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. Author manuscript; available in PMC: 2016 Mar 9.
Published in final edited form as: Cell Rep. 2016 Jan 28;14(5):1169–1180. doi: 10.1016/j.celrep.2015.12.104

Tead and AP1 coordinate transcription and motility

Xiangfan Liu 1,5, Huapeng Li 1,5, Mihir Rajurkar 1,6, Qi Li 2, Jennifer L Cotton 1, Jianhong Ou 1, Lihua J Zhu 1, Hira L Goel 1, Arthur M Mercurio 1, Joo-Seop Park 3, Roger J Davis 2,4, Junhao Mao 1,*
PMCID: PMC4749442  NIHMSID: NIHMS749926  PMID: 26832411

SUMMARY

The Tead family transcription factors are the major intracellular mediators of the Hippo-Yap pathway. Despite the importance of Hippo signaling in tumorigenesis, Tead-dependent downstream oncogenic programs and target genes in cancer cells remain poorly understood. Here we characterize Tead4-mediated transcriptional networks in a diverse range of cancer cells, including neuroblastoma, colorectal, lung, and endometrial carcinomas. By intersecting genome-wide chromatin occupancy analyses of Tead4, JunD and Fra1/2, we find that Tead4 cooperates with AP1 transcription factors to coordinate target gene transcription. We find that Tead-AP1 interaction is JNK independent, but engages the SRC1-3 coactivators to promote downstream transcription. Furthermore we show that Tead-AP1 cooperation regulates the activity of the Dock-Rac/CDC42 module and drives the expression of a unique core set of target genes, thereby directing cell migration and invasion. Together, our data unveil a critical regulatory mechanism underlying Tead- and AP1-controlled transcriptional and functional outputs in cancer cells.

Keywords: Tead, AP1, transcriptional co-regulation, invasion

INTRODUCTION

The Tead family transcription factors are a family of evolutionary conserved proteins carrying a TEA DNA binding domain that recognizes the GGAATG consensus sequence (Kaneko and DePamphilis, 1998; Pobbati and Hong, 2013). Scalloped (Sd) is the only Tead family proteins in Drosophila (Halder et al., 1998; Wu et al., 2008a). In mammals, four Tead family members, Tead1-4, were originally identified by their various roles in early embryonic development (Chen et al., 1994; Nishioka et al., 2008; Sawada et al., 2008). Tead proteins require additional transcriptional co-activators to activate transcription, and recent studies have established the YAP family transcriptional regulators (Yki in fly and YAP/TAZ in mammals) as the major co-activator for Tead proteins (Nishioka et al., 2008; Wu et al., 2008a; Zhang et al., 2009a; Zhao et al., 2008), although other Tead upstream regulators have been reported (Gupta et al., 1997; Halder et al., 1998; Pobbati et al., 2012). YAP and TAZ are the key intracellular effectors of Hippo signaling, and dysregulation of the Hippo-YAP/TAZ pathway has been implicated in a variety of human cancers (Halder and Camargo, 2013; Hong and Guan, 2012; Moroishi et al., 2015; Pan, 2010). Despite the potential importance of Tead proteins in tumorigenesis, the molecular mechanism underlying Tead-mediated transcriptional regulation is not well understood and the Tead-controlled downstream target network in cancer cells remains poorly characterized.

RESULTS

Functional requirement and genomic occupancy of Tead proteins in neuroblastoma, lung, colon, and endometrial cancer cells

To gain insight into Tead-dependent oncogenic programs, we first examined the expression of Tead proteins in four distinct types of human cancers; lung adenocarcinoma, colorectal carcinoma, endometrial cancer, and neuroblastoma. Immunohistochemistry (IHC) revealed that nuclear Tead4 expression was readily detected in all four cancer types (Figure 1A). Although mis-regulation of the Hippo-YAP pathway in lung, colon and endometrial cancers has been previously reported (Moroishi et al., 2015; Tsujiura et al., 2014), its connection to neuroblastoma, a common infant and childhood tumor arising from the neural crest lineage (Louis and Shohet, 2015), was not known. We found that Tead4 was highly expressed in the majority of human neuroblastoma samples we examined, in comparison to low or no expression in normal peripheral nerve tissues (Figure 1A; Figure S1), pointing to a potential Tead involvement in neuroblastoma pathogenesis. Interestingly, Tead4 and overall Tead proteins, detected by the Tead4 and pan-Tead antibodies respectively, exhibited distinct expression patterns in human A549 (lung adenocarcinoma), HCT116 (colon cancer), SK-N-SH (neuroblastoma) and ECC1 (endometrial cancer) cells (Figure 1B), suggesting potential functional redundancy among Tead proteins in cancer cells. To block the activity of all Tead proteins, we generated lentiviral-based constructs, Teads KD/KO, which enable both shRNA-mediated knockdown of human Tead1/3/4 (Zhao et al., 2008) and Crispr-mediated knockout of human Tead2 (Figure 1C; Figure S1). Further, we showed that Teads KD/KO effectively blocked YAP/TAZ-induced transcriptional activation, and inhibited the ability of A549, HCT116, SK-N-SH, and ECC1 cells to form anchorage-independent colony (Figure 1D, E), highlighting the critical functional requirement for Tead proteins in these cancer cells.

