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The Transcriptional Regulators TAZ and YAP Direct Transforming Growth Factor β-induced Tumorigenic Phenotypes in Breast Cancer Cells * ♦

The Transcriptional Regulators TAZ and YAP Direct Transforming Growth Factor β-induced... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 19, pp. 13461–13474, May 9, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. The Transcriptional Regulators TAZ and YAP Direct Transforming Growth Factor -induced Tumorigenic Phenotypes in Breast Cancer Cells Received for publication, October 21, 2013, and in revised form, March 9, 2014 Published, JBC Papers in Press, March 19, 2014, DOI 10.1074/jbc.M113.529115 Samantha E. Hiemer, Aleksander D. Szymaniak, and Xaralabos Varelas From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118 Background: The TGF and Hippo pathways are dysregulated in metastatic breast cancers. Results: TGF-induced cues and nuclear TAZ/YAP converge at the transcriptional level to control gene expression important for tumorigenesis. Conclusion: TAZ/YAP are required to promote TGF-induced tumorigenic phenotypes in breast cancer cells. Significance: Our study reveals novel cross-talk between the TGF pathway and TAZ/YAP in late-stage breast cancers. Uncontrolled transforming growth factor- (TGF) signaling age-independent conditions (5–9). Moreover, high nuclear promotes aggressive metastatic properties in late-stage breast TAZ levels induce cancer stem cell-like activity (2, 10) and pro- cancers. However, how TGF-mediated cues are directed to mote evasion of certain breast cancer drug therapies (2, 11). induce tumorigenic events is poorly understood, particularly Thus, understanding the roles of TAZ/YAP is critical for direct- given that TGF has clear tumor suppressing activity in other ing efficient breast cancer therapies. contexts. Here, we demonstrate that the transcriptional regula- The tumor initiating activity of TAZ/YAP relies on their tors TAZ and YAP (TAZ/YAP), key effectors of the Hippo path- binding to the TEAD family of transcription factors way, are necessary to promote and maintain TGF-induced (TEAD1–4) (10, 12, 13), indicating that together these factors tumorigenic phenotypes in breast cancer cells. Interactions direct a tumorigenic transcriptional program. Supporting this between TAZ/YAP, TGF-activated SMAD2/3, and TEAD premise, TAZ/YAPTEAD complexes directly promote the transcription factors reveal convergent roles for these factors in expression of genes encoding oncogenic factors, such as CTGF the nucleus. Genome-wide expression analyses indicate that (also known as CCN2) and CYR61 (also known as CCN1) (12, TAZ/YAP, TEADs, and TGF-induced signals coordinate a spe- 13), which contribute to human breast cancer progression (14). cific pro-tumorigenic transcriptional program. Importantly, Nuclear TAZ/YAP activity is highly regulated and governed in genes cooperatively regulated by TAZ/YAP, TEAD, and TGF, large part by the Hippo pathway-regulated LATS1 and LATS2 such as the novel targets NEGR1 and UCA1, are necessary for kinases (15). LATS1/2 kinases phosphorylate TAZ/YAP on maintaining tumorigenic activity in metastatic breast cancer conserved serine residues, which promote 14-3-3 binding and cells. Nuclear TAZ/YAP also cooperate with TGF signaling to subsequent sequestration in the cytoplasm (16, 17), and also promote phenotypic and transcriptional changes in nontumori- prime TAZ/YAP for further phosphorylation by CK1/ genic cells to overcome TGF-repressive effects. Our work thus kinases that evoke TAZ/YAP degradation via proteasome-de- identifies cross-talk between nuclear TAZ/YAP and TGF sig- pendent mechanisms (18, 19). Additional phosphorylation naling in breast cancer cells, revealing novel insight into late- stage disease-driving mechanisms. events destabilize TAZ, including those regulated by Wnt, phosphatidylinositol 3-kinase, and GSK3 (20, 21). Thus, dys- regulation of multiple upstream signals likely contributes to the Elevated nuclear levels of the transcriptional regulators TAZ aberrant nuclear TAZ/YAP activity that is observed in cancers. and YAP (TAZ/YAP) are associated with a broad range of TAZ/YAP modify the activity of other transcription factors aggressive cancers (1). For instance, the extent of nuclear TAZ besides TEADs, including the transforming growth factor- or YAP levels corresponds with breast cancer tumor grade (TGF)-activated SMAD complexes (22). TGF is the proto- (2–4). In breast cancer cells, enhanced nuclear TAZ and YAP typic member of a family of secreted factors that regulates levels promote oncogenic transformation and endow cells with numerous developmental and homeostatic processes (23). tumorigenic properties, including the ability to proliferate, sub- SMAD2 and SMAD3 (SMAD2/3) are the primary mediators of vert apoptotic cues, migrate, invade, and grow under anchor- TGF-induced transcription. SMAD2/3 are phosphorylated by TGF-bound membrane receptors, which induce binding to * This work was supported in part by funds from the Concern Cancer Foun- SMAD4 (24, 25), forming active transcriptional complexes that dation, the Karin Grunebaum Cancer Research Foundation, and Research accumulate in the nucleus upon binding to TAZ/YAP (26). In Grant 5-FY11-578 from the March of Dimes Foundation. cancer, the role of TGF is complex, as it can suppress early This article was selected as a Paper of the Week. □ S This article contains supplemental Tables S1–S4. oncogenic events but also promote aggressive late-stage meta- To whom correspondence should be addressed: Dept. of Biochemistry, Bos- static phenotypes (27, 28). What mechanistically distinguishes ton University School of Medicine, Rm. K225, Boston, MA 02118. Tel.: 617- 638-4182; Fax: 617-638-5339; E-mail: [email protected]. the different TGF-dependent responses is poorly understood. This is an open access article under the CC BY license. MAY 9, 2014 • VOLUME 289 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13461 Convergent TAZ/YAP-TGF Signals in Breast Cancer Several lines of evidence indicate that TGF, like TAZ/YAP, 0.1% Triton X-100/PBS, blocked in 2% BSA/PBS, and probed promotes aggressive tumorigenic properties in late-stage breast with primary and secondary antibodies outlined in supplemen- carcinomas (29, 30). Given that TAZ/YAP bind to SMAD tran- tal Table S2. LM2-4 cells were treated with or without TGF1 scription factors and direct TGF signaling in other contexts (500 pM, R&D Systems) or SB-431542 (5 M, Sigma) for 24 h (26, 31, 32), we sought to characterize whether TAZ/YAP before fixing. For the PLA, LM2-4 cells were plated on 96-well define TGF-mediated tumorigenic cues in breast cancer cells. microplates (Falcon) and treated with or without TGF1 for Our observations indicate that TGF-induced tumorigenic 24 h. Cells were fixed and permeabilized as described, blocked events, such as increased cell migration, invasion, and anchor- according to the manufacturer’s protocol (Duolink), and age-independent growth, require TAZ/YAP. Our data also probed with the primary antibodies outlined in supplemental indicate that, like TAZ/YAP, the TEAD transcription factors Table S2. Anti-mouse MINUS and anti-rabbit PLUS PLA interact with TGF-induced SMAD2/3 in the nucleus, suggest- probes (Duolink) were used. Nuclei were stained with Hoechst. ing that TAZ/YAPTEADSMAD2/3 complexes coordinate All immunofluorescence was visualized by confocal micros- transcriptional events in a concerted manner. Genome-wide copy (LSM 700), and images were processed using Volocity microarray analysis of gene expression changes that occur upon software (PerkinElmer Life Sciences). Images were quantitated knockdown of TAZ/YAP or TEADs, or inhibition of TGF sig- using ImageJ software. naling, revealed that TAZ/YAP, TEAD, and TGF regulate Mammospheres—LM2-4 cells were transfected with siRNA, overlapping target genes. Interestingly, the direct gene targets dissociated 24 h later, and resuspended in Mammary Epithe- NEGR1 and UCA1, which are synergistically regulated by TAZ/ lium Growth Medium (MEGM; Lonza) supplemented with B27 YAP, TEAD, and TGF, are necessary for maintaining tumor- (Invitrogen), 20 ng/ml epidermal growth factor (EGF; Pepro- igenic activity in metastatic breast cancer cells, suggesting that Tech), 20 ng/ml basic fibroblast growth factor (bFGF; Pepro- the convergence of TAZ/YAPTEAD-TGF signals is critical Tech). Single cells were seeded at 5  10 cells/ml in 6-well for driving late-stage breast cancer phenotypes. Supporting this ultra-low attachment plates (Corning Glass) and treated with premise, expression of nuclear-localized TAZ or YAP mutants or without TGF1 or SB-431542. Primary spheres were photo- direct transcriptional events that sensitize untransformed graphed after 7 days and either lysed for RNA (Quick-RNA breast cancer cells to adopt tumorigenic phenotypes in MiniPrep, Zymo Research) to examine knockdown or dissoci- response to TGF, while also suppressing TGF-induced cyto- ated in 0.05% trypsin for 10 min and resuspended as single cells stasis. These findings reveal novel cross-talk between TGF in MEGM for passage. Secondary spheres were photographed and Hippo signaling that we propose is important for late stage after an additional 14 days. Images were analyzed using ImageJ tumorigenic events in breast cancer. software, and statistics were calculated using Prism software (GraphPad) using a two-tailed unpaired Student’s t test. EXPERIMENTAL PROCEDURES Immunoprecipitation and Immunoblots—LM2-4 cells exam- Cell Culture, Plasmids, and Transfections—MCF10A, MCF- ined for endogenous protein expression were treated with or 12A, HMLE, and MCF7 cells were cultured using DMEM/F-12 without TGF1 or SB-431542 for 2 h and were lysed and exam- media (1:1) supplemented with 5% horse serum, 20 ng/ml ined by immunoblotting. Transfected HEK293T cells express- epithelial growth factor (EGF; PeproTech), 0.5 g/ml hydro- ing the indicated proteins were lysed, subjected to immunopre- cortisone (Sigma), 100 ng/ml cholera toxin (Sigma), 10 g/ml cipitation using anti-FLAG-conjugated protein-G beads insulin (Sigma). MDA-MB-231 (MDA-231) and MDA-MB- (Sigma), and analyzed by immunoblotting. MCF10A doxycy- 231-LM2-4 (LM2-4) cells were cultured using RPMI media cline-inducible cells were treated with or without doxycycline supplemented with 10% FBS. SUM-149 cells were cultured (0.1 to 100 ng/ml) or TGF1 for 24 h and were lysed and exam- using Ham’s F-12 media supplemented with 5% FBS, 10 g/ml ined by immunoblotting. Antibodies are outlined in supple- insulin (Sigma), 0.5 g/ml hydrocortisone (Sigma). BT20, mental Table S2. HS578T, SKBR3, and HEK293T cells were cultured using Cell Morphology Analysis, Wound Healing, and Transwell DMEM supplemented with 10% FBS. HEK293T cells were Migration—Low density MCF10A doxycycline-inducible cells transfected using TurboFect (Thermo Scientific) according to were pretreated with doxycycline (100 ng/ml, Clontech) for the manufacturer’s protocol. MCF10A doxycycline-inducible 24 h and then treated with or without TGF1 for an additional stable cell lines were generated using the lentiviral Tet-On sys- 24 h. For the wound-healing scratch assays, LM2-4 cells were tem (Clontech). 3FLAG-tagged mutants of TAZ (4SA: S66A, transfected with siRNA and 24 h later were treated with or S89A, S117A, and S311A) or YAP (5SA: S61A, S109A, S127A, without TGF1 or SB-431542 for an additional 24 h. MCF10A S164A, and S397A) were generated by site-directed mutagene- doxycycline-inducible cells were treated with or without doxy- sis and cloned into the pLVX-Tight-Puro plasmid (catalog no. cycline or TGF1 for 24 h. Monolayers were wounded and pho- 632162, Clontech). Tet-On cells were selected with 1 mg/ml tographed after an additional 24 h (LM2-4) or 12 h (MCF10A). G-418 sulfate (Gold Biotechnology) and 1 g/ml puromycin Images were analyzed using ImageJ software, and statistics (American Bioanalytical). RNA interference was performed by were calculated using Prism software (GraphPad) using a two- transfecting siRNA using Dharmafect 1 (Thermo Scientific) tailed unpaired Student’s t test. Cells used in the transwell assay according to manufacturer’s protocol. Sequences for the were transfected with siRNA, trypsinized 24 h later, and resus- siRNAs used are outlined in supplemental Table S1. Immunofluorescence and Proximity Ligation Assay (PLA)— The abbreviations used are: PLA, proximity ligation assay; qPCR, quantita- Cells were fixed with 4% paraformaldehyde, permeabilized with tive real time PCR; OCLN, occludin. 