TEAD Transcription Factors Mediate the Function of TAZ in Cell Growth and Epithelial-Mesenchymal Transition

The TAZ transcription co-activator has been shown to promote cell proliferation and to induce epithelial-mesenchymal transition. Recently we have demonstrated that TAZ is phosphorylated and inhibited by the Hippo tumor suppressor pathway, which is altered in human cancer. The mechanism of TAZ-mediated transcription is unclear. We demonstrate here that TEAD is a key downstream transcription factor mediating the function of TAZ. Disruption of TEAD-TAZ binding or silencing of TEAD expression blocked the function of TAZ to promote cell proliferation and to induce epithelial-mesenchymal transition, demonstrating TEAD as a key downstream effector of TAZ. We also identified CTGF, a gene that regulates cell adhesion, proliferation, and migration, as a direct target of TAZ and TEAD. Our study establishes a functional partnership between TAZ and TEAD under negative regulation by the Hippo signaling pathway. The TAZ transcription co-activator has been shown to promote cell proliferation and to induce epithelial-mesenchymal transition. Recently we have demonstrated that TAZ is phosphorylated and inhibited by the Hippo tumor suppressor pathway, which is altered in human cancer. The mechanism of TAZ-mediated transcription is unclear. We demonstrate here that TEAD is a key downstream transcription factor mediating the function of TAZ. Disruption of TEAD-TAZ binding or silencing of TEAD expression blocked the function of TAZ to promote cell proliferation and to induce epithelial-mesenchymal transition, demonstrating TEAD as a key downstream effector of TAZ. We also identified CTGF, a gene that regulates cell adhesion, proliferation, and migration, as a direct target of TAZ and TEAD. Our study establishes a functional partnership between TAZ and TEAD under negative regulation by the Hippo signaling pathway. Correction: TEAD transcription factors mediate the function of TAZ in cell growth and epithelial-mesenchymal transition.Journal of Biological ChemistryVol. 294Issue 15PreviewVOLUME 284 (2009) PAGES 13355–13362 Full-Text PDF Open Access TAZ (transcriptional co-activator with PDZ binding motif) is a transcription co-activator that was initially identified as a 14-3-3-binding protein (1Kanai F. Marignani P.A. Sarbassova D. Yagi R. Hall R.A. Donowitz M. Hisaminato A. Fujiwara T. Ito Y. Cantley L.C. Yaffe M.B. EMBO J. 2000; 19: 6778-6791Crossref PubMed Scopus (582) Google Scholar). TAZ contains a conserved WW domain, a coil-coil domain, a transactivation domain, and a C-terminal PDZ binding motif (2Hong J.H. Yaffe M.B. Cell Cycle. 2006; 5: 176-179Crossref PubMed Scopus (146) Google Scholar). It is involved in the development of multiple organs such as lung, fat, muscle, bone, limb, and heart tissues (2Hong J.H. Yaffe M.B. Cell Cycle. 2006; 5: 176-179Crossref PubMed Scopus (146) Google Scholar, 3Hong J.H. Hwang E.S. McManus M.T. Amsterdam A. Tian Y. Kalmukova R. Mueller E. Benjamin T. Spiegelman B.M. Sharp P.A. Hopkins N. Yaffe M.B. Science. 2005; 309: 1074-1078Crossref PubMed Scopus (814) Google Scholar, 4Murakami M. Tominaga J. Makita R. Uchijima Y. Kurihara Y. Nakagawa O. Asano T. Kurihara H. Biochem. Biophys. Res. Commun. 2006; 339: 533-539Crossref PubMed Scopus (94) Google Scholar, 5Murakami M. Nakagawa M. Olson E.N. Nakagawa O. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 18034-18039Crossref PubMed Scopus (219) Google Scholar). TAZ also modulates mesenchymal stem cell differentiation by promoting Runx-2-dependent transcription while repressing peroxisome proliferator-activated receptor γ-dependent transcription (3Hong J.H. Hwang E.S. McManus M.T. Amsterdam A. Tian Y. Kalmukova R. Mueller E. Benjamin T. Spiegelman B.M. Sharp P.A. Hopkins N. Yaffe M.B. Science. 