Figure 1. Functional requirement and genomic occupancy of Tead proteins in A549, HCT116, SK-N-SH and ECC1 cancer cells.

Figure 1

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(A) Representative IHC images of Tead4 staining showing nuclear expression of Tead4 proteins in human lung adenocarcinoma, colorectal carcinoma, endometrial cancer, and neuroblastoma. (B) Expression of YAP, TAZ and Tead factors in A549, HCT116, SK-N-SH and ECC1 cells. Immunoblot analysis of YAP, TAZ, Tead4, and overall Tead protein expression using the antibodies against YAP, TAZ, Tead4 and pan-Tead. (C) Immunoblot analysis of overall Tead (pan-Tead) protein and Tead2 expression in HCT116 cells expressing shRNA against Tead1/3/4 (shTead1/3/4), Crispr-mediated Tead2 knockout construct (Crispr-Tead2), or both (Teads KD/KO). (D) Tead1-4 knockdown/knockout (Teads KD/KO) blocks YAP- or TAZ-induced Tead-luciferase reporter (Tead-Luc) activity in 293T cells, and Tead-dependent transcriptional activity and colony formation in A549, HCT116, SK-N-SH, and ECC1 cells. (E) Representative images of anchorage-independent colony formation in control and Teads KD/KO-expressing HCT116 cells. (F) Venn diagram showing overlapping of Tead4 binding sites in A549, HCT116, SK-N-SH, and ECC1 cells identified by Tead4 ChIP-Seq. (G) ChIP-qPCR analysis of selected Tead4 binding sites in the known target genes and the genes involved in pathway feedback regulation. Mean fold enrichment in ChIP is expressed relative to a control βActin genomic region. Sites are named according to the nearest locus. (H) qPCR analysis of the known YAP target genes, ANKRD1, CTGF and Cyr61, as well as the target genes involved in pathway feedback regulation in HCT116 cells with and without Teads KD/KO. (I) Enrichment of AP1 motif on Tead4-occupied cis-regulatory regions in the genomes of A549, HCT116, SK-N-SH, and ECC1 cells. De novo motif analysis of Tead4 binding sites revealed the presence of the two most enriched motifs of Tead and AP1 in all four genomes. *P<0.01, error bars indicate mean ± s.d. See also Figure S1, S2 and Table S1.

Next we performed the analysis of genome-wide Tead4 ChIP-seq data sets of A549, HCT116, SK-N-SH and ECC1 cells that are available at the ENCODE project (http://genome.ucsc.edu/ENCODE/downloads.html). After intersecting the Tead4 ChIP-seq data from these four cancer cell lines (Figure 1F; Table S1), we found that, in addition to the known direct YAP target genes, CTGF, Cyr61, and ANKRD1 (Dupont et al., 2011; Lai et al., 2011; Zhao et al., 2008), many genes carrying the Tead4 binding peaks encoded the Hippo pathway components and regulators, such as AmotL2, Ajuba, Tead1 and Tead4 (Figure 1G). Transcription of many of these genes could also be modulated by Tead inhibition (Figure 1H), suggesting an active feedback regulation of the Hippo pathway in cancer cells.

To identify the regulatory mechanism of Tead-mediated transcription, we performed de novo motif analysis of the Tead4 binding regions identified in our ChIP-seq data analysis. Interestingly, in addition to the core Tead motif, the activating protein-1 (AP1) motif was always amongst the two most enriched sequences within the Tead4 peaks in all four genomes (Figure 1I; Figure S2), suggesting a possible engagement of AP1 in Tead-regulated transcription.

AP1 and Tead4 co-occupancy at cis-regulatory regions of cancer genomes

The AP1 family transcription factor is a collection of dimeric complexes composed of members of the Jun, Fos and ATF/CREB families, and has been implicated in a broad range of diseases, including cancers (Meixner et al., 2010; Sarkar et al., 2011; Schonthaler et al., 2011; Verde et al., 2007). However, the molecular mechanism underlying AP1 activity in tumor cells remains poorly understood. Immunofluorescence staining and western blot analysis showed that many AP1 proteins, including JunD, cJun, Fos, Fra1 and Fra2, were expressed in the cancer cells (Figure 2A, B; data not shown), suggesting potential redundancy among AP1 proteins. Consistent with this idea, we showed that Crispr-mediated knockout of JunD and cJun only partially blocked the activity of an AP1-controlled luciferase reporter (AP1-Luc) (Figure 2C, D); however, DN-JunD was able to effectively inhibit AP1-Luc activation (Figure 2D). DN-JunD is a dominant repressor form of JunD that lacks the N-terminal transcriptional activation domain but retains the DNA binding domain and the ability to dimerize with other AP1 proteins, thereby inhibiting AP1 activation (Figure S1).