13462 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 19 • MAY 9, 2014 Convergent TAZ/YAP-TGF Signals in Breast Cancer pended in low serum media (0.25% FBS). Cells were plated at enode). Immunoprecipitations were performed using antibod- cells/ml on 0.4-M transwell filters (BD Biosciences) pre- ies outlined in supplemental Table S2 (note: anti-TEAD4 also treated for 24 h with 1 g/ml fibronectin (Millipore). Media  recognizes TEAD1 and -3 (35)) followed by incubation with 10% FBS were used in the bottom chamber. Cells were allowed protein-G Dynabeads (Invitrogen), and then washing sequen- to migrate for 24 h in the presence of TGF1 and were subse- tially in buffer A (20 mM Tris/HCl, pH 7.6, 140 mM NaCl, 1 mM quently stained with 0.5% crystal violet. EDTA, pH 8.0, 0.1% sodium deoxycholate, 0.1% SDS, 1% Triton Three-dimensional Invasion—Stable knockdown of TAZ and X-100), buffer B (20 mM Tris/HCl, pH 7.6, 500 mM NaCl, 1 mM YAP in LM2-4 cells was accomplished by lentivirus-mediated EDTA, pH 8.0, 0.5% sodium deoxycholate, 1% Triton X-100), transduction of shRNA using the pLKO1-puro and pLKO1-neo buffer C (20 mM Tris/HCl, pH 7.6, 1 mM EDTA, pH 8.0, 0.5% vectors and subsequent selection with 2 g/ml puromycin and sodium deoxycholate, 1% Triton X-100, 250 mM LiCl), and 1.5 mg/ml G418. The shRNA sequences used are listed in sup- TBS. Samples were eluted in Elution buffer (50 mM NaHCO , plemental Table S1. Single cells were plated on 100% growth 50 mM Tris/HCl, pH 8.0, 2 mM EDTA, pH 8.0, 1% SDS). Cross- factor-reduced Matrigel (BD Biosciences) using the overlay links were reversed overnight at 65 °C in 0.2 M NaCl in Elution method (33). Assay media contained 2% Matrigel added to sup- buffer, and DNA was purified using QIAquick PCR purification plemented MEGM, and cells were cultured with puromycin columns (Qiagen). Samples were then analyzed by qPCR using and G418 with medium changes every 3 days. TGF1 and the primers outlined in supplemental Table S3. SB-431542 were added after 9 days and then cultured for an Cell Proliferation and Cell Cycle Analysis—MCF10A doxycy- additional 3 days before being photographed. cline-inducible cells were plated (5 10 cells) and treated with Microarrays—LM2-4 cells were transfected with control doxycycline with or without TGF1 (day 0). Cells were counted siRNA or siRNAs targeting TAZ/YAP or all four TEADs (out- each day for 6 consecutive days (day 1–6). For cell cycle analy- lined in supplemental Table S1) and were treated 24 h later sis, MCF10A doxycycline-inducible cells were treated with with 1 or SB-431542 for an additional 24 h. Total RNA doxycycline with or without TGF1 for 48 h. 1 10 TGF cells were was isolated and purified by Quick-RNA MiniPrep (Zymo fixed overnight in 100% ethanol and stained using 50 g/ml Research). Twelve microarrays in total were performed, with propidium iodide (Sigma) and 100 g/ml RNase A (Sigma). each condition carried out three times on separate days. The Samples were acquired on the FACScan (BD Biosciences), col- Boston University Microarray Core generated the data using lecting 1  10 events, and analyzed using FlowJo software the Affymetrix Human Gene 1.0 St Array, which covers 27,300 (Tree Star). Statistical analysis was conducted using a two- probe sets. The data were filtered using a moderated p value of tailed unpaired Student’s t test. less than 0.01, and the average fold change in expression of each RESULTS gene, for each condition, relative to the siCTL  TGF sample was calculated. Fold expression changes relative to siCTL  Nuclear TAZ/YAP Are Required to Promote TGF-induced TGF-treated cells were calculated, and statistical significance Tumorigenic Phenotypes in Breast Cancer Cells—In cancer, the was assessed using a moderated t test and p values. Hierarchical role of TGF is complex, as it can suppress early oncogenic gene clustering was performed on overlapping genes displaying events, such as cell cycle progression, but can also promote a p value of 0.01 with the open source program Cluster 3.0 late-stage metastatic phenotypes (27, 28). What distinguishes (34). these different TGF-dependent responses is poorly under- Quantitative Real Time PCR (qPCR)—LM2-4 cells were stood. Several lines of evidence indicate that nuclear TAZ/YAP, transfected with siRNA and were treated 24 h later with or like TGF, induce tumorigenic properties in late-stage breast without TGF1 or SB-431542 for an additional 24 h. MCF10A carcinomas (29, 30). In untransformed mammary epithelium, doxycycline-inducible cells were treated with or without doxy- TAZ/YAP localization is restricted to the cytoplasm by cell cycline (0.1 to 100 ng/ml) or TGF1 for 24 h. Total RNA was compaction/polarity-regulated cues (9, 32). Dysregulation of purified using Quick-RNA MiniPrep kit, and cDNA synthesis cell polarity cues, which is a hallmark of cancer progression was performed using 1 g RNA and iScript cDNA synthesis kit (36), induces nuclear TAZ/YAP localizations. Given our prior (Bio-Rad) according to manufacturer’s protocol. qPCR was per- work showing that TAZ/YAP bind to and regulate the localiza- formed using Fast SYBR Green enzyme (Applied Biosystems) tion and activity of TGF-activated SMAD transcription and measured on ViiA 7 real time PCR system (Applied Biosys- factors (26, 32), we sought to test whether TAZ and/or YAP tems). Transcript levels were analyzed using the C method promote TGF-induced tumorigenic events. We began our and normalized to GAPDH. Primer sequences are indicated in analysis by examining the relationship between TAZ and YAP supplemental Table S3. localizations and the TGF-induced cytostatic response in a Chromatin Immunoprecipitation (ChIP)—LM2-4 cells were panel of mammary epithelial and breast cancer cell lines. Based fixed with 1 mM EGS (Thermo Scientific) for 30 min, 1% form- on published data, we divided the panel into cells that are aldehyde for 10 min, and quenched in 0.125 M glycine in PBS. responsive to TGF-induced cytostasis (MCF10A, BT20, Cells were collected and lysed in Cell Lysis buffer (10 mM KOH/ HMLE, HS578T, MCF7, and MCF-12) and cells in which TGF HEPES, pH 7.8, 85 mM KCl, 1 mM EDTA, pH 8.0, 1% Nonidet induces pro-tumorigenic signals but not growth arrest (MDA- P-40) with a protease inhibitor mixture. Nuclei were lysed in MB-231, MDA-MB-231-LM2-4, SKRB3, and SUM149) (37– Nuclear Lysis buffer (50 mM Tris/HCl, pH 7.4, 1% SDS, 10 mM 44). Interestingly, we observed that cells displaying high levels EDTA, pH 8.0) with protease inhibitors, and genomic DNA was of nuclear TAZ/YAP correlate with those in which TGF fragmented to 400 bp using Bioruptor bath sonicator (Diag- induces tumorigenic cues (Fig. 1A). MAY 9, 2014 • VOLUME 289 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13463 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 1. TAZ/YAP are required for TGF-induced tumorigenic events. A, panel of breast cancer cell lines was divided by TGF-induced tumor suppression and promoting responses and examined by immunofluorescence for endogenous TAZ and YAP localization. B, LM2-4 cells were transiently transfected with control siRNA (siCTL) or siRNA targeting TAZ (siTAZ), YAP (siYAP), or TAZ and YAP (siTAZ/YAP). Cells were left untreated, treated with TGF or SB-431542 TGF, and grown in anchorage-independent conditions. Primary mammospheres were examined for knockdown or were passaged into secondary spheres. Sec- ondary mammospheres following SB-431542 (SB) treatment, or transfection with siTAZ, siYAP, or siTAZ/YAP, were unable to be determined due to low numbers. Representative images are shown, and three independent experiments from each condition were quantitated, measuring the number of colonies formed and the size of each colony. Black error bars represent the average S.E., and red error bars represent the average S.E., *, p 0.025; **, p 0.005; ***, p  0.0001 (t test). C, LM2-4 cell lysates were immunoblotted to examine endogenous levels of the indicated proteins upon TGF or SB-431542 treatment compared with GAPDH (loading control). D, LM2-4 cells were transiently transfected with siCTL or siTAZ/YAP. Cells were left untreated, treated with TGF,or SB-431542 TGF. Monolayers were wounded and analyzed for cell migration. E, LM2-4 cells stably expressing control shRNA (shCTL) or shRNA targeting TAZ and YAP (shTAZ/YAP) were treated with TGF or SB-431542  TGF and incubated in three-dimensional Matrigel culture conditions. Representative images from three independent experiments are shown. To further investigate this relationship, we sought to deter- mospheres under anchorage-independent conditions (Fig. 1B), mine the roles of nuclear TAZ/YAP in the human MDA-MB- which is often used as a measure of the self-renewing potential 231-LM2-4 (herein referred to as LM2-4) metastatic breast of tumorigenic cells in vitro (47). TGF treatment of LM2-4 cancer cell line (45), a highly aggressive derivative of triple- cells led to dramatic increases in the number and size of mam- negative basal subtype MDA-MB-231 cells (46). A fraction of mospheres observed (Fig. 1B), similar to that observed with LM2-4 cells in culture are capable of generating clonal mam- TGF treatment of other mammary cells (30). The self-renew- 13464 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 19 • MAY 9, 2014 Convergent TAZ/YAP-TGF Signals in Breast Cancer ing properties of the cells within the mammospheres were other) by microscopy (52). Using PLA, we observed TAZ/ assessed by their ability to form secondary clonal spheres (47), YAPSMAD2/3 interactions in both the nucleus and cytoplasm and we found that TGF also promoted secondary mammo- of untreated LM2-4 cells (Fig. 2C). Upon TGF treatment, sphere formation. Co-treatment of the cells with the TGF nuclear TAZ/YAP-SMAD2/3 binding became much more receptor antagonist SB-431542 abolished the formation of pri- apparent in the nucleus (Fig. 2C), consistent with nuclear TAZ/ mary mammospheres, validating that the observed effects are YAPSMAD2/3 complexes directing transcriptional events (26, indeed generated via canonical TGF receptor-mediated sig- 32). We also detected endogenous TAZ/TEAD1 interactions in nals (Fig. 1B) (48, 49). As expected, SB-431542 treatment elim- the nucleus of LM2-4 cells with or without TGF stimulation inated the TGF-induced phosphorylation of SMAD2 and (Fig. 2D), which were increased slightly upon TGF treatment SMAD3 in these cells (Fig. 1C). Individual TAZ or YAP knock- (Fig. 2D). TEAD1SMAD2/3 interactions were readily detected down also repressed the number and size of TGF-induced in the nucleus of LM2-4 cells, particularly after TGF treat- mammospheres (Fig. 1B). However, simultaneous knockdown ment (Fig. 2E), suggesting these complexes stabilize upon of both TAZ and YAP dramatically reduced mammosphere for- nuclear accumulation of SMADs. Taken together, our observa- mation (Fig. 1B), indicating redundant roles for TAZ and YAP tions indicate that TAZ/YAP, TEAD, and SMAD interact in transducing TGF-mediated cues required for anchorage- in TGF-stimulated metastatic breast cancer cells and suggest independent growth. that they may form transcriptional complexes that function We further investigated other hallmark tumorigenic proper- together in the nucleus. ties that may be mediated by TGF and TAZ/YAP in metastatic To explore the possible overlap in transcriptional activity breast cancers, including cell migration and invasion (36). We by TAZ/YAP, TEAD, and SMAD complexes, we used microar- found that treatment of LM2-4 cells with TGF led to increases rays to compare the global expression profiles of LM2-4 cells in cell migration in an in vitro wound-healing scratch assay (Fig. treated as follows (complete data available in supplemental 1D), similar to previous work (50). As expected, co-treatment Table S4): 1) transfected with control siRNA (siCTL) and with the TGF receptor antagonist SB-431542 blocked TGF- treated with TGF; 2) transfected with siRNA targeting both induced cell migration (Fig. 1D). Simultaneous knockdown of TAZ/YAP (siTAZ/YAP) and treated with TGF; 3) transfected TAZ/YAP using siRNA also abolished TGF-induced LM2-4 with siRNA targeting all four TEAD (TEAD1–4) family mem- cell migration (Fig. 1D). Similarly, SB-431542 treatment or bers (siTEAD) and treated with TGF; and 4) transfected with shRNA-mediated TAZ/YAP knockdown abolished the ability control siRNA (siCTL) and treated simultaneously with TGF of three-dimensional colonies of LM2-4 cells to invade into the and SB-431542. In terms of significant gene expression differ- surrounding Matrigel matrix in the presence of TGF (Fig. 1E). ences (p value 0.01) relative to siCTL TGF treatment, 461 Taken together, our observations indicate that TAZ/YAP are genes overlapped between siTAZ/YAP and siTEAD conditions critical mediators of TGF-induced tumorigenic events, (Fig. 3A). This gene set displayed a high degree of correlation in including mammosphere formation, cell migration, and expression (R  0.86). The expression of 594 genes changed invasion. following SB-431542 treatment, and of these, 176 genes over- TAZ/YAP, TEADs, and SMADs Converge to Regulate a lapped with siTAZ/YAP conditions. Of these 176 genes, 80 TGF-induced Transcriptional Program in Breast Cancer Cells— were also altered following TEAD knockdown (Fig. 3A). Studies indicate that TAZ/YAP-induced cell transformation Interestingly, genes for which expression was altered among relies on the recruitment of TAZ/YAP to DNA by the TEAD all three experimental conditions exhibited distinct expression family of transcription factors (TEAD1–4) (12, 13). TAZ and correlations. Unbiased clustering segregated TAZ/YAPTEAD- YAP also bind TGF-activated SMAD complexes to control TGF-regulated genes into four different groups as follows: SMAD localization and activity in a variety of cell types, includ- group 1, repressed following siTAZ/YAP, siTEADs, or TGF ing mammary epithelial cells (26, 32). Recent work has shown inhibition (therefore normally induced by the presence of these that TAZ/YAPTEADSMAD2/3 complexes control transcrip- factors); group 2, repressed following siTAZ/YAP or siTEAD tional events important for maintaining human embryonic treatment but induced by TGF inhibition; group 3, induced stem cell pluripotency (35). Thus, we hypothesized that similar following siTAZ/YAP, siTEADs, or TGF inhibition (therefore