2005; 309: 1074-1078Crossref PubMed Scopus (814) Google Scholar). TAZ knock-out mice have minor skeletal defects, but pathological changes in the kidney and lung resemble polycystic kidney disease and pulmonary emphysema, respectively (6Makita R. Uchijima Y. Nishiyama K. Amano T. Chen Q. Takeuchi T. Mitani A. Nagase T. Yatomi Y. Aburatani H. Nakagawa O. Small E.V. Cobo-Stark P. Igarashi P. Murakami M. Tominaga J. Sato T. Asano T. Kurihara Y. Kurihara H. Am. J. Physiol. Renal Physiol. 2008; 294: 542-553Crossref PubMed Scopus (220) Google Scholar, 7Hossain Z. Ali S.M. Ko H.L. Xu J. Ng C.P. Guo K. Qi Z. Ponniah S. Hong W. Hunziker W. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1631-1636Crossref PubMed Scopus (225) Google Scholar). We recently reported that TAZ is negatively regulated by the Lats tumor suppressor kinase (8Lei Q.Y. Zhang H. Zhao B. Zha Z.Y. Bai F. Pei X.H. Zhao S. Xiong Y. Guan K.L. Mol. Cell. Biol. 2008; 28: 2426-2436Crossref PubMed Scopus (720) Google Scholar), which is a component of the Hippo tumor suppressor pathway initially defined by genetic studies in Drosophila. Phosphorylation of TAZ by Lats leads to 14-3-3 binding and translocation from nucleus to cytoplasm, resulting in functional inactivation of this transcription co-activator (8Lei Q.Y. Zhang H. Zhao B. Zha Z.Y. Bai F. Pei X.H. Zhao S. Xiong Y. Guan K.L. Mol. Cell. Biol. 2008; 28: 2426-2436Crossref PubMed Scopus (720) Google Scholar). This phosphorylation-dependent inactivation of TAZ by Lats is similar to the mechanism of YAP regulation (9Zhao B. Ye X. Yu J. Li L. Li W. Li S. Yu J. Lin J.D. Wang C.Y. Chinnaiyan A.M. Lai Z.C. Guan K.L. Genes Dev. 2008; 22: 1962-1971Crossref PubMed Scopus (1684) Google Scholar), which is a co-activator with similar structure to TAZ, by the Hippo pathway. Accumulating evidence suggests an important and evolutionary conserved function of the Hippo pathway in control of tissue growth and cancer development. Both genetic and cell biological studies from Drosophila indicated that merlin, a product of the well established human tumor suppressor gene NF2 (10McClatchey A.I. Giovannini M. Genes Dev. 2005; 19: 2265-2277Crossref PubMed Scopus (211) Google Scholar), acts through the Hippo pathway to inhibit cell proliferation (11Hamaratoglu F. Willecke M. Kango-Singh M. Nolo R. Hyun E. Tao C. Jafar-Nejad H. Halder G. Nat. Cell Biol. 2006; 8: 27-36Crossref PubMed Scopus (591) Google Scholar). Mutations in Sav and Mob, two other components in the Hippo pathway, have also been identified in human tumor cell lines (12Lai Z.C. Wei X. Shimizu T. Ramos E. Rohrbaugh M. Nikolaidis N. Ho L.L. Li Y. Cell. 2005; 120: 675-685Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 13Tapon N. Harvey K.F. Bell D.W. Wahrer D.C. Schiripo T.A. Haber D.A. Hariharan I.K. Cell. 2002; 110: 467-478Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar), further supporting the importance of the Hippo pathway in human cancer. Interestingly, TAZ is shown to be overexpressed in ∼20% of breast cancer samples and plays an important role in breast tumorigenesis, migration, and invasion (14Chan S.W. Lim C.J. Guo K. Ng C.P. Lee I. Hunziker W. Zeng Q. Hong W.J. Cancer Res. 2008; 68: 2592-2598Crossref PubMed Scopus (381) Google Scholar). Consistent with this observation, TAZ overexpression promotes cell proliferation and induces epithelial-mesenchymal transition (EMT). 4The abbreviations used are: EMT, epithelial-mesenchymal transition; SBP, streptavidin-binding peptide; ChIP, chromatin immunoprecipitation; CTGF, connective tissue growth factor; MS, mass spectroscopy; shRNA, short hairpin RNA; YAP, Yes-associated protein; TEAD, TEA domain. Together, these studies suggest an important role of TAZ in mediating the Hippo pathway signaling to regulate cell growth and tumorigenesis. TAZ itself has no DNA binding domain; therefore, it must bind to DNA binding transcription factors to stimulate downstream target gene expression. TAZ has been reported to bind many transcription factors such as RUNX family, thyroid TF1 (TTF-1), TBX5, PAX3, PAX8, peroxisome proliferator-activated receptor γ, and TEAD (2Hong J.H. Yaffe M.B. Cell Cycle. 