. We also discuss a possible mechanism underlying the cell-specific enhancer activity of the 117-bp sequence.

Mammals express four highly conserved TEAD/TEF transcription factors that bind the same DNA sequence, but serve different functions during development. TEAD-2/TEF-4 protein purified from mouse cells was associated predominantly with a novel TEAD-binding domain at the amino terminus of YAP65, a powerful transcriptional coactivator. YAP65 interacted specifically with the carboxyl terminus of all four TEAD proteins. Both this interaction and sequence-specific DNA binding by TEAD were required for transcriptional activation in mouse cells. Expression of YAP in lymphocytic cells that normally do not support TEAD-dependent transcription (e.g., MPC11) resulted in up to 300-fold induction of TEAD activity. Conversely, TEAD overexpression squelched YAP activity. Therefore, the carboxy-terminal acidic activation domain in YAP is the transcriptional activation domain for TEAD transcription factors. However, whereas TEAD was concentrated in the nucleus, excess YAP65 accumulated in the cytoplasm as a complex with the cytoplasmic localization protein, 14-3-3. Because TEAD-dependent transcription was limited by YAP65, and YAP65 also binds Src/Yes protein tyrosine kinases, we propose that YAP65 regulates TEAD-dependent transcription in response to mitogenic signals.

The cellular changes during an epithelial-mesenchymal transition (EMT) largely rely on global changes in gene expression orchestrated by transcription factors. Tead transcription factors and their transcriptional co-activators Yap and Taz have been previously implicated in promoting an EMT, however, their direct transcriptional target genes and their functional role during EMT have remained widely elusive. We have uncovered a previously unanticipated role of the transcription factor Tead2 during EMT. During EMT in mammary gland epithelial cells and breast cancer cells, levels of Tead2 increase in the nucleus of cells, thereby directing a predominant nuclear localization of its co-factors Yap and Taz via the formation of Tead2/Yap/Taz complexes. Genome-wide chromatin immunoprecipitation/next generation sequencing in combination with gene expression profiling reveals the transcriptional targets of Tead2 during EMT. Among these, zyxin contributes to the migratory and invasive phenotype evoked by Tead2. The results demonstrate that Tead transcription factors are critical regulators of Yap/Taz cellular distribution and together they control the expression of genes critical for EMT and metastasis.

Recent molecular classification of glioblastoma (GBM) has shown that patients with a mesenchymal (MES) gene expression signature exhibit poor overall survival and treatment resistance. Using regulatory network analysis of available expression microarray data sets of GBM, including The Cancer Genome Atlas (TCGA), we identified the transcriptional coactivator with PDZ-binding motif (TAZ), to be highly associated with the MES network. TAZ expression was lower in proneural (PN) GBMs and lower-grade gliomas, which correlated with CpG island hypermethylation of the TAZ promoter compared with MES GBMs. Silencing of TAZ in MES glioma stem cells (GSCs) decreased expression of MES markers, invasion, self-renewal, and tumor formation. Conversely, overexpression of TAZ in PN GSCs as well as murine neural stem cells (NSCs) induced MES marker expression and aberrant osteoblastic and chondrocytic differentiation in a TEAD-dependent fashion. Using chromatin immunoprecipitation (ChIP), we show that TAZ is directly recruited to a majority of MES gene promoters in a complex with TEAD2. The coexpression of TAZ, but not a mutated form of TAZ that lacks TEAD binding, with platelet-derived growth factor-B (PDGF-B) resulted in high-grade tumors with MES features in a murine model of glioma. Our studies uncover a direct role for TAZ and TEAD in driving the MES differentiation of malignant glioma.