complexes may also be present in late stage breast cancers such normally repressed by the presence of these factors); and group that TEAD and SMAD transcription factors can cooperatively 4, induced by siTAZ/YAP and siTEADs but repressed by TGF facilitate TAZ/YAP-mediated tumorigenic activity. We found inhibition. The top five genes with altered expression in each that TEAD2 and TEAD4 associate with SMAD3, as well as YAP group are listed in Fig. 3A. Quantitative PCR analysis confirmed (Fig. 2A), and these interactions were unaffected by stimulation the respective knockdown of TAZ/YAP and TEADs knock- with a constitutively active TGF receptor (TGFR1-T240D down in each sample (Fig. 3B), as well as the expression changes (51)). Given that TAZ/YAP exhibit a predominantly nuclear observed from our microarray results for each group (Fig. 3, localization in LM2-4 cells (Fig. 2B), we speculated that TAZ/ C–F). Notable genes for group 1 included the following: neuro- YAPTEAD might be interacting with TGF-activated nal growth regulator 1 (NEGR1), urothelial cancer associated 1 SMAD2/3 to specify pro-tumorigenic transcriptional events. (UCA1), and CTGF. Elevated expression of the group 1 genes To acquire both protein interaction and localization informa- NEGR1, UCA1, and CTGF relied on the presence of TAZ/YAP, tion, we performed in situ PLA. PLA is a sensitive technique TEADs, and active TGF signaling (Fig. 3C), suggesting that used to visualize the localization and association of endogenous TAZ/YAPTEAD-TGF synergize to promote the expression protein complexes (proteins localized within 40 nm of each of these genes. In agreement with our observations, CTGF has MAY 9, 2014 • VOLUME 289 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13465 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 2. TAZ/YAP, TEADs, and SMAD2/3 interact endogenously. A, HEK293T cells expressing the indicated proteins were lysed and subjected to immu- noprecipitation (IP) with a FLAG antibody followed by immunoblotting with the indicated antibodies. B, LM2-4 cells were left untreated or treated with SB-431542 (SB)orTGF and examined by immunofluorescence for endogenous TAZ or YAP localization. C and D, LM2-4 cells left untreated or treated with TGF were probed with primary antibodies recognizing TAZ/YAP and SMAD2/3 (C), TEAD1 and TAZ (D), or TEAD1 and SMAD2/3 (E). In situ PLA followed by confocal microscopy were performed using mouse and rabbit secondary probes. Red dots indicate endogenous interactions, and nuclei were visualized with Hoechst stain. Representative images are shown, and three fields from each condition were quantitated, measuring the nuclear-cytoplasmic localization of the interactions and the number of interactions per nucleus. Black error bars either represent the average  S.E. or the average  S.E. recently been confirmed as an important transcriptional target gesting that although TAZ/YAPTEAD complexes synergize of YAPTEADSMAD complexes that promotes tumorigenesis with some TGF-mediated signals (group 1 and 3 targets), they in human malignant mesothelioma (31). NEGR1, UCA1, and repress others (group 2 and 4 targets). CTGF expression was abolished following TAZ/YAP or TEAD NEGR1 and UCA1 Are Direct Targets of TEADs and Are Nec- knockdown in the absence of TGF (Fig. 3C), suggesting that essary to Maintain Tumorigenic Breast Cancer Phenotypes— although specific TGF signals rely on TAZ/YAPTEAD, the Our analysis of LM2-4 cells indicates that TAZ/YAP, TEAD, basal level of TAZ/YAPTEAD activity does not require TGF, and TGF co-regulate the expression of a distinct subset of and therefore TAZ/YAPTEAD complexes may function dom- genes. To examine the importance of these genes in tumorigen- inantly to TGF signals. esis, we focused our attention on group 1 genes, as these are The group 2 genes we confirmed by qPCR included the fol- synergistically induced by TAZ/YAP, TEAD, and TGF and lowing: Occludin (OCLN) and cytoplasmic FMR1-interacting include CTGF, a defined mediator of TAZ/YAP-induced protein 2 (CYFIP2) (Fig. 3D). The group 3 genes confirmed tumorigenesis and cancer stem cell-like phenotypes (2, 31). The include the following: killer cell lectin-like receptor subfamily C top two genes synergistically induced by TAZ/YAPTEAD and protein (KLRC3) and serine palmitoyltransferase long chain TGF identified in our analysis were NEGR1 and UCA1. base subunit 3 (SPTLC3) (Fig. 3E). The group 4 genes we con- NEGR1 encodes a cell adhesion molecule that plays a role in firmed include the following: limb bud and heart development neuronal growth and development (53–59). UCA1 encodes a (LBH) and prostate transmembrane protein androgen-induced long noncoding RNA that is expressed in development, is 1(PMEPA1) (Fig. 3F). Notably, many genes were found to be turned off in homeostatic tissues, and has been found to be differentially regulated by TAZ/YAPTEADs and TGF, sug- highly expressed in bladder carcinomas (60). To determine 13466 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 19 • MAY 9, 2014 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 3. TAZ/YAP, TEADs, and TGF direct different and overlapping transcriptional events. A, LM2-4 cells were transfected with control siRNA (siCTL), siRNA targeting TAZ and YAP (siTAZ/YAP), or siRNA targeting all four TEADs(siTEAD1– 4), and then treated with TGF or SB-431542 TGF. RNA from cell lysates was harvested, and global gene expression profiles were examined using Affymetrix microarrays. The Venn diagram highlights the number of genes with significant expression changes occurring for the indicated condition relative to the siCTL  TGF sample. Hierarchical clustering was performed on the significantly changing genes, which revealed four major clusters as indicated. Top significantly changing genes of interest are highlighted in each of the four clustered groups. B–F, LM2-4 cells were transiently transfected with siCTL, siTAZ, siYAP, siTAZ/YAP, or siTEADs and treated with or without TGF or SB-431542 TGF. Relative expression of genes indicated in the microarray analysis was determined by qPCR. All data are shown as the average of three independent experiments S.E. B, confirmation of knockdown. C, group 1, genes repressed by siTAZ/YAP, siTEAD1– 4, and SB-431542 treatment. D, group 2, genes repressed by siTAZ/YAP and siTEAD1– 4 but induced by SB-431542 treatment. E, group 3, genes induced by siTAZ/YAP, siTEAD1– 4, and SB-431542 treatment. F, group 4, genes induced by siTAZ/YAP and siTEAD1– 4 but repressed by SB-431542 treatment. whether these are direct transcriptional targets of TAZ/YAP, of these experiments reflect our observations with TGF inhi- TEAD, and SMAD2/3, we performed chromatin immunopre- bition (SB-431542 treatment) or TAZ/YAP knockdown, sug- cipitation (ChIP). Examination of the promoter regions of gesting that cooperative regulation of NEGR1 and UCA1 NEGR1, UCA1, and CTGF revealed consensus TEAD binding expression by TAZ/YAPTEADSMAD complexes is necessary (61) and SMAD-binding motifs (62). ChIP of TAZ/YAP, TEAD, to promote tumorigenic phenotypes. and SMAD2/3 from LM2-4 cell lysates revealed enrichment of Nuclear TAZ and YAP Cooperate with TGF to Promote Phe- these factors at the NEGR1, UCA1, and CTGF promoters, with notypic and Transcriptional Changes in Nontumorigenic Cells— SMAD2/3 recruitment only apparent after TGF treatment Based on the results uncovered from our gene expression stud- (Fig. 4, A–C). ies, we decided to test whether ectopic expression of nuclear To further investigate the role of NEGR1 and UCA1 in TAZ/YAP in nontumorigenic human mammary MCF10A cells TGF-mediated tumorigenesis, we examined the conse- would lead to the induction of TGF-dependent transcrip- quences of reducing their expression following siRNA- tional events similar to those we characterized in the malignant mediated knockdown. Knockdown of NEGR1 or UCA1 LM2-4 cells. Stable expression of nuclear TAZ or YAP mutants repressed the migration of LM2-4 cells treated with TGF in can transform epithelial cells (2, 5, 7, 8), but this occurs follow- wound-healing scratch assays (Fig. 5A) and in transwell migra- ing weeks of stable selection. Similarly, treatment of cells with tion assays (Fig. 5B). Knockdown of either NEGR1 or UCA1 also TGF for several days to weeks is required to observe tumori- suppressed the ability of LM2-4 cells to form large mammo- genic events in mammary epithelial cells (30, 63). To prevent sphere colonies in the presence of TGF (Fig. 5C), consistent confounding issues with long term culture conditions, we gen- with pro-tumorigenic roles for NEGR1 and UCA1. The results erated MCF10A cells that express a nuclear-localized and sta- MAY 9, 2014 • VOLUME 289 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13467 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 4. NEGR1 and UCA1 are direct transcriptional targets of TAZ/YAP, TEADs, and SMADs. LM2-4 cells treated with TGF or SB-431542 (SB) were subjected to ChIP analysis using control rabbit IgG, TAZ/YAP, TEAD4, or SMAD2/3 antibodies. Samples were analyzed by qPCR using primers recognizing the indicated regions in the promoter of NEGR1 (A), UCA1 (B), or CTGF (C). Normalized values are shown as the average of three independent experiments  S.E. ble TAZ mutant (TAZ(4SA)) (7) or YAP mutant (YAP(5SA)) In accordance with our expression analysis of LM2-4 cells, (9) in a doxycycline-inducible manner. These TAZ/YAP we found that nuclear TAZ or YAP function in concert with mutants have the LATS kinase-induced phosphorylation sites TGF to control transcriptional events in MCF10A cells. For substituted to alanines, preventing their cytoplasmic sequestra- example, TAZ or YAP synergized with TGF to promote the tion and proteasomal degradation (7, 9). Titration of increasing transcription of group 1 genes in an inducible fashion, includ- amounts of doxycycline evoked subtle to high expression of ing the expression of NEGR1, UCA1, and CTGF (Fig. 6D). TAZ(4SA) or YAP(5SA) in these cells (Fig. 6A). High levels of Increased TAZ(4SA) or YAP(5SA) levels also induced the TAZ(4SA) or YAP(5SA) expression for short time frames (24 h) expression of group 2 genes (e.g. OCLN and CYFIP2), whereas had minimal effects on the morphology of these cells (Fig. 6B). TGF repressed this group of genes (Fig. 6E). Conversely, Short treatments of TGF led to flattening of cells (Fig. 6B), a group 4 genes, such as LBH and PMEPA1, were induced by morphology indicative of cells undergoing cell cycle arrest, as TGF but repressed in an inducible fashion by nuclear TAZ or has been described for MCF10A cells post-TGF treatment YAP (Fig. 6F). Intriguingly, group 3 genes were undetectable in (64). Strikingly, simultaneous doxycycline and TGF treatment MCF10A cells, which may reflect the more differentiated state led to rapid cell morphology changes that differed from either of these cells compared with LM2-4 cells. Together, our data condition alone, with the cells becoming more spindle-like and indicate that the relationship between TAZ/YAP and TGF is elongated (Fig. 6B). Furthermore, TAZ(4SA)- or YAP(5SA)- conserved in mammary-derived cells, and our observations expressing cells treated with TGF displayed much more rapid support the idea that dysregulated TAZ/YAP and TGF work cell migration in a wound-healing scratch assay, as compared in concert to control transcriptional events. with either condition alone (Fig. 6C), indicating that nuclear Nuclear TAZ and YAP Overcome TGF-induced Cytostasis TAZ/YAP synergize with TGF to promote cell morphology in Nontumorigenic Cells—A hallmark trait of TGF is its ability and cell migration changes. to suppress tumorigenesis in normal epithelium and early stage 13468 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 19 • MAY 9, 2014 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 5. NEGR1 and UCA1 are necessary for TGF-induced tumorigenic events. A, LM2-4 cells were transiently transfected with control siRNA (siCTL) or siRNA targeting NEGR1 (siNEGR1)or UCA1 (siUCA1) and treated with TGF. Monolayers were wounded and analyzed for cell migration. Representative images of three independent experiments are shown. B, LM2-4 cells transfected with siCTL, siNEGR1, or siUCA1 were plated on transwell filters to assess cell migration. Migrated cells are shown as the average number in 10 random fields over two independent experiments  S.E. C, LM2-4 cells were transfected with siCTL, siNEGR1, or siUCA1 and then grown under anchorage-independent conditions in the presence of TGF to examine primary mammosphere formation. Representative images are shown, and three independent experiments from each condition were quantitated, measuring the number of colonies formed and the size of each colony. Black error bars represent the average S.E., and red error bars represent the average S.E., **, p  0.005; ***, p  0.0001 (t test). cancers, particularly through cell cycle inhibition. However, cancer cells, such as clonal anchorage-independent growth, cell TGF signals lose their ability to induce cytostasis in late stage migration, and invasion. Interactions between endogenous cancers via poorly understood mechanisms (27, 28). TGF-in- TAZ/YAP, TEAD, and SMAD2/3 in the nucleus suggest that duced cell cycle arrest has been previously described in these complexes coordinate their activities at the transcrip- MCF10A cells (64), so we sought to explore the relationship tional level. Through genome-wide expression analysis, we