2006; 5: 176-179Crossref PubMed Scopus (146) Google Scholar, 4Murakami M. Tominaga J. Makita R. Uchijima Y. Kurihara Y. Nakagawa O. Asano T. Kurihara H. Biochem. Biophys. Res. Commun. 2006; 339: 533-539Crossref PubMed Scopus (94) Google Scholar, 5Murakami M. Nakagawa M. Olson E.N. Nakagawa O. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 18034-18039Crossref PubMed Scopus (219) Google Scholar, 15Park K.S. Whitsett J.A. Di Palma T. Hong J.H. Yaffe M.B. Zannini M. J. Biol. Chem. 2004; 279: 17384-17390Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). However, the relevance of these putative TAZ target transcription factors in mediating the function of TAZ in promoting cell proliferation and tumorigenesis is not clear. Here we report that using unbiased biochemical purification, we identified TEAD family transcription factors as the major TAZ interacting transcription factors in HEK293T cells. At the same time, by screening a human transcription factor library, we also identified TEAD family transcription factors as the targets most potently activated by TAZ. We further demonstrated that TEAD is indeed indispensable for TAZ to stimulate cell proliferation, migration, and EMT induction. Cell Culture and Transfection—HEK293T cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum (HyClone) and 100 units/ml penicillin and streptomycin (Invitrogen). MCF10A cells were maintained in Dulbecco's modified Eagle's medium/F-12 medium (Invitrogen) supplemented with 5% horse serum (Invitrogen), 20 ng/ml epidermal growth factor, 0.5 μg/ml hydrocortisone, 10 μg/ml insulin, 100 ng/ml cholera toxin, 100 units/ml penicillin, and streptomycin (Invitrogen). Cell transfection was performed using Lipofectamine 2000 (Invitrogen) or calcium phosphate methods. Cells were harvested at 24 h post-transfection for protein analysis or luciferase activity assay. To establish stable TAZ-expressing cells, pBabe-TAZ retroviruses were generated and used to infect MCF10A cells. Stable pools were selected with puromycin for 5 days. TAZ stable pool cells were seeded in 6-well plates with 1 × 105 cells/well in triplicates. Cell growth was counted every day for 7 days. Streptavidin-binding Peptide (SBP) Purification of TAZ Protein Complex—SBP-TAZ stable 293T cells from ten 150-mm plates were collected and lysed in 40 ml of 0.3% Nonidet P-40 buffer (50 mm Tris-HCl, 150 mm NaCl, pH 7.5) containing protease and phosphatase inhibitors. SBP-TAZ in the supernatant was precipitated for 3 h with 100 μl of streptavidin resin (GE HealthCare, 17-5113-01), which was then washed 3 times with 0.3% Nonidet P-40 buffer followed by washing with 50 mm NH4HCO3 3 times. The precipitated protein was digested with trypsin (22Rybak J.N. Ettorre A. Kaissling B. Giavazzi R. Neri D. Elia G. Nat. Methods. 2005; 2: 291-298Crossref PubMed Google Scholar). The supernatant was collected, dried, and dissolved in 10% acetonitrile, 0.8% formic acid solution. The peptides were analyzed by MS/MS. Luciferase Activity Assay—For the luciferase reporter assay, 293T cells were seeded in 24-well plates. A mixture of 5× upstream activating sequence-luciferase reporter, Renilla, and the indicated plasmids was cotransfected. Twenty-four hours after transfection, cells were lysed, and luciferase activity was measured using a dual-luciferase reporter assay system (#E1960; Promega) following the manufacturer's instructions. The luciferase activity was measured by a luminometer (model TD-20/20). Transfection efficiency was normalized to thymidine kinase-driven Renilla luciferase activity as the internal control. RNA Isolation and Real-time PCR—Total RNA was isolated from cultured cells using Trizol reagent (Invitrogen). cDNA was synthesized by reverse transcription using oligo(dT) as the primer and proceeded to real-time PCR with