G A 0 C A 2. Giuili, G., Roechel, N., Scholl, U., Mattei, M-G., and Guellaen, G. (1993). Colocalization of the genes coding for the a3 and b3 subunits of soluble guanylyl cyclase to human chromosome 4 at q31.3–q33. Hum. Genet. 91: 257–260. 3. Gupta, G., Azam, M., Yang, L., and Danziger, R. S. (1997). The b2 subunit inhibits stimulation of the a1/b2 form of soluble guanylyl cyclase by nitric oxide. J. Clin. Invest. 100: 1488–1492. 4. Yu, F., Warburton, D., Wellington, S., and Danziger, R. S. (1996). Assignment of GUCIA2, the gene coding for the a2 subunit of soluble guanylyl cyclase to position 11q21–q22 on human chromosome 11. Genomics 33: 334–336.

Activating mutations in KRAS are among the most frequent events in diverse human carcinomas and are particularly prominent in human pancreatic ductal adenocarcinoma (PDAC). An inducible KrasG12D-driven mouse model of PDAC has established a critical role for sustained KrasG12D expression in tumor maintenance, providing a model to determine the potential for, and underlying mechanisms of, KrasG12D–independent PDAC recurrence. We utilized MRI imaging to monitor regression of advanced pancreatic tumors measuring at least 8 mm in diameter at time of doxycycline withdrawal. Here we show that some tumors undergo spontaneous relapse and are devoid of KrasG12D expression, downstream canonical MAPK signaling but instead acquired amplification and overexpression of the transcriptional co-activator Yap1. Enforced Yap1 expression can substitute for oncogenic Kras-driven tumor maintenance and associated tumor cell proliferation without activation of canonical Kras signaling surrogates. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving KrasG12D–independent tumor maintenance. The Yap1/Tead2 complex acts cooperatively with E2F transcription factors to activate a cell cycle and DNA replication program. Our studies, along with corroborating evidence from human PDAC models indicates that the oncogenic Kras-independent relapse tumors generated in response to KrasG12D extinction closely resemble the quasimesenchymal-subtype of human PDAC and rely on alternative oncogenic mechanisms including Yap1 for their growth. Citation Format: Avnish Kapoor, Wantong Yao, Haoqiang Ying, Sujun Hua, Alison Liewen, Anguraj Sadanandam, Ramsey Al-Khalil, Andrea Viale, Piergiorgio Pettazzoni, Nora Sanchez, Alexei Protopopov, Jianhua Zhang, Timothy Heffernan, Randy L. Johnson, Lynda Chin, Y. Alan Wang, Giulio Draetta, Ronald A. DePinho. Yap1 activation enables bypass of oncogenic Kras addiction in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A94.