between TGF, nuclear TAZ/YAP, and cell cycle progression. show that TAZ/YAP, TEAD, and TGF regulate individual and We performed proliferation assays using control MCF10A cells common gene targets both positively and negatively, implying a or cells with doxycycline-inducible nuclear TAZ(4SA) or complex level of transcriptional regulation and cross-talk YAP(5SA) expression. TGF-induced cytostasis was evident in between these factors. Of those gene targets we identified, control MCF10A cells (Fig. 7A). Strikingly, we found that many have yet to be characterized in breast cancer, and there- expression of TAZ(4SA) or YAP(5SA) overcomes TGF fore our work may highlight previously unrecognized factors growth arrest, as cells treated simultaneously with doxycycline contributing to tumorigenesis. Of note, epithelial-mesenchy- and TGF proliferated similarly to control cells (Fig. 7A). To mal transition-related genes were not enriched among the investigate whether the proliferative differences were due to overlapping TAZ/YAPTEAD-TGF-regulated subset, indi- cell cycle alterations, we used fluorescence-activated cell sort- cating that the TAZ/YAPTEADSMAD2/3 complex drives ing analysis (FACS) to examine the DNA content of these cells. aggressive behaviors of metastatic breast cancer cells down- We found that TGF treatment arrests cells in the G phase of stream from the loss of epithelial cell polarity. Our transcrip- the cell cycle, and that TAZ(4SA) or YAP(5SA) expression tional signature may thus reveal insight into the TAZ/YAP- reverses the G phase arrest (Fig. 7, B and C). Our data therefore mediated tumorigenic program occurring in late-stage cancers, suggest that nuclear TAZ/YAP are responsible for the switch in as MDA-MB-231 cells, and their LM2-4 derivatives possess activity from tumor-suppressive to tumorigenic in later TGF mesenchymal properties. Indeed, the two genes that we char- stage breast cancers by converging to direct a distinct transcrip- acterized, NEGR1 and UCA1, proved to be necessary for the tional program (see model in Fig. 8). anchorage-independent growth and migratory properties of DISCUSSION LM2-4 cells. TAZ/YAP and TGF synergistically induce the expression of NEGR1 and UCA1 (group 1 genes), and given that We have found TAZ/YAP to be necessary for transduction of TGF-induced tumorigenic phenotypes in metastatic breast TAZ/YAP, TEADs, and SMAD2/3 are enriched at the promot- MAY 9, 2014 • VOLUME 289 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13469 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 6. TAZ and YAP synergize with TGF to promote distinct morphological changes and gene transcription. A, doxycycline (Dox)-inducible MCF10A cells expressing 3FLAG-TAZ(4SA) or 3FLAG-YAP(5SA) were treated with increasing levels of doxycycline with or without TGF. Expression of TAZ or YAP was determined by immunoblotting along with GAPDH (loading control). B, doxycycline-inducible MCF10A control cells or cells expressing 3FLAG-TAZ(4SA) or 3FLAG-YAP(5SA) were treated with doxycycline with or without TGF and examined for cell morphology. Representative images of three independent experiments are shown. C, doxycycline-inducible MCF10A cells expressing 3FLAG-TAZ(4SA) or 3FLAG-YAP(5SA) were treated with or without doxycycline and/or TGF. Monolayers were wounded and analyzed for cell migration. Representative images are shown, and three independent experiments were quantitated. Error bars represent the average  S.E., *, p  0.05; **, p  0.01; ***, p  0.0005 (t test). D–F, doxycycline-inducible MCF10A cells expressing 3FLAG-TAZ(4SA) or 3FLAG-YAP(5SA) were treated with increasing levels of doxycycline with or without TGF. Relative expression of group 1 genes (D), group 2 genes (E), and group 4 genes (F) was analyzed by qPCR and is shown as the average of three independent experiments  S.E. ers of these genes, direct transcriptional synergy between TAZ/ and thus we propose that cross-talk between TAZ/YAPTEAD YAPTEADSMAD complexes likely promotes their expression and TGF signals demarcate a distinct local cellular environ- in breast cancer. ment that may promote a tumor-initiating niche. The well doc- Out of the 80 genes co-regulated by TAZ/YAP, TEAD, and umented TAZ/YAPTEAD target CTGF best highlights a TGF, 21 of them encode membrane proteins, several of which secreted factor that is cooperatively induced by TGF. CTGF is function as cell surface receptors, and 13 of them encode a well established target of TGF-activated SMAD2/3 tran- secreted proteins. The enrichment of such genes may reflect scription factors (67) but is also an important driver of TAZ/ important non-cell autonomous alterations that are regulated YAP-induced tumorigenic events (2, 13). We observe that by TAZ/YAPTEAD and TGF signals. Such signals are impor- CTGF expression relies on the presence of TAZ/YAP, TEADs, tant for the pro-tumorigenic activity of TAZ and YAP (65, 66), and TGF signaling, and nuclear TAZ or YAP mutants syner- 13470 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 19 • MAY 9, 2014 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 7. Nuclear TAZ and YAP overcome TGF-induced cell cycle arrest. A, doxycycline-inducible MCF10A control cells or cells expressing 3FLAG- TAZ(4SA) or 3FLAG-YAP(5SA) were treated with doxycycline (Dox) with or without TGF. Cells were counted over 6 days and graphed to determine their rate of proliferation. Cell number counts are shown as the average of three independent experiments S.E. B, doxycycline-inducible MCF10A control cells or cells expressing 3FLAG-TAZ(4SA) or 3FLAG-YAP(5SA) were treated with doxycycline with or without TGF. Cells were subject to propidium iodide staining and flow cytometry analysis to determine DNA content. Data from a representative experiment are shown. C, cell cycle phase quantitation from the data in B is represented as the ratio of cells in S  G to cells in G . The average of three independent experiments  S.E. is shown, *, p  0.015 (t test). 2 1 function to overcome the induced expression of this gene to sustain pro-tumorigenic TGF signals. Historically, TAZ and YAP have been considered to be acti- vators of gene transcription. However, our data indicate that TAZ/YAP play repressive roles as well (group 3 and 4 genes). We hypothesize TAZ/YAPTEAD complexes execute this repressive function by various means. Recent work has shown that TAZ/YAP recruit the nucleosome remodeling and deacetylation (NuRD) complex to repress gene expression (35). Yorkie (Yki), the homolog of TAZ/YAP in Drosophila melano- gaster, is also known to associate with chromatin-modifying proteins (69, 70). Thus, TAZ/YAPTEAD complexes likely function directly to inhibit transcription in breast cancers through similar recruitment of repressive factors to control local chromatin remodeling at promoters. However, TAZ/ YAPTEAD complexes may also function in an indirect man- FIGURE 8. Model for how TAZ/YAP direct TGF-induced tumorigenic events. We propose that increased nuclear TAZ/YAP, resulting from defects ner, particularly in conjunction with TGF signaling, by bind- in upstream Hippo pathway signals, overcome TGF-mediated tumor sup- ing, and re-localizing SMAD complexes (26, 32). SMAD pressive functions (e.g. cytostasis) and concomitantly drive tumorigenic tran- redistribution by TAZ/YAP may explain why nuclear TAZ or scriptional events by promoting the activity of TEADSMAD complexes. YAP affects the expression of certain target genes (group 2 and 4) more dramatically in MCF10A cells in the presence of TGF. gize with TGF to strongly induce CTGF expression. There- Moreover, TAZ/YAP binding to SMADs is evident in the fore, as in malignant mesotheliomas (31), the synergistic regu- nucleus and in the cytoplasm (Fig. 2C), suggesting that interac- lation of the CTGF promoter likely promotes aggressive breast tions between these proteins in different localizations may cancer phenotypes. direct distinct events. We have additionally identified genes that are activated by Of interest, nuclear TAZ or YAP is capable of overcoming both TAZ and YAP but repressed by TGF signaling (group 2 TGF-induced cytostasis (Fig. 7), which is a major mechanism genes) and, reciprocally, genes repressed by TAZ/YAP but by which TGF functions as a tumor suppressor in early stage induced by TGF (group 4 genes). These groups of genes were cancers (27). Consistent with this, we find that constitutively somewhat surprising as they indicate that TAZ/YAP and TGF nuclear TAZ/YAP is evident in breast cancer cell lines where direct opposing transcriptional events, and therefore suggest TGF has lost its ability to induce cytostatic signals (Fig. 1A). that a subset of TGF-activated SMAD activity does not rely on TAZ/YAP drive the expression of cell cycle regulators (6), TAZ/YAP and vice versa. Based on the products encoded by which may account for the ability of these factors to overcome several of these genes, we speculate that nuclear TAZ/YAP may override tumor-suppressive or negative feedback mechanisms cell cycle arrest. Indeed, our gene expression analysis in LM2-4 initiated by TGF. For example, PMEPA1, which we found is cells identified several cell cycle regulators as TAZ/YAP-regu- induced by TGF and inhibited by TAZ/YAP (group 4 gene), lated genes (e.g. CDKL1, CCNA1, CCNB1, and CCND3). How- encodes a transmembrane protein that sequesters SMAD com- ever, given that TAZ/YAP bind SMAD complexes, we also plexes in the cytoplasm (68). Thus, nuclear TAZ/YAP may speculate that TAZ/YAP may be capable of redirecting TGF- MAY 9, 2014 • VOLUME 289 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13471 Convergent TAZ/YAP-TGF Signals in Breast Cancer 36, 375–384 induced SMADs away from their cell cycle-repressive tran- 4. Wang, X., Su, L., and Ou, Q. (2012) Yes-associated protein promotes scriptional roles toward those that promote tumorigenesis. tumour development in luminal epithelial derived breast cancer. Eur. J. Our phenotypic and transcriptional analysis revealed redun- Cancer 48, 1227–1234 dant functions for TAZ and YAP. For example, TAZ and YAP 5. Chan, S. W., Lim, C. J., Guo, K., Ng, C. P., Lee, I., Hunziker, W., Zeng, Q., have redundant roles in mediating TGF-induced mammo- and Hong, W. (2008) A role for TAZ in migration, invasion, and tumori- sphere formation. Additionally, TAZ and YAP redundantly genesis of breast cancer cells. Cancer Res. 68, 2592–2598 6. Dong, J., Feldmann, G., Huang, J., Wu, S., Zhang, N., Comerford, S. A., regulate the expression of group 1 genes NEGR1 and UCA1 Gayyed, M. F., Anders, R. A., Maitra, A., and Pan, D. (2007) Elucidation of (Fig. 3C). Interestingly, TAZ knockdown alone led to increases a universal size-control mechanism in Drosophila and mammals. Cell in UCA1 expression, which may reflect compensatory YAP 130, 1120–1133 hyperactivity in this context. A redundant role for these factors 7. Lei, Q. Y., Zhang, H., Zhao, B., Zha, Z. Y., Bai, F., Pei, X. H., Zhao, S., Xiong, is further implied on account of similar effects resulting from Y., and Guan, K. L. (2008) TAZ promotes cell proliferation and epithelial- nuclear TAZ or YAP mutant expression in MCF10A cells. Such mesenchymal transition and is inhibited by the hippo pathway. Mol. Cell. Biol. 28, 2426–2436 redundancy is consistent with the overlapping roles of TAZ/ 8. Overholtzer, M., Zhang, J., Smolen, G. A., Muir, B., Li, W., Sgroi, D. C., YAP in early development (71). However, we also present evi- Deng, C. X., Brugge, J. S., and Haber, D. A. (2006) Transforming properties dence for divergent transcriptional activity, based on specific of YAP, a candidate oncogene on the chromosome 11q22 amplicon. Proc. gene expression reliance on either TAZ or YAP exclusively. For Natl. 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Dev. 26, 1300–1305 13474 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 19 • MAY 9, 2014 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

The Transcriptional Regulators TAZ and YAP Direct Transforming Growth Factor β-induced Tumorigenic Phenotypes in Breast Cancer Cells * ♦

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American Society for Biochemistry and Molecular Biology
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0021-9258
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10.1074/jbc.m113.529115