gene-specific primers in the presence of SYBR Premix Ex Taq (#DRR041A; TaKaRa). The relative abundance of mRNA was calculated by normalization to glyceraldehyde-3-phosphate dehydrogenase mRNA. Western Blotting Analysis—Protein lysates were prepared from MCF10A, MCF10A TAZ, and mutants stable pool cells in a buffer containing 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% SDS, 0.5% deoxycholate, 1% Nonidet P-40, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 25 mm NaF, and mixture protease inhibitors (Roche Applied Science). Tissue lysate was by followed by Western to or and Cell were of cell lysate was with for h at were washed 3 times with buffer and at × for 5 between Protein was from with 50 μl of buffer were and for Western analysis was performed as G. Y. H. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). was to cell to a of 1% at was to a of The cells were washed with and collected in followed by of and with DNA were and was digested and for PCR The DNA was to PCR using primers tissue growth factor cells were with a Cell was h by were performed as reported with minor M. Nakagawa M. Olson E.N. Nakagawa O. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 18034-18039Crossref PubMed Scopus (219) Google Scholar). cell plates were with Cells were the and cultured with cell growth medium supplemented with hydrocortisone, insulin, epidermal growth factor, fetal and was by TAZ a transcription TAZ must with a downstream transcription factor to induce gene expression. To TAZ target transcription we performed TAZ TAZ was as a protein with the SBP, which has streptavidin and be by streptavidin To by expression we used to establish 293T stable cell with expression of SBP-TAZ was by and the were by Western and SBP-TAZ and other were in cells SBP-TAZ but not in control cells purification, we performed trypsin and the mixture was analyzed by and mass This identified multiple putative TEAD family and were identified with multiple peptides for TEAD protein were identified peptides were from control from cells with the Western of the SBP-TAZ further that was with SBP-TAZ TEAD also performed an for TAZ target transcription This is the of TAZ to target transcription factors in a luciferase reporter assay. of the of putative human transcription factors with DNA binding and 5× upstream activating sequence-luciferase reporter by binding were with or TAZ. a transcription factor is activated by TAZ, of TAZ luciferase This unbiased identified and as the transcription factors that most potently activated by TAZ is not in the but was also to be activated by TAZ and were potently activated by TAZ and the TAZ to establish as major transcription factors activated by TAZ. We that the between YAP and TEAD (9Zhao B. Ye X. Yu J. Li L. Li W. Li S. Yu J. Lin J.D. Wang C.Y. Chinnaiyan A.M. Lai Z.C. Guan K.L. Genes Dev. 2008; 22: 1962-1971Crossref PubMed Scopus (1684) Google Scholar). Interestingly, this for TEAD binding is conserved in TAZ. therefore, generated which is the to We that or the between TAZ and was also in that the is important for TAZ to TEAD family transcription To further the mechanism of TAZ in gene mRNA expression from or MCF10A cells, which is an but human cell were by many we a of CTGF, a of the family of that regulates a of adhesion, proliferation, migration, and differentiation C.Y. E. J. Sci. 2008; PubMed Scopus Google Scholar, O. P.A. S. 2007; PubMed Scopus Google Scholar). has also been in cell growth and cancer. We further TAZ expression and the role of TEAD in this We that TAZ expression activated the luciferase reporter by the of a negative which has a of the C-terminal TAZ interacting but the DNA binding domain, blocked the of TAZ to stimulate in a as a negative by with TEAD for binding to target gene that TEAD is for TAZ to the To a direct of TAZ with was performed in or 293T cells with We that bind to the the TEAD not bind to supporting that TEAD the between