Four members of the Transcriptional Enhancer Factor (TEF) family of transcription factors have been identified in human and mouse (TEF-1, TEF-3, TEF-4, and TEF-5; approved gene symbols TEAD1, TEAD4, TEAD2, and TEAD3 respectively; Yasunami et al. 1995, 1996; Jacquemin et al. 1996, 1997; Yockey et al. 1996; Hsu et al. 1996; Kaneko et al. 1997; reviewed in Jacquemin and Davidson 1997). These factors possess the TEA DNA binding domain which recognizes degenerate sites in the enhancers and promoters of several viral and cellular genes. The TEFs comprise a short variable N-terminal region preceding the highly conserved TEA domain, a variable hydrophobic region immediately after the TEA domain, and a large, well-conserved C-terminal domain (see Fig. 1A). Expression of the murine (m)Tead factors is differentially regulated during development, the principal sites of expression being mitotic neuroblasts, skeletal and cardiac muscle, the placenta, lung, and several other viscera (see Jacquemin and Davidson 1997). Insertional mutagenesis showed that Tead1 is essential for cardiac development (Chen et al. 1994). Tead3 is first expressed in extraembryonic tissues such as the giant trophoblastic cells, and expression persists in the giant cells and labyrinthine region of the placenta throughout gestation (Jacquemin et al. 1998). Tead3 expression is limited to the extraembryonic layers until around 9.5 days post coitum, when further expression in epithelia and developing viscera is also observed. As gene targeting experiments will be required to further define the role of Tead3 in placental development, we report here as a first step the isolation and characterization of the Tead3 gene and the determination of its chromosomal location. A lEMBL3 mouse genomic DNA library (kindly provided by J.-M. Garnier) was screened with a mix of the cDNAs encoding the Tead1, -2, -3 and -4 TEA domains as probes. After isolation, phages containing Tead3 were identified by PCR using Tead3specific primers, and their DNA was analyzed by Southern blotting with the full-length Tead3 cDNA. Following digestion with PvuII, 4 hybridizing fragments ranging from 1.1 to 3.2 kb were cloned. These fragments were entirely sequenced and comparison with the Tead3 cDNA allowed identification of the introns and exons. Junctions between the fragments, and sequences upstream and downstream of the coding region were obtained by direct sequencing on the phage DNA. A total sequence of 12,622 base pairs (bp) was obtained. Analysis of this sequence indicates that the Tead3 gene comprises 13 exons (the smallest is 12 bp) and 12 introns (from 91 bp to 3582 bp; Fig. 1A). All of the intron-exon junctions conform with the GT/AG rule. The TEA DBD is encoded by three exons (II–IVA), each coding for one of the three putative a-helices. In chicken (c) two Tead3 isoforms have been characterized with differences in the C-terminal a-helix of the TEA DBD encoded by exon IV. These isoforms may be generated by differential use of a duplication of this exon. Indeed, downstream of exon IVA, we found a variant copy of this exon (IVB, Fig. 1B). So far only exon IVA has been found in mammalian Tead3 cDNAs. In contrast, in TEAD1, only exon IVB has been found (Fig. 1B). However, in one exceptional case a TEAD1 cDNA from HeLa cells that we characterized previously contained a duplicated region at the end of the TEA domain corresponding to a splice variant in which both exons IVA and IVB were present (our unpublished data and see Fig. 1B). This TEAD1 variant was still able to specifically bind DNA in electrophoretic mobility shift assays (our unpublished data). Alternative splicing of the four amino acids encoded by exon V was also observed. This exon was present in all the isolated mouse or human TEAD3 cDNAs, but was variable in TEAD1. Comparison of the Tead3 locus with that of Tead2 (Suzuki et al. 1996) showed that the overall organization of the genes is conserved. The location of the intron-exon junctions for Tead3 relative to the peptide sequence is shown in Fig. 2A. All the junctions are in analogous position to those of Tead2 with the exception of the upstream and downstream borders of exon VII. A comparison of the exon VII frontiers for Tead2 and -3 is shown in Fig. 2B. This exon encodes part of the most variable region of the TEAD family. We further determined the chromosomal localization of Tead3, using an interspecific backcross DNA panel (The Jackson Laboratory; Bar Harbor, Me; Rowe et al. 1994). The panel was generated by using DNA from 94 backcross animals from the reciprocal cross (C57BL/6JEi × SPRET/Ei)F1 × SPRET/Ei (“Jackson BSS” backcross panel). Southern blot analysis with a Tead3 cDNA probe detected a restriction fragment length variation (RFLV) between DNAs of the C57BL/6J and M. spretus. The probe detected a 2-kb BamHI fragment in C57BL/6 DNA and a 5-kb fragment in M. spretus DNA (data not shown). Therefore, the RFLV identified was used to follow the segregation of the Tead3 locus in the Jackson BSS backcross panel. The resulting data show that Tead3 is 1.1 centimorgans (cM) distal to the D17WSU92e locus, 2.1 cM and 3.2 cM proximal to the Tff3 (intestinal trefoil factor 3) or D17Mit16 locus respectively, near the proximal end of Chr 17 (Fig. 3). The Fkbp5 gene (FK506 binding protein 5) is also present in this region of Chr 17. In fact, Tead3 co-segregates with Fkbp5 gene in all 94 animals. Thus, we *Present address: Unite HORM, UCL-ICP, 75 Av. Hippocrate, B-1200 Bruxelles, Belgique.