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 19, pp. 13461–13474, May 9, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. The Transcriptional Regulators TAZ and YAP Direct Transforming Growth Factor -induced Tumorigenic Phenotypes in Breast Cancer Cells Received for publication, October 21, 2013, and in revised form, March 9, 2014 Published, JBC Papers in Press, March 19, 2014, DOI 10.1074/jbc.M113.529115 Samantha E. Hiemer, Aleksander D. Szymaniak, and Xaralabos Varelas From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118 Background: The TGF and Hippo pathways are dysregulated in metastatic breast cancers. Results: TGF-induced cues and nuclear TAZ/YAP converge at the transcriptional level to control gene expression important for tumorigenesis. Conclusion: TAZ/YAP are required to promote TGF-induced tumorigenic phenotypes in breast cancer cells. Significance: Our study reveals novel cross-talk between the TGF pathway and TAZ/YAP in late-stage breast cancers. Uncontrolled transforming growth factor- (TGF) signaling age-independent conditions (5–9). Moreover, high nuclear promotes aggressive metastatic properties in late-stage breast TAZ levels induce cancer stem cell-like activity (2, 10) and pro- cancers. However, how TGF-mediated cues are directed to mote evasion of certain breast cancer drug therapies (2, 11). induce tumorigenic events is poorly understood, particularly Thus, understanding the roles of TAZ/YAP is critical for direct- given that TGF has clear tumor suppressing activity in other ing efficient breast cancer therapies. contexts. Here, we demonstrate that the transcriptional regula- The tumor initiating activity of TAZ/YAP relies on their tors TAZ and YAP (TAZ/YAP), key effectors of the Hippo path- binding to the TEAD family of transcription factors way, are necessary to promote and maintain TGF-induced (TEAD1–4) (10, 12, 13), indicating that together these factors tumorigenic phenotypes in breast cancer cells. Interactions direct a tumorigenic transcriptional program. Supporting this between TAZ/YAP, TGF-activated SMAD2/3, and TEAD premise, TAZ/YAPTEAD complexes directly promote the transcription factors reveal convergent roles for these factors in expression of genes encoding oncogenic factors, such as CTGF the nucleus. Genome-wide expression analyses indicate that (also known as CCN2) and CYR61 (also known as CCN1) (12, TAZ/YAP, TEADs, and TGF-induced signals coordinate a spe- 13), which contribute to human breast cancer progression (14). cific pro-tumorigenic transcriptional program. Importantly, Nuclear TAZ/YAP activity is highly regulated and governed in genes cooperatively regulated by TAZ/YAP, TEAD, and TGF, large part by the Hippo pathway-regulated LATS1 and LATS2 such as the novel targets NEGR1 and UCA1, are necessary for kinases (15). LATS1/2 kinases phosphorylate TAZ/YAP on maintaining tumorigenic activity in metastatic breast cancer conserved serine residues, which promote 14-3-3 binding and cells. Nuclear TAZ/YAP also cooperate with TGF signaling to subsequent sequestration in the cytoplasm (16, 17), and also promote phenotypic and transcriptional changes in nontumori- prime TAZ/YAP for further phosphorylation by CK1/ genic cells to overcome TGF-repressive effects. Our work thus kinases that evoke TAZ/YAP degradation via proteasome-de- identifies cross-talk between nuclear TAZ/YAP and TGF sig- pendent mechanisms (18, 19). Additional phosphorylation naling in breast cancer cells, revealing novel insight into late- stage disease-driving mechanisms. events destabilize TAZ, including those regulated by Wnt, phosphatidylinositol 3-kinase, and GSK3 (20, 21). Thus, dys- regulation of multiple upstream signals likely contributes to the Elevated nuclear levels of the transcriptional regulators TAZ aberrant nuclear TAZ/YAP activity that is observed in cancers. and YAP (TAZ/YAP) are associated with a broad range of TAZ/YAP modify the activity of other transcription factors aggressive cancers (1). For instance, the extent of nuclear TAZ besides TEADs, including the transforming growth factor- or YAP levels corresponds with breast cancer tumor grade (TGF)-activated SMAD complexes (22). TGF is the proto- (2–4). In breast cancer cells, enhanced nuclear TAZ and YAP typic member of a family of secreted factors that regulates levels promote oncogenic transformation and endow cells with numerous developmental and homeostatic processes (23). tumorigenic properties, including the ability to proliferate, sub- SMAD2 and SMAD3 (SMAD2/3) are the primary mediators of vert apoptotic cues, migrate, invade, and grow under anchor- TGF-induced transcription. SMAD2/3 are phosphorylated by TGF-bound membrane receptors, which induce binding to * This work was supported in part by funds from the Concern Cancer Foun- SMAD4 (24, 25), forming active transcriptional complexes that dation, the Karin Grunebaum Cancer Research Foundation, and Research accumulate in the nucleus upon binding to TAZ/YAP (26). In Grant 5-FY11-578 from the March of Dimes Foundation. cancer, the role of TGF is complex, as it can suppress early This article was selected as a Paper of the Week. □ S This article contains supplemental Tables S1–S4. oncogenic events but also promote aggressive late-stage meta- To whom correspondence should be addressed: Dept. of Biochemistry, Bos- static phenotypes (27, 28). What mechanistically distinguishes ton University School of Medicine, Rm. K225, Boston, MA 02118. Tel.: 617- 638-4182; Fax: 617-638-5339; E-mail: [email protected]. the different TGF-dependent responses is poorly understood. This is an open access article under the CC BY license. MAY 9, 2014 • VOLUME 289 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13461 Convergent TAZ/YAP-TGF Signals in Breast Cancer Several lines of evidence indicate that TGF, like TAZ/YAP, 0.1% Triton X-100/PBS, blocked in 2% BSA/PBS, and probed promotes aggressive tumorigenic properties in late-stage breast with primary and secondary antibodies outlined in supplemen- carcinomas (29, 30). Given that TAZ/YAP bind to SMAD tran- tal Table S2. LM2-4 cells were treated with or without TGF1 scription factors and direct TGF signaling in other contexts (500 pM, R&D Systems) or SB-431542 (5 M, Sigma) for 24 h (26, 31, 32), we sought to characterize whether TAZ/YAP before fixing. For the PLA, LM2-4 cells were plated on 96-well define TGF-mediated tumorigenic cues in breast cancer cells. microplates (Falcon) and treated with or without TGF1 for Our observations indicate that TGF-induced tumorigenic 24 h. Cells were fixed and permeabilized as described, blocked events, such as increased cell migration, invasion, and anchor- according to the manufacturer’s protocol (Duolink), and age-independent growth, require TAZ/YAP. Our data also probed with the primary antibodies outlined in supplemental indicate that, like TAZ/YAP, the TEAD transcription factors Table S2. Anti-mouse MINUS and anti-rabbit PLUS PLA interact with TGF-induced SMAD2/3 in the nucleus, suggest- probes (Duolink) were used. Nuclei were stained with Hoechst. ing that TAZ/YAPTEADSMAD2/3 complexes coordinate All immunofluorescence was visualized by confocal micros- transcriptional events in a concerted manner. Genome-wide copy (LSM 700), and images were processed using Volocity microarray analysis of gene expression changes that occur upon software (PerkinElmer Life Sciences). Images were quantitated knockdown of TAZ/YAP or TEADs, or inhibition of TGF sig- using ImageJ software. naling, revealed that TAZ/YAP, TEAD, and TGF regulate Mammospheres—LM2-4 cells were transfected with siRNA, overlapping target genes. Interestingly, the direct gene targets dissociated 24 h later, and resuspended in Mammary Epithe- NEGR1 and UCA1, which are synergistically regulated by TAZ/ lium Growth Medium (MEGM; Lonza) supplemented with B27 YAP, TEAD, and TGF, are necessary for maintaining tumor- (Invitrogen), 20 ng/ml epidermal growth factor (EGF; Pepro- igenic activity in metastatic breast cancer cells, suggesting that Tech), 20 ng/ml basic fibroblast growth factor (bFGF; Pepro- the convergence of TAZ/YAPTEAD-TGF signals is critical Tech). Single cells were seeded at 5  10 cells/ml in 6-well for driving late-stage breast cancer phenotypes. Supporting this ultra-low attachment plates (Corning Glass) and treated with premise, expression of nuclear-localized TAZ or YAP mutants or without TGF1 or SB-431542. Primary spheres were photo- direct transcriptional events that sensitize untransformed graphed after 7 days and either lysed for RNA (Quick-RNA breast cancer cells to adopt tumorigenic phenotypes in MiniPrep, Zymo Research) to examine knockdown or dissoci- response to TGF, while also suppressing TGF-induced cyto- ated in 0.05% trypsin for 10 min and resuspended as single cells stasis. These findings reveal novel cross-talk between TGF in MEGM for passage. Secondary spheres were photographed and Hippo signaling that we propose is important for late stage after an additional 14 days. Images were analyzed using ImageJ tumorigenic events in breast cancer. software, and statistics were calculated using Prism software (GraphPad) using a two-tailed unpaired Student’s t test. EXPERIMENTAL PROCEDURES Immunoprecipitation and Immunoblots—LM2-4 cells exam- Cell Culture, Plasmids, and Transfections—MCF10A, MCF- ined for endogenous protein expression were treated with or 12A, HMLE, and MCF7 cells were cultured using DMEM/F-12 without TGF1 or SB-431542 for 2 h and were lysed and exam- media (1:1) supplemented with 5% horse serum, 20 ng/ml ined by immunoblotting. Transfected HEK293T cells express- epithelial growth factor (EGF; PeproTech), 0.5 g/ml hydro- ing the indicated proteins were lysed, subjected to immunopre- cortisone (Sigma), 100 ng/ml cholera toxin (Sigma), 10 g/ml cipitation using anti-FLAG-conjugated protein-G beads insulin (Sigma). MDA-MB-231 (MDA-231) and MDA-MB- (Sigma), and analyzed by immunoblotting. MCF10A doxycy- 231-LM2-4 (LM2-4) cells were cultured using RPMI media cline-inducible cells were treated with or without doxycycline supplemented with 10% FBS. SUM-149 cells were cultured (0.1 to 100 ng/ml) or TGF1 for 24 h and were lysed and exam- using Ham’s F-12 media supplemented with 5% FBS, 10 g/ml ined by immunoblotting. Antibodies are outlined in supple- insulin (Sigma), 0.5 g/ml hydrocortisone (Sigma). BT20, mental Table S2. HS578T, SKBR3, and HEK293T cells were cultured using Cell Morphology Analysis, Wound Healing, and Transwell DMEM supplemented with 10% FBS. HEK293T cells were Migration—Low density MCF10A doxycycline-inducible cells transfected using TurboFect (Thermo Scientific) according to were pretreated with doxycycline (100 ng/ml, Clontech) for the manufacturer’s protocol. MCF10A doxycycline-inducible 24 h and then treated with or without TGF1 for an additional stable cell lines were generated using the lentiviral Tet-On sys- 24 h. For the wound-healing scratch assays, LM2-4 cells were tem (Clontech). 3FLAG-tagged mutants of TAZ (4SA: S66A, transfected with siRNA and 24 h later were treated with or S89A, S117A, and S311A) or YAP (5SA: S61A, S109A, S127A, without TGF1 or SB-431542 for an additional 24 h. MCF10A S164A, and S397A) were generated by site-directed mutagene- doxycycline-inducible cells were treated with or without doxy- sis and cloned into the pLVX-Tight-Puro plasmid (catalog no. cycline or TGF1 for 24 h. Monolayers were wounded and pho- 632162, Clontech). Tet-On cells were selected with 1 mg/ml tographed after an additional 24 h (LM2-4) or 12 h (MCF10A). G-418 sulfate (Gold Biotechnology) and 1 g/ml puromycin Images were analyzed using ImageJ software, and statistics (American Bioanalytical). RNA interference was performed by were calculated using Prism software (GraphPad) using a two- transfecting siRNA using Dharmafect 1 (Thermo Scientific) tailed unpaired Student’s t test. Cells used in the transwell assay according to manufacturer’s protocol. Sequences for the were transfected with siRNA, trypsinized 24 h later, and resus- siRNAs used are outlined in supplemental Table S1. Immunofluorescence and Proximity Ligation Assay (PLA)— The abbreviations used are: PLA, proximity ligation assay; qPCR, quantita- Cells were fixed with 4% paraformaldehyde, permeabilized with tive real time PCR; OCLN, occludin. 13462 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 19 • MAY 9, 2014 Convergent TAZ/YAP-TGF Signals in Breast Cancer pended in low serum media (0.25% FBS). Cells were plated at enode). Immunoprecipitations were performed using antibod- cells/ml on 0.4-M transwell filters (BD Biosciences) pre- ies outlined in supplemental Table S2 (note: anti-TEAD4 also treated for 24 h with 1 g/ml fibronectin (Millipore). Media  recognizes TEAD1 and -3 (35)) followed by incubation with 10% FBS were used in the bottom chamber. Cells were allowed protein-G Dynabeads (Invitrogen), and then washing sequen- to migrate for 24 h in the presence of TGF1 and were subse- tially in buffer A (20 mM Tris/HCl, pH 7.6, 140 mM NaCl, 1 mM quently stained with 0.5% crystal violet. EDTA, pH 8.0, 0.1% sodium deoxycholate, 0.1% SDS, 1% Triton Three-dimensional Invasion—Stable knockdown of TAZ and X-100), buffer B (20 mM Tris/HCl, pH 7.6, 500 mM NaCl, 1 mM YAP in LM2-4 cells was accomplished by lentivirus-mediated EDTA, pH 8.0, 0.5% sodium deoxycholate, 1% Triton X-100), transduction of shRNA using the pLKO1-puro and pLKO1-neo buffer C (20 mM Tris/HCl, pH 7.6, 1 mM EDTA, pH 8.0, 0.5% vectors and subsequent selection with 2 g/ml puromycin and sodium deoxycholate, 1% Triton X-100, 250 mM LiCl), and 1.5 mg/ml G418. The shRNA sequences used are listed in sup- TBS. Samples were eluted in Elution buffer (50 mM NaHCO , plemental Table S1. Single cells were plated on 100% growth 50 mM Tris/HCl, pH 8.0, 2 mM EDTA, pH 8.0, 1% SDS). Cross- factor-reduced Matrigel (BD Biosciences) using the overlay links were reversed overnight at 65 °C in 0.2 M NaCl in Elution method (33). Assay media contained 2% Matrigel added to sup- buffer, and DNA was purified using QIAquick PCR purification plemented MEGM, and cells were cultured with puromycin columns (Qiagen). Samples were then analyzed by qPCR using and G418 with medium changes every 3 days. TGF1 and the primers outlined in supplemental Table S3. SB-431542 were added after 9 days and then cultured for an Cell Proliferation and Cell Cycle Analysis—MCF10A doxycy- additional 3 days before being photographed. cline-inducible cells were plated (5 10 cells) and treated with Microarrays—LM2-4 cells were transfected with control doxycycline with or without TGF1 (day 0). Cells were counted siRNA or siRNAs targeting TAZ/YAP or all four TEADs (out- each day for 6 consecutive days (day 1–6). For cell cycle analy- lined in supplemental Table S1) and were treated 24 h later sis, MCF10A doxycycline-inducible cells were treated with with 1 or SB-431542 for an additional 24 h. Total RNA doxycycline with or without TGF1 for 48 h. 1 10 TGF cells were was isolated and purified by Quick-RNA MiniPrep (Zymo fixed overnight in 100% ethanol and stained using 50 g/ml Research). Twelve microarrays in total were performed, with propidium iodide (Sigma) and 100 g/ml RNase A (Sigma). each condition carried out three times on separate days. The Samples were acquired on the FACScan (BD Biosciences), col- Boston University Microarray Core generated the data using lecting 1  10 events, and analyzed using FlowJo software the Affymetrix Human Gene 1.0 St Array, which covers 27,300 (Tree Star). Statistical analysis was conducted using a two- probe sets. The data were filtered using a moderated p value of tailed unpaired Student’s t test. less than 0.01, and the average fold change in expression of each RESULTS gene, for each condition, relative to the siCTL  TGF sample was calculated. Fold expression changes relative to siCTL  Nuclear TAZ/YAP Are Required to Promote TGF-induced TGF-treated cells were calculated, and statistical significance Tumorigenic Phenotypes in Breast Cancer Cells—In cancer, the was assessed using a moderated t test and p values. Hierarchical role of TGF is complex, as it can suppress early oncogenic gene clustering was performed on overlapping genes displaying events, such as cell cycle progression, but can also promote a p value of 0.01 with the open source program Cluster 3.0 late-stage metastatic phenotypes (27, 28). What distinguishes (34). these different TGF-dependent responses is poorly under- Quantitative Real Time PCR (qPCR)—LM2-4 cells were stood. Several lines of evidence indicate that nuclear TAZ/YAP, transfected with siRNA and were treated 24 h later with or like TGF, induce tumorigenic properties in late-stage breast without TGF1 or SB-431542 for an additional 24 h. MCF10A carcinomas (29, 30). In untransformed mammary epithelium, doxycycline-inducible cells were treated with or without doxy- TAZ/YAP localization is restricted to the cytoplasm by cell cycline (0.1 to 100 ng/ml) or TGF1 for 24 h. Total RNA was compaction/polarity-regulated cues (9, 32). Dysregulation of purified using Quick-RNA MiniPrep kit, and cDNA synthesis cell polarity cues, which is a hallmark of cancer progression was performed using 1 g RNA and iScript cDNA synthesis kit (36), induces nuclear TAZ/YAP localizations. Given our prior (Bio-Rad) according to manufacturer’s protocol. qPCR was per- work showing that TAZ/YAP bind to and regulate the localiza- formed using Fast SYBR Green enzyme (Applied Biosystems) tion and activity of TGF-activated SMAD transcription and measured on ViiA 7 real time PCR system (Applied Biosys- factors (26, 32), we sought to test whether TAZ and/or YAP tems). Transcript levels were analyzed using the C method promote TGF-induced tumorigenic events. We began our and normalized to GAPDH. Primer sequences are indicated in analysis by examining the relationship between TAZ and YAP supplemental Table S3. localizations and the TGF-induced cytostatic response in a Chromatin Immunoprecipitation (ChIP)—LM2-4 cells were panel of mammary epithelial and breast cancer cell lines. Based fixed with 1 mM EGS (Thermo Scientific) for 30 min, 1% form- on published data, we divided the panel into cells that are aldehyde for 10 min, and quenched in 0.125 M glycine in PBS. responsive to TGF-induced cytostasis (MCF10A, BT20, Cells were collected and lysed in Cell Lysis buffer (10 mM KOH/ HMLE, HS578T, MCF7, and MCF-12) and cells in which TGF HEPES, pH 7.8, 85 mM KCl, 1 mM EDTA, pH 8.0, 1% Nonidet induces pro-tumorigenic signals but not growth arrest (MDA- P-40) with a protease inhibitor mixture. Nuclei were lysed in MB-231, MDA-MB-231-LM2-4, SKRB3, and SUM149) (37– Nuclear Lysis buffer (50 mM Tris/HCl, pH 7.4, 1% SDS, 10 mM 44). Interestingly, we observed that cells displaying high levels EDTA, pH 8.0) with protease inhibitors, and genomic DNA was of nuclear TAZ/YAP correlate with those in which TGF fragmented to 400 bp using Bioruptor bath sonicator (Diag- induces tumorigenic cues (Fig. 1A). MAY 9, 2014 • VOLUME 289 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13463 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 1. TAZ/YAP are required for TGF-induced tumorigenic events. A, panel of breast cancer cell lines was divided by TGF-induced tumor suppression and promoting responses and examined by immunofluorescence for endogenous TAZ and YAP localization. B, LM2-4 cells were transiently transfected with control siRNA (siCTL) or siRNA targeting TAZ (siTAZ), YAP (siYAP), or TAZ and YAP (siTAZ/YAP). Cells were left untreated, treated with TGF or SB-431542 TGF, and grown in anchorage-independent conditions. Primary mammospheres were examined for knockdown or were passaged into secondary spheres. Sec- ondary mammospheres following SB-431542 (SB) treatment, or transfection with siTAZ, siYAP, or siTAZ/YAP, were unable to be determined due to low numbers. Representative images are shown, and three independent experiments from each condition were quantitated, measuring the number of colonies formed and the size of each colony. Black error bars represent the average S.E., and red error bars represent the average S.E., *, p 0.025; **, p 0.005; ***, p  0.0001 (t test). C, LM2-4 cell lysates were immunoblotted to examine endogenous levels of the indicated proteins upon TGF or SB-431542 treatment compared with GAPDH (loading control). D, LM2-4 cells were transiently transfected with siCTL or siTAZ/YAP. Cells were left untreated, treated with TGF,or SB-431542 TGF. Monolayers were wounded and analyzed for cell migration. E, LM2-4 cells stably expressing control shRNA (shCTL) or shRNA targeting TAZ and YAP (shTAZ/YAP) were treated with TGF or SB-431542  TGF and incubated in three-dimensional Matrigel culture conditions. Representative images from three independent experiments are shown. To further investigate this relationship, we sought to deter- mospheres under anchorage-independent conditions (Fig. 1B), mine the roles of nuclear TAZ/YAP in the human MDA-MB- which is often used as a measure of the self-renewing potential 231-LM2-4 (herein referred to as LM2-4) metastatic breast of tumorigenic cells in vitro (47). TGF treatment of LM2-4 cancer cell line (45), a highly aggressive derivative of triple- cells led to dramatic increases in the number and size of mam- negative basal subtype MDA-MB-231 cells (46). A fraction of mospheres observed (Fig. 1B), similar to that observed with LM2-4 cells in culture are capable of generating clonal mam- TGF treatment of other mammary cells (30). The self-renew- 13464 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 19 • MAY 9, 2014 Convergent TAZ/YAP-TGF Signals in Breast Cancer ing properties of the cells within the mammospheres were other) by microscopy (52). Using PLA, we observed TAZ/ assessed by their ability to form secondary clonal spheres (47), YAPSMAD2/3 interactions in both the nucleus and cytoplasm and we found that TGF also promoted secondary mammo- of untreated LM2-4 cells (Fig. 2C). Upon TGF treatment, sphere formation. Co-treatment of the cells with the TGF nuclear TAZ/YAP-SMAD2/3 binding became much more receptor antagonist SB-431542 abolished the formation of pri- apparent in the nucleus (Fig. 2C), consistent with nuclear TAZ/ mary mammospheres, validating that the observed effects are YAPSMAD2/3 complexes directing transcriptional events (26, indeed generated via canonical TGF receptor-mediated sig- 32). We also detected endogenous TAZ/TEAD1 interactions in nals (Fig. 1B) (48, 49). As expected, SB-431542 treatment elim- the nucleus of LM2-4 cells with or without TGF stimulation inated the TGF-induced phosphorylation of SMAD2 and (Fig. 2D), which were increased slightly upon TGF treatment SMAD3 in these cells (Fig. 1C). Individual TAZ or YAP knock- (Fig. 2D). TEAD1SMAD2/3 interactions were readily detected down also repressed the number and size of TGF-induced in the nucleus of LM2-4 cells, particularly after TGF treat- mammospheres (Fig. 1B). However, simultaneous knockdown ment (Fig. 2E), suggesting these complexes stabilize upon of both TAZ and YAP dramatically reduced mammosphere for- nuclear accumulation of SMADs. Taken together, our observa- mation (Fig. 1B), indicating redundant roles for TAZ and YAP tions indicate that TAZ/YAP, TEAD, and SMAD interact in transducing TGF-mediated cues required for anchorage- in TGF-stimulated metastatic breast cancer cells and suggest independent growth. that they may form transcriptional complexes that function We further investigated other hallmark tumorigenic proper- together in the nucleus. ties that may be mediated by TGF and TAZ/YAP in metastatic To explore the possible overlap in transcriptional activity breast cancers, including cell migration and invasion (36). We by TAZ/YAP, TEAD, and SMAD complexes, we used microar- found that treatment of LM2-4 cells with TGF led to increases rays to compare the global expression profiles of LM2-4 cells in cell migration in an in vitro wound-healing scratch assay (Fig. treated as follows (complete data available in supplemental 1D), similar to previous work (50). As expected, co-treatment Table S4): 1) transfected with control siRNA (siCTL) and with the TGF receptor antagonist SB-431542 blocked TGF- treated with TGF; 2) transfected with siRNA targeting both induced cell migration (Fig. 1D). Simultaneous knockdown of TAZ/YAP (siTAZ/YAP) and treated with TGF; 3) transfected TAZ/YAP using siRNA also abolished TGF-induced LM2-4 with siRNA targeting all four TEAD (TEAD1–4) family mem- cell migration (Fig. 1D). Similarly, SB-431542 treatment or bers (siTEAD) and treated with TGF; and 4) transfected with shRNA-mediated TAZ/YAP knockdown abolished the ability control siRNA (siCTL) and treated simultaneously with TGF of three-dimensional colonies of LM2-4 cells to invade into the and SB-431542. In terms of significant gene expression differ- surrounding Matrigel matrix in the presence of TGF (Fig. 1E). ences (p value 0.01) relative to siCTL TGF treatment, 461 Taken together, our observations indicate that TAZ/YAP are genes overlapped between siTAZ/YAP and siTEAD conditions critical mediators of TGF-induced tumorigenic events, (Fig. 3A). This gene set displayed a high degree of correlation in including mammosphere formation, cell migration, and expression (R  0.86). The expression of 594 genes changed invasion. following SB-431542 treatment, and of these, 176 genes over- TAZ/YAP, TEADs, and SMADs Converge to Regulate a lapped with siTAZ/YAP conditions. Of these 176 genes, 80 TGF-induced Transcriptional Program in Breast Cancer Cells— were also altered following TEAD knockdown (Fig. 3A). Studies indicate that TAZ/YAP-induced cell transformation Interestingly, genes for which expression was altered among relies on the recruitment of TAZ/YAP to DNA by the TEAD all three experimental conditions exhibited distinct expression family of transcription factors (TEAD1–4) (12, 13). TAZ and correlations. Unbiased clustering segregated TAZ/YAPTEAD- YAP also bind TGF-activated SMAD complexes to control TGF-regulated genes into four different groups as follows: SMAD localization and activity in a variety of cell types, includ- group 1, repressed following siTAZ/YAP, siTEADs, or TGF ing mammary epithelial cells (26, 32). Recent work has shown inhibition (therefore normally induced by the presence of these that TAZ/YAPTEADSMAD2/3 complexes control transcrip- factors); group 2, repressed following siTAZ/YAP or siTEAD tional events important for maintaining human embryonic treatment but induced by TGF inhibition; group 3, induced stem cell pluripotency (35). Thus, we hypothesized that similar following siTAZ/YAP, siTEADs, or TGF inhibition (therefore complexes may also be present in late stage breast cancers such normally repressed by the presence of these factors); and group that TEAD and SMAD transcription factors can cooperatively 4, induced by siTAZ/YAP and siTEADs but repressed by TGF facilitate TAZ/YAP-mediated tumorigenic activity. We found inhibition. The top five genes with altered expression in each that TEAD2 and TEAD4 associate with SMAD3, as well as YAP group are listed in Fig. 3A. Quantitative PCR analysis confirmed (Fig. 2A), and these interactions were unaffected by stimulation the respective knockdown of TAZ/YAP and TEADs knock- with a constitutively active TGF receptor (TGFR1-T240D down in each sample (Fig. 3B), as well as the expression changes (51)). Given that TAZ/YAP exhibit a predominantly nuclear observed from our microarray results for each group (Fig. 3, localization in LM2-4 cells (Fig. 2B), we speculated that TAZ/ C–F). Notable genes for group 1 included the following: neuro- YAPTEAD might be interacting with TGF-activated nal growth regulator 1 (NEGR1), urothelial cancer associated 1 SMAD2/3 to specify pro-tumorigenic transcriptional events. (UCA1), and CTGF. Elevated expression of the group 1 genes To acquire both protein interaction and localization informa- NEGR1, UCA1, and CTGF relied on the presence of TAZ/YAP, tion, we performed in situ PLA. PLA is a sensitive technique TEADs, and active TGF signaling (Fig. 3C), suggesting that used to visualize the localization and association of endogenous TAZ/YAPTEAD-TGF synergize to promote the expression protein complexes (proteins localized within 40 nm of each of these genes. In agreement with our observations, CTGF has MAY 9, 2014 • VOLUME 289 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13465 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 2. TAZ/YAP, TEADs, and SMAD2/3 interact endogenously. A, HEK293T cells expressing the indicated proteins were lysed and subjected to immu- noprecipitation (IP) with a FLAG antibody followed by immunoblotting with the indicated antibodies. B, LM2-4 cells were left untreated or treated with SB-431542 (SB)orTGF and examined by immunofluorescence for endogenous TAZ or YAP localization. C and D, LM2-4 cells left untreated or treated with TGF were probed with primary antibodies recognizing TAZ/YAP and SMAD2/3 (C), TEAD1 and TAZ (D), or TEAD1 and SMAD2/3 (E). In situ PLA followed by confocal microscopy were performed using mouse and rabbit secondary probes. Red dots indicate endogenous interactions, and nuclei were visualized with Hoechst stain. Representative images are shown, and three fields from each condition were quantitated, measuring the nuclear-cytoplasmic localization of the interactions and the number of interactions per nucleus. Black error bars either represent the average  S.E. or the average  S.E. recently been confirmed as an important transcriptional target gesting that although TAZ/YAPTEAD complexes synergize of YAPTEADSMAD complexes that promotes tumorigenesis with some TGF-mediated signals (group 1 and 3 targets), they in human malignant mesothelioma (31). NEGR1, UCA1, and repress others (group 2 and 4 targets). CTGF expression was abolished following TAZ/YAP or TEAD NEGR1 and UCA1 Are Direct Targets of TEADs and Are Nec- knockdown in the absence of TGF (Fig. 3C), suggesting that essary to Maintain Tumorigenic Breast Cancer Phenotypes— although specific TGF signals rely on TAZ/YAPTEAD, the Our analysis of LM2-4 cells indicates that TAZ/YAP, TEAD, basal level of TAZ/YAPTEAD activity does not require TGF, and TGF co-regulate the expression of a distinct subset of and therefore TAZ/YAPTEAD complexes may function dom- genes. To examine the importance of these genes in tumorigen- inantly to TGF signals. esis, we focused our attention on group 1 genes, as these are The group 2 genes we confirmed by qPCR included the fol- synergistically induced by TAZ/YAP, TEAD, and TGF and lowing: Occludin (OCLN) and cytoplasmic FMR1-interacting include CTGF, a defined mediator of TAZ/YAP-induced protein 2 (CYFIP2) (Fig. 3D). The group 3 genes confirmed tumorigenesis and cancer stem cell-like phenotypes (2, 31). The include the following: killer cell lectin-like receptor subfamily C top two genes synergistically induced by TAZ/YAPTEAD and protein (KLRC3) and serine palmitoyltransferase long chain TGF identified in our analysis were NEGR1 and UCA1. base subunit 3 (SPTLC3) (Fig. 3E). The group 4 genes we con- NEGR1 encodes a cell adhesion molecule that plays a role in firmed include the following: limb bud and heart development neuronal growth and development (53–59). UCA1 encodes a (LBH) and prostate transmembrane protein androgen-induced long noncoding RNA that is expressed in development, is 1(PMEPA1) (Fig. 3F). Notably, many genes were found to be turned off in homeostatic tissues, and has been found to be differentially regulated by TAZ/YAPTEADs and TGF, sug- highly expressed in bladder carcinomas (60). To determine 13466 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 19 • MAY 9, 2014 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 3. TAZ/YAP, TEADs, and TGF direct different and overlapping transcriptional events. A, LM2-4 cells were transfected with control siRNA (siCTL), siRNA targeting TAZ and YAP (siTAZ/YAP), or siRNA targeting all four TEADs(siTEAD1– 4), and then treated with TGF or SB-431542 TGF. RNA from cell lysates was harvested, and global gene expression profiles were examined using Affymetrix microarrays. The Venn diagram highlights the number of genes with significant expression changes occurring for the indicated condition relative to the siCTL  TGF sample. Hierarchical clustering was performed on the significantly changing genes, which revealed four major clusters as indicated. Top significantly changing genes of interest are highlighted in each of the four clustered groups. B–F, LM2-4 cells were transiently transfected with siCTL, siTAZ, siYAP, siTAZ/YAP, or siTEADs and treated with or without TGF or SB-431542 TGF. Relative expression of genes indicated in the microarray analysis was determined by qPCR. All data are shown as the average of three independent experiments S.E. B, confirmation of knockdown. C, group 1, genes repressed by siTAZ/YAP, siTEAD1– 4, and SB-431542 treatment. D, group 2, genes repressed by siTAZ/YAP and siTEAD1– 4 but induced by SB-431542 treatment. E, group 3, genes induced by siTAZ/YAP, siTEAD1– 4, and SB-431542 treatment. F, group 4, genes induced by siTAZ/YAP and siTEAD1– 4 but repressed by SB-431542 treatment. whether these are direct transcriptional targets of TAZ/YAP, of these experiments reflect our observations with TGF inhi- TEAD, and SMAD2/3, we performed chromatin immunopre- bition (SB-431542 treatment) or TAZ/YAP knockdown, sug- cipitation (ChIP). Examination of the promoter regions of gesting that cooperative regulation of NEGR1 and UCA1 NEGR1, UCA1, and CTGF revealed consensus TEAD binding expression by TAZ/YAPTEADSMAD complexes is necessary (61) and SMAD-binding motifs (62). ChIP of TAZ/YAP, TEAD, to promote tumorigenic phenotypes. and SMAD2/3 from LM2-4 cell lysates revealed enrichment of Nuclear TAZ and YAP Cooperate with TGF to Promote Phe- these factors at the NEGR1, UCA1, and CTGF promoters, with notypic and Transcriptional Changes in Nontumorigenic Cells— SMAD2/3 recruitment only apparent after TGF treatment Based on the results uncovered from our gene expression stud- (Fig. 4, A–C). ies, we decided to test whether ectopic expression of nuclear To further investigate the role of NEGR1 and UCA1 in TAZ/YAP in nontumorigenic human mammary MCF10A cells TGF-mediated tumorigenesis, we examined the conse- would lead to the induction of TGF-dependent transcrip- quences of reducing their expression following siRNA- tional events similar to those we characterized in the malignant mediated knockdown. Knockdown of NEGR1 or UCA1 LM2-4 cells. Stable expression of nuclear TAZ or YAP mutants repressed the migration of LM2-4 cells treated with TGF in can transform epithelial cells (2, 5, 7, 8), but this occurs follow- wound-healing scratch assays (Fig. 5A) and in transwell migra- ing weeks of stable selection. Similarly, treatment of cells with tion assays (Fig. 5B). Knockdown of either NEGR1 or UCA1 also TGF for several days to weeks is required to observe tumori- suppressed the ability of LM2-4 cells to form large mammo- genic events in mammary epithelial cells (30, 63). To prevent sphere colonies in the presence of TGF (Fig. 5C), consistent confounding issues with long term culture conditions, we gen- with pro-tumorigenic roles for NEGR1 and UCA1. The results erated MCF10A cells that express a nuclear-localized and sta- MAY 9, 2014 • VOLUME 289 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13467 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 4. NEGR1 and UCA1 are direct transcriptional targets of TAZ/YAP, TEADs, and SMADs. LM2-4 cells treated with TGF or SB-431542 (SB) were subjected to ChIP analysis using control rabbit IgG, TAZ/YAP, TEAD4, or SMAD2/3 antibodies. Samples were analyzed by qPCR using primers recognizing the indicated regions in the promoter of NEGR1 (A), UCA1 (B), or CTGF (C). Normalized values are shown as the average of three independent experiments  S.E. ble TAZ mutant (TAZ(4SA)) (7) or YAP mutant (YAP(5SA)) In accordance with our expression analysis of LM2-4 cells, (9) in a doxycycline-inducible manner. These TAZ/YAP we found that nuclear TAZ or YAP function in concert with mutants have the LATS kinase-induced phosphorylation sites TGF to control transcriptional events in MCF10A cells. For substituted to alanines, preventing their cytoplasmic sequestra- example, TAZ or YAP synergized with TGF to promote the tion and proteasomal degradation (7, 9). Titration of increasing transcription of group 1 genes in an inducible fashion, includ- amounts of doxycycline evoked subtle to high expression of ing the expression of NEGR1, UCA1, and CTGF (Fig. 6D). TAZ(4SA) or YAP(5SA) in these cells (Fig. 6A). High levels of Increased TAZ(4SA) or YAP(5SA) levels also induced the TAZ(4SA) or YAP(5SA) expression for short time frames (24 h) expression of group 2 genes (e.g. OCLN and CYFIP2), whereas had minimal effects on the morphology of these cells (Fig. 6B). TGF repressed this group of genes (Fig. 6E). Conversely, Short treatments of TGF led to flattening of cells (Fig. 6B), a group 4 genes, such as LBH and PMEPA1, were induced by morphology indicative of cells undergoing cell cycle arrest, as TGF but repressed in an inducible fashion by nuclear TAZ or has been described for MCF10A cells post-TGF treatment YAP (Fig. 6F). Intriguingly, group 3 genes were undetectable in (64). Strikingly, simultaneous doxycycline and TGF treatment MCF10A cells, which may reflect the more differentiated state led to rapid cell morphology changes that differed from either of these cells compared with LM2-4 cells. Together, our data condition alone, with the cells becoming more spindle-like and indicate that the relationship between TAZ/YAP and TGF is elongated (Fig. 6B). Furthermore, TAZ(4SA)- or YAP(5SA)- conserved in mammary-derived cells, and our observations expressing cells treated with TGF displayed much more rapid support the idea that dysregulated TAZ/YAP and TGF work cell migration in a wound-healing scratch assay, as compared in concert to control transcriptional events. with either condition alone (Fig. 6C), indicating that nuclear Nuclear TAZ and YAP Overcome TGF-induced Cytostasis TAZ/YAP synergize with TGF to promote cell morphology in Nontumorigenic Cells—A hallmark trait of TGF is its ability and cell migration changes. to suppress tumorigenesis in normal epithelium and early stage 13468 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 19 • MAY 9, 2014 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 5. NEGR1 and UCA1 are necessary for TGF-induced tumorigenic events. A, LM2-4 cells were transiently transfected with control siRNA (siCTL) or siRNA targeting NEGR1 (siNEGR1)or UCA1 (siUCA1) and treated with TGF. Monolayers were wounded and analyzed for cell migration. Representative images of three independent experiments are shown. B, LM2-4 cells transfected with siCTL, siNEGR1, or siUCA1 were plated on transwell filters to assess cell migration. Migrated cells are shown as the average number in 10 random fields over two independent experiments  S.E. C, LM2-4 cells were transfected with siCTL, siNEGR1, or siUCA1 and then grown under anchorage-independent conditions in the presence of TGF to examine primary mammosphere formation. Representative images are shown, and three independent experiments from each condition were quantitated, measuring the number of colonies formed and the size of each colony. Black error bars represent the average S.E., and red error bars represent the average S.E., **, p  0.005; ***, p  0.0001 (t test). cancers, particularly through cell cycle inhibition. However, cancer cells, such as clonal anchorage-independent growth, cell TGF signals lose their ability to induce cytostasis in late stage migration, and invasion. Interactions between endogenous cancers via poorly understood mechanisms (27, 28). TGF-in- TAZ/YAP, TEAD, and SMAD2/3 in the nucleus suggest that duced cell cycle arrest has been previously described in these complexes coordinate their activities at the transcrip- MCF10A cells (64), so we sought to explore the relationship tional level. Through genome-wide expression analysis, we between TGF, nuclear TAZ/YAP, and cell cycle progression. show that TAZ/YAP, TEAD, and TGF regulate individual and We performed proliferation assays using control MCF10A cells common gene targets both positively and negatively, implying a or cells with doxycycline-inducible nuclear TAZ(4SA) or complex level of transcriptional regulation and cross-talk YAP(5SA) expression. TGF-induced cytostasis was evident in between these factors. Of those gene targets we identified, control MCF10A cells (Fig. 7A). Strikingly, we found that many have yet to be characterized in breast cancer, and there- expression of TAZ(4SA) or YAP(5SA) overcomes TGF fore our work may highlight previously unrecognized factors growth arrest, as cells treated simultaneously with doxycycline contributing to tumorigenesis. Of note, epithelial-mesenchy- and TGF proliferated similarly to control cells (Fig. 7A). To mal transition-related genes were not enriched among the investigate whether the proliferative differences were due to overlapping TAZ/YAPTEAD-TGF-regulated subset, indi- cell cycle alterations, we used fluorescence-activated cell sort- cating that the TAZ/YAPTEADSMAD2/3 complex drives ing analysis (FACS) to examine the DNA content of these cells. aggressive behaviors of metastatic breast cancer cells down- We found that TGF treatment arrests cells in the G phase of stream from the loss of epithelial cell polarity. Our transcrip- the cell cycle, and that