TAZ and with a study demonstrating that the contains putative TEAD binding and expression is regulated by TEAD (9Zhao B. Ye X. Yu J. Li L. Li W. Li S. Yu J. Lin J.D. Wang C.Y. Chinnaiyan A.M. Lai Z.C. Guan K.L. Genes Dev. 2008; 22: 1962-1971Crossref PubMed Scopus (1684) Google Scholar). We further the of TAZ expression mRNA mRNA expression is by TAZ and by which is a the Lats in this However, the TEAD or not induce expression Together, we that TEAD plays an role in the of by TAZ. TEAD for TAZ to Cell we have established that TAZ acts as a downstream component of the Hippo pathway to promote cell proliferation (8Lei Q.Y. Zhang H. Zhao B. Zha Z.Y. Bai F. Pei X.H. Zhao S. Xiong Y. Guan K.L. Mol. Cell. Biol. 2008; 28: 2426-2436Crossref PubMed Scopus (720) Google Scholar). To the role of TEAD in the of TAZ to stimulate cell we established MCF10A stable cells TAZ or MCF10A cells TAZ the control cells of to which TEAD binding the of TAZ to stimulate MCF10A cell proliferation the importance of TEAD binding for the of TAZ to stimulate cell of the putative Lats in a that is in cell proliferation the TAZ TAZ with and was to promote MCF10A proliferation further supporting the that TEAD binding is important for TAZ to stimulate cell proliferation and that TEAD is a downstream effector of the Hippo pathway. We further the of TEAD binding for TAZ to stimulate MCF10A cell proliferation in a MCF10A cells in with defined a is to the and of We that the of MCF10A cell was by expression of TAZ and potently by expression of However, the expression of the or TEAD not TEAD binding is for TAZ to promote cell To further evidence for the functional importance of TEAD-TAZ we to in MCF10A cells. We have generated a which and (9Zhao B. Ye X. Yu J. Li L. Li W. Li S. Yu J. Lin J.D. Wang C.Y. Chinnaiyan A.M. Lai Z.C. Guan K.L. Genes Dev. 2008; 22: 1962-1971Crossref PubMed Scopus (1684) Google Scholar). We the of cell Our further an important role of TEAD in TAZ-mediated of cell proliferation as TEAD proliferation of the cells and these evidence that TEAD is for the function of TAZ in promoting cell TEAD for TAZ to reported that expression of TAZ or the in MCF10A cells induces We TEAD binding is for TAZ to promote EMT in MCF10A cells. TAZ or expression induces a of and cell However, TEAD and were to promote such We also performed to the of Cells TAZ of cell and the cells a of with of cell with the EMT the and cells of as in the MCF10A control cells A and that TEAD binding is important for TAZ to induce changes in MCF10A cells. Western for EMT was performed to further TEAD-TAZ binding plays a role in and were indeed in cells TAZ and in cells Consistent with the of expression of mesenchymal and were in cells but not in the TEAD cells The of and mRNA were also regulated by TAZ but not by the TEAD mutants in a similar to protein of cells with this inhibited the of in promoting cell proliferation and Together, that TEAD-TAZ binding is for TAZ to induce EMT in MCF10A cells. TEAD for TAZ to Cell plays an important role in cancer development. To the of TEAD binding in mediating TAZ function in promoting cell migration, we performed of TAZ promote the in cultured MCF10A cells of the Lats the of TAZ to promote However, cells and that TEAD binding is for TAZ to promote cell This is with a of or in We also the of TEAD the activity of TAZ to promote cell of TEAD the of in with cells with these the that TEAD is for the function of TAZ in promoting cell cancer is the of in in K. P. M. K. D. G. Nat. 2007; PubMed Scopus Google Scholar). the the development and of breast cancer is for and TAZ has been reported to promote cell proliferation, EMT, and cell in cells (8Lei Q.Y. Zhang H. Zhao B. Zha Z.Y. Bai F. Pei X.H. Zhao S. Xiong Y. Guan K.L. Mol. Cell. Biol. 2008; 28: 2426-2436Crossref PubMed Scopus (720) Google Scholar). TAZ expression has also been reported to with breast cancer cell (14Chan S.W. Lim C.J. Guo K. Ng