Figure 2. AP1 and Tead4 co-occupancy at cis-regulatory regions of cancer genomes.

Figure 2

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(A) Immunoblot analysis of JunD, cJun, Fra1 and Fra2 protein expression in A549, HCT116 and SK-N-SH cells. (B) Immunofluorecence staining of JunD and Fra1 in HCT116 cells. DAPI labels the nuclei. (C) Immunoblot analysis of JunD and cJun expression in HCT116 cells with and without expression of Cripsr knockout constructs against JunD or cJun. (D) The activity of AP1-dependent luciferase reporter (AP1-Luc) in HCT116 cells with the Cripsr knockout (KO) constructs against JunD and cJun, or the expression construct of dominant negative JunD (DN-JunD). (E–G) Intersection of Tead4, JunD and Fra1/2 ChIP-seq in the A549 (E), HCT116 (F) and SK-N-SH (G) genomes showing significant co-occupancy of Tead4 and JunD/Fra1/2 in all three cell lines. (H) Heatmap representing Tead4/JunD/Fra2-coocupied peaks located within promoter or enhancer regions of A549 genome. The headmap is sorted by density of Tead4 signals in each category. 0: peak center; −/+1000: 1kb upstream or downstream of the center. (I) Percentile of Tead4/JunD/Fra2-coocupied peaks in the categories of active promoters (H3K4me3+;H3K27ac+), active enhancers (H3K4me1+;H3K27ac+), and inactive enhancers (H3K4me1+;H3K27ac−). (J) Bimodal distribution of H3K4me1signal around the summit of the Tead4/JunD/Fra2-coocupied peaks. *P<0.01, error bars indicate mean ± s.d. See also Figure S1, S2, Table S2 and S3.

To explore AP1-mediated transcriptional programs in cancer cells, we performed the analysis of the JunD, Fra1 and Fra2 ChIP-seq datasets of the A549, HCT116 and SK-N-SH genomes from the ENCODE project (http://genome.ucsc.edu/ENCODE/downloads.html) (Table S2, S3). We then intersected these data with the Tead4 ChIP-seq data sets, and found significant overlaps of Tead4 peaks with AP1 (JunD, Fra1 and Fra2) peaks in three cell lines (Figure 2E–G). Statistical analysis of Tead4 peaks overlapping JunD or Fra1/2 peaks or random genomic regions showed significant enrichment of the AP1 overlapping peaks with the empirical p-values at 0 in all cases. More strikingly, more than half of the Tead4 binding regions overlapped with the JunD or Fra1/2-occupied sites in all three genomes (Figure 2E–G). Furthermore, we performed de novo motif analysis of the JunD and Fra1/2 peaks identified in three cell lines and showed that Tead motif was also among the top enriched motifs within the JunD or Fra1/2 binding regions (Figure S2). Thus, our analyses of both Tead4 and AP1 genome-wide ChIP-seq data identified the intensive interactions between Tead4 and AP1-controlled transcriptional networks in a broad range of human cancer cells.

To further characterize Tead4 and AP1 genomic co-occupany, we examined the histone modification status of Tead4/AP1 peaks in A549 cells by analysis of genome-wide H3K4me1, H3k4me3 and H3K27ac ChIP-seq data (http://genome.ucsc.edu/ENCODE/downloads.html). The Tead-AP1 peaks with histone H3 monomethylation at Lys 4 (H3K4me1) were considered as enhancer regions. Active enhancers were defined by co-presence of H3K4me1 and H3K27ac (histone H3 acetylation at Lys 27), and promoter regions were defined as H3K4me3 (histone H3 trimethylation at Lys 4) and H3K27ac overlapping peaks that have minimal overlapping of 1000bp and are close to a transcriptional start site (TSS) (2000bp upstream to 500bp downstream). Our analysis revealed that the majority of Tead4-AP1 co-occupied peaks were the active enhancer regions (86%), and 12% of them were active promoters (Figure 2H, I). In addition, the bimodal distribution of H3K4me1 signal around the peak center (Figure 2J) supported the notion that these Tead-AP1 peaks were indeed in an active state. Further analysis showed that the Tead and AP1 binding motifs were located with close proximity around the peak summit (Figure 2J), and the median space between them was about 70bp. Taken together, these data suggest that Tead and AP1 factors may operate closely in active enhancer/promoter regions to regulate downstream transcription in cancer cells.

Tead and AP1 coordinate downstream gene transcription

To examine Tead-AP1 mediated co