TAZ(4SA) or YAP(5SA) expression tional signature may thus reveal insight into the TAZ/YAP- reverses the G phase arrest (Fig. 7, B and C). Our data therefore mediated tumorigenic program occurring in late-stage cancers, suggest that nuclear TAZ/YAP are responsible for the switch in as MDA-MB-231 cells, and their LM2-4 derivatives possess activity from tumor-suppressive to tumorigenic in later TGF mesenchymal properties. Indeed, the two genes that we char- stage breast cancers by converging to direct a distinct transcrip- acterized, NEGR1 and UCA1, proved to be necessary for the tional program (see model in Fig. 8). anchorage-independent growth and migratory properties of DISCUSSION LM2-4 cells. TAZ/YAP and TGF synergistically induce the expression of NEGR1 and UCA1 (group 1 genes), and given that We have found TAZ/YAP to be necessary for transduction of TGF-induced tumorigenic phenotypes in metastatic breast TAZ/YAP, TEADs, and SMAD2/3 are enriched at the promot- MAY 9, 2014 • VOLUME 289 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13469 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 6. TAZ and YAP synergize with TGF to promote distinct morphological changes and gene transcription. A, doxycycline (Dox)-inducible MCF10A cells expressing 3FLAG-TAZ(4SA) or 3FLAG-YAP(5SA) were treated with increasing levels of doxycycline with or without TGF. Expression of TAZ or YAP was determined by immunoblotting along with GAPDH (loading control). B, doxycycline-inducible MCF10A control cells or cells expressing 3FLAG-TAZ(4SA) or 3FLAG-YAP(5SA) were treated with doxycycline with or without TGF and examined for cell morphology. Representative images of three independent experiments are shown. C, doxycycline-inducible MCF10A cells expressing 3FLAG-TAZ(4SA) or 3FLAG-YAP(5SA) were treated with or without doxycycline and/or TGF. Monolayers were wounded and analyzed for cell migration. Representative images are shown, and three independent experiments were quantitated. Error bars represent the average  S.E., *, p  0.05; **, p  0.01; ***, p  0.0005 (t test). D–F, doxycycline-inducible MCF10A cells expressing 3FLAG-TAZ(4SA) or 3FLAG-YAP(5SA) were treated with increasing levels of doxycycline with or without TGF. Relative expression of group 1 genes (D), group 2 genes (E), and group 4 genes (F) was analyzed by qPCR and is shown as the average of three independent experiments  S.E. ers of these genes, direct transcriptional synergy between TAZ/ and thus we propose that cross-talk between TAZ/YAPTEAD YAPTEADSMAD complexes likely promotes their expression and TGF signals demarcate a distinct local cellular environ- in breast cancer. ment that may promote a tumor-initiating niche. The well doc- Out of the 80 genes co-regulated by TAZ/YAP, TEAD, and umented TAZ/YAPTEAD target CTGF best highlights a TGF, 21 of them encode membrane proteins, several of which secreted factor that is cooperatively induced by TGF. CTGF is function as cell surface receptors, and 13 of them encode a well established target of TGF-activated SMAD2/3 tran- secreted proteins. The enrichment of such genes may reflect scription factors (67) but is also an important driver of TAZ/ important non-cell autonomous alterations that are regulated YAP-induced tumorigenic events (2, 13). We observe that by TAZ/YAPTEAD and TGF signals. Such signals are impor- CTGF expression relies on the presence of TAZ/YAP, TEADs, tant for the pro-tumorigenic activity of TAZ and YAP (65, 66), and TGF signaling, and nuclear TAZ or YAP mutants syner- 13470 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 19 • MAY 9, 2014 Convergent TAZ/YAP-TGF Signals in Breast Cancer FIGURE 7. Nuclear TAZ and YAP overcome TGF-induced cell cycle arrest. A, doxycycline-inducible MCF10A control cells or cells expressing 3FLAG- TAZ(4SA) or 3FLAG-YAP(5SA) were treated with doxycycline (Dox) with or without TGF. Cells were counted over 6 days and graphed to determine their rate of proliferation. Cell number counts are shown as the average of three independent experiments S.E. B, doxycycline-inducible MCF10A control cells or cells expressing 3FLAG-TAZ(4SA) or 3FLAG-YAP(5SA) were treated with doxycycline with or without TGF. Cells were subject to propidium iodide staining and flow cytometry analysis to determine DNA content. Data from a representative experiment are shown. C, cell cycle phase quantitation from the data in B is represented as the ratio of cells in S  G to cells in G . The average of three independent experiments  S.E. is shown, *, p  0.015 (t test). 2 1 function to overcome the induced expression of this gene to sustain pro-tumorigenic TGF signals. Historically, TAZ and YAP have been considered to be acti- vators of gene transcription. However, our data indicate that TAZ/YAP play repressive roles as well (group 3 and 4 genes). We hypothesize TAZ/YAPTEAD complexes execute this repressive function by various means. Recent work has shown that TAZ/YAP recruit the nucleosome remodeling and deacetylation (NuRD) complex to repress gene expression (35). Yorkie (Yki), the homolog of TAZ/YAP in Drosophila melano- gaster, is also known to associate with chromatin-modifying proteins (69, 70). Thus, TAZ/YAPTEAD complexes likely function directly to inhibit transcription in breast cancers through similar recruitment of repressive factors to control local chromatin remodeling at promoters. However, TAZ/ YAPTEAD complexes may also function in an indirect man- FIGURE 8. Model for how TAZ/YAP direct TGF-induced tumorigenic events. We propose that increased nuclear TAZ/YAP, resulting from defects ner, particularly in conjunction with TGF signaling, by bind- in upstream Hippo pathway signals, overcome TGF-mediated tumor sup- ing, and re-localizing SMAD complexes (26, 32). SMAD pressive functions (e.g. cytostasis) and concomitantly drive tumorigenic tran- redistribution by TAZ/YAP may explain why nuclear TAZ or scriptional events by promoting the activity of TEADSMAD complexes. YAP affects the expression of certain target genes (group 2 and 4) more dramatically in MCF10A cells in the presence of TGF. gize with TGF to strongly induce CTGF expression. There- Moreover, TAZ/YAP binding to SMADs is evident in the fore, as in malignant mesotheliomas (31), the synergistic regu- nucleus and in the cytoplasm (Fig. 2C), suggesting that interac- lation of the CTGF promoter likely promotes aggressive breast tions between these proteins in different localizations may cancer phenotypes. direct distinct events. We have additionally identified genes that are activated by Of interest, nuclear TAZ or YAP is capable of overcoming both TAZ and YAP but repressed by TGF signaling (group 2 TGF-induced cytostasis (Fig. 7), which is a major mechanism genes) and, reciprocally, genes repressed by TAZ/YAP but by which TGF functions as a tumor suppressor in early stage induced by TGF (group 4 genes). These groups of genes were cancers (27). Consistent with this, we find that constitutively somewhat surprising as they indicate that TAZ/YAP and TGF nuclear TAZ/YAP is evident in breast cancer cell lines where direct opposing transcriptional events, and therefore suggest TGF has lost its ability to induce cytostatic signals (Fig. 1A). that a subset of TGF-activated SMAD activity does not rely on TAZ/YAP drive the expression of cell cycle regulators (6), TAZ/YAP and vice versa. Based on the products encoded by which may account for the ability of these factors to overcome several of these genes, we speculate that nuclear TAZ/YAP may override tumor-suppressive or negative feedback mechanisms cell cycle arrest. Indeed, our gene expression analysis in LM2-4 initiated by TGF. For example, PMEPA1, which we found is cells identified several cell cycle regulators as TAZ/YAP-regu- induced by TGF and inhibited by TAZ/YAP (group 4 gene), lated genes (e.g. CDKL1, CCNA1, CCNB1, and CCND3). How- encodes a transmembrane protein that sequesters SMAD com- ever, given that TAZ/YAP bind SMAD complexes, we also plexes in the cytoplasm (68). Thus, nuclear TAZ/YAP may speculate that TAZ/YAP may be capable of redirecting TGF- MAY 9, 2014 • VOLUME 289 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13471 Convergent TAZ/YAP-TGF Signals in Breast Cancer 36, 375–384 induced SMADs away from their cell cycle-repressive tran- 4. Wang, X., Su, L., and Ou, Q. (2012) Yes-associated protein promotes scriptional roles toward those that promote tumorigenesis. tumour development in luminal epithelial derived breast cancer. Eur. J. Our phenotypic and transcriptional analysis revealed redun- Cancer 48, 1227–1234 dant functions for TAZ and YAP. For example, TAZ and YAP 5. Chan, S. W., Lim, C. J., Guo, K., Ng, C. P., Lee, I., Hunziker, W., Zeng, Q., have redundant roles in mediating TGF-induced mammo- and Hong, W. (2008) A role for TAZ in migration, invasion, and tumori- sphere formation. Additionally, TAZ and YAP redundantly genesis of breast cancer cells. Cancer Res. 68, 2592–2598 6. Dong, J., Feldmann, G., Huang, J., Wu, S., Zhang, N., Comerford, S. A., regulate the expression of group 1 genes NEGR1 and UCA1 Gayyed, M. F., Anders, R. A., Maitra, A., and Pan, D. (2007) Elucidation of (Fig. 3C). Interestingly, TAZ knockdown alone led to increases a universal size-control mechanism in Drosophila and mammals. Cell in UCA1 expression, which may reflect compensatory YAP 130, 1120–1133 hyperactivity in this context. A redundant role for these factors 7. Lei, Q. Y., Zhang, H., Zhao, B., Zha, Z. Y., Bai, F., Pei, X. H., Zhao, S., Xiong, is further implied on account of similar effects resulting from Y., and Guan, K. L. (2008) TAZ promotes cell proliferation and epithelial- nuclear TAZ or YAP mutant expression in MCF10A cells. Such mesenchymal transition and is inhibited by the hippo pathway. Mol. Cell. Biol. 28, 2426–2436 redundancy is consistent with the overlapping roles of TAZ/ 8. Overholtzer, M., Zhang, J., Smolen, G. A., Muir, B., Li, W., Sgroi, D. C., YAP in early development (71). However, we also present evi- Deng, C. X., Brugge, J. S., and Haber, D. A. (2006) Transforming properties dence for divergent transcriptional activity, based on specific of YAP, a candidate oncogene on the chromosome 11q22 amplicon. Proc. gene expression reliance on either TAZ or YAP exclusively. For Natl. Acad. Sci. U.S.A. 103, 12405–12410 example, the expression of CTGF was repressed by TAZ or 9. Zhao, B., Wei, X., Li, W., Udan, R. S., Yang, Q., Kim, J., Xie, J., Ikenoue, T., TAZ/YAP knockdown in LM2-4 cells but not by YAP knock- Yu, J., Li, L., Zheng, P., Ye, K., Chinnaiyan, A., Halder, G., Lai, Z. C., and Guan, K. L. (2007) Inactivation of YAP oncoprotein by the Hippo pathway down alone (Fig. 3C). Thus, TAZ appears to have a dominant is involved in cell contact inhibition and tissue growth control. Genes Dev. role in regulating CTGF expression in LM2-4 cells. Interest- 21, 2747–2761 ingly, recent work has revealed that YAP, in cooperation with 10. Lamar, J. M., Stern, P., Liu, H., Schindler, J. W., Jiang, Z. G., and Hynes, TGF, has critical roles in controlling the expression of CTGF R. O. (2012) The hippo pathway target, YAP, promotes metastasis through in malignant mesotheliomas (31). Thus, it appears that context its TEAD-interaction domain. Proc. Natl. Acad. Sci. U.S.A. 109, E2441–2450 defines dominance of TAZ or YAP. 11. Lai, D., Ho, K. C., Hao, Y., and Yang, X. (2011) Taxol resistance in breast Effective treatments of late-stage breast cancers are lacking, cancer cells is mediated by the hippo pathway component TAZ and its and our current understanding of the important signals driving downstream transcriptional targets Cyr61 and CTGF. Cancer Res. 71, and maintaining proliferation and metastasis is unclear. Our 2728–2738 work has revealed critical intersections between TAZ/YAP, 12. Zhang, H., Liu, C. Y., Zha, Z. Y., Zhao, B., Yao, J., Zhao, S., Xiong, Y., Lei, TEAD, and TGF signaling in directing pro-tumorigenic phe- Q. Y., and Guan, K. L. (2009) TEAD transcription factors mediate the function of TAZ in cell growth and epithelial-mesenchymal transition. notypes in breast cancer, and provides novel mechanisms by J. Biol. Chem. 284, 13355–13362 which the TGF program may be directed toward aggressive 13. Zhao, B., Ye, X., Yu, J., Li, L., Li, W., Li, S., Yu, J., Lin, J. D., Wang, C. Y., tumorigenic phenotypes. Given the well documented roles of Chinnaiyan, A. M., Lai, Z. C., and Guan, K. L. (2008) TEAD mediates TGF in late-stage cancers, recent efforts have been focused on YAP-dependent gene induction and growth control. Genes Dev. 22, optimizing new TGF signaling inhibitors, which are currently 1962–1971 in pre-clinical and clinical trials (72). Although advancement 14. Xie, D., Nakachi, K., Wang, H., Elashoff, R., and Koeffler, H. P. (2001) Elevated levels of connective tissue growth factor, WISP-1, and CYR61 in with such treatments is logical, our work suggests that primary breast cancers associated with more advanced features. Cancer enhanced efficacy may be achieved by treatment or co-treat- Res. 61, 8917–8923 ment with current (73) or future TAZ/YAPTEAD inhibitors. 15. Pan, D. (2010) The hippo signaling pathway in development and cancer. Dev. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: May 9, 2014

Keywords: Breast Cancer; Cell Migration; Cell Signaling; Coregulator Transcription; Signal Transduction; Transforming Growth Factor β (TGFβ); Hippo Pathway; Signaling Cross-talk; YAP/TAZ

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