C.P. Lee I. Hunziker W. Zeng Q. Hong W.J. Cancer Res. 2008; 68: 2592-2598Crossref PubMed Scopus (381) Google Scholar). TAZ expression is in of breast (14Chan S.W. Lim C.J. Guo K. Ng C.P. Lee I. Hunziker W. Zeng Q. Hong W.J. Cancer Res. 2008; 68: 2592-2598Crossref PubMed Scopus (381) Google Scholar). TAZ has also been shown to be inhibited by the Hippo tumor suppressor pathway, which contains well established human tumor suppressor NF2 and other in human such as Sav and However, the key transcription factors mediating TAZ function in promoting cell proliferation, migration, and EMT have not been transcription such as peroxisome proliferator-activated receptor γ, and have been to downstream of TAZ (3Hong J.H. Hwang E.S. McManus M.T. Amsterdam A. Tian Y. Kalmukova R. Mueller E. Benjamin T. Spiegelman B.M. Sharp P.A. Hopkins N. Yaffe M.B. Science. 2005; 309: 1074-1078Crossref PubMed Scopus (814) Google Scholar, 4Murakami M. Tominaga J. Makita R. Uchijima Y. Kurihara Y. Nakagawa O. Asano T. Kurihara H. Biochem. Biophys. Res. Commun. 2006; 339: 533-539Crossref PubMed Scopus (94) Google Scholar, 5Murakami M. Nakagawa M. Olson E.N. Nakagawa O. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 18034-18039Crossref PubMed Scopus (219) Google Scholar). The of study TAZ transcription factor targets is a for the of this therefore, we this by a of biochemical and functional Interestingly, TAZ identified of the TEAD family transcription factors as the TEAD two transcription factors were identified in but with mass not This suggests as the major TAZ transcription factor at in HEK293T cells. an of a human transcription factor identified of the TEAD family transcription factors as the most targets of TAZ by the reporter assay, the other TEAD family was not in the Together, these the that TAZ target transcription other transcription factors be used for the of TAZ in other cell We demonstrated in this report that TEAD TAZ function in promoting cell proliferation, EMT, and cell migration, which involved in cancer and We by two that a TAZ that and of TEAD. Both that of or the of TAZ promoting cell proliferation in or changes and EMT and promoting in cell assay. be a target of for TAZ expression or Hippo pathway of the function of in EMT is in stem cell A report a between mesenchymal and the of stem cell using human cells as a system Guo W. E.N. A. M. F. Zhang M. L.L. K. C. J. R.A. Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, TAZ has recently been in stem cell this function of TAZ has been to to regulate X. R. P. R. B.M. J. Yaffe M.B. Nat. Cell Biol. 2008; PubMed Scopus Google Scholar). It be to TEAD also to the of TAZ to stem cell or cancer stem cell in breast cancer. To the regulation of gene expression by we used gene expression to by TAZ expression. for a transcription expression of TAZ induces expression of many We the gene expression of or MCF10A cells. Interestingly, TAZ and YAP and of by TAZ and YAP, also many by TAZ or YAP 1 and We reported that many YAP target by at the of YAP target by and protein but not TEAD we that of and of by TAZ and YAP TEAD family transcription factors to induce of as well as target which be for the and of TAZ and To further the function of TEAD in gene we that is a direct target gene of in cells and TEAD is for TAZ to induce expression. Interestingly, has been in multiple human cancer development and with the role of TAZ in tumorigenesis C.Y. E. J. Sci. 2008; PubMed Scopus Google Scholar, O. P.A. S. 2007; PubMed Scopus Google Scholar, N. S. K. A. S. N. C. F. P. R. M. Q. M. G. A. A. Cancer Res. 2006; PubMed Scopus Google Scholar). study identified TEAD family transcription factors as major TAZ target transcription factors and established a functional partnership between TAZ and TEAD in cell proliferation, EMT, and cell of the role of TAZ in cancer development and a target for human breast cancer We the of the and Cell for this study and the of for with