We describe the molecular cloning of two novel human and murine transcription factors containing the TEA/ATTS DNA binding domain and related to transcriptional enhancer factor-1 (TEF-1). These factors bind to the consensus TEA/ATTS cognate binding site exemplified by the GT-IIC and Sph enhansons of the SV40 enhancer but differ in their ability to bind cooperatively to tandemly repeated sites. The human TEFs are differentially expressed in cultured cell lines and the mouse (m)TEFs are differentially expressed in embryonic and extra-embryonic tissues in early post-implantation embryos. Strikingly, at later stages of embryogenesis, mTEF-3 is specifically expressed in skeletal muscle precursors, whereas mTEF-1 is expressed not only in developing skeletal muscle but also in the myocardium. Together with previous data, these results point to important, partially redundant, roles for these TEF proteins in myogenesis and cardiogenesis. In addition, mTEF-1 is strongly coexpressed with mTEF-4 in mitotic neuroblasts, while accentuated mTEF-4 expression is also observed in the gut and the nephrogenic region of the kidney. These observations suggest additional roles for the TEF proteins in central nervous system development and organogenesis.

The Salvador/Warts/Hippo (SWH) pathway plays an important role in organ size control, and deregulation of the pathway can lead to organ overgrowth and human cancer as well. The SWH pathway regulates tissue growth through inhibiting the activity of the downstream regulator, Yorkie (Yki) in D. melanogaster and Yes-associated protein (YAP) in mammals, which act as transcriptional co-activators. Yki and YAP have a conserved N-terminal domain, through which they associate and cooperate with the transcription factors, Scalloped (Sd)/TEAD, to promote tissue growth. In addition, Yki and YAP possess two conserved WW domains that mediate interaction between several proteins that either promote or inhibit YAP's ability to drive transcription. Furthermore, the C-terminus of YAP includes three further motifs: a SH3 domain-binding motif, transcriptional activation domain and a PDZ domain-binding motif, none of which are conserved in Yki. To study the role of the Yki/YAP WW domains and the YAP C-terminus, several Yki/YAP mutants were constructed and their function was analyzed with assays for tissue growth in Drosophila, or with assays for colony growth, cell transformation, migration and invasion in MCF10A or NIH-3T3 cells. Results showed that: 1. WW domain mutant YAP promoted transformation, migration and invasive ability of MCF10A cells, suggesting that WW domains impart inhibitory regulation of YAP in these cells. In contrast, the YAP WW domains were essential to promote NIH-3T3 cell growth and transformation, and for the capability of Yki to drive tissue growth in D. melanogaster. These results also suggested that YAP WW domains are required for tissue growth but inhibit invasion. 2. Deletion of YAP's transcriptional activation domain abolished its ability to enhance transcriptional activity of TEAD2, as well as YAP's ability to stimulate invasion of MCF10A cells. However, this mutant promoted growth and transformation of both MCF10A and NIH-3T3 cells. These results suggest that the transcriptional activation domain is required for YAP-mediated invasion but inhibits its ability to stimulate colony growth and cell transformation. 3. Mutation of YAP's PDZ domain-binding motif lowered its ability to activate TEAD2 and promote invasive behavior of MCF10A cells. In contrast, PDZ binding motif-deleted YAP promoted 3D growth of MCF10A cells and transformation in both MCF10A and NIH-3T3 cells. These results suggest that YAP's PDZ domain-binding motif is required for YAP-mediated invasion but has an inhibitory effect on growth and transformation. Taken together, these results suggest that YAP is able to mediate tissue growth, transformation and invasion through different domains and the total effect of YAP on growth and invasion is dependent on the coordination of protein interactions via its WW and transcriptional activation domains, as well as its PDZ domain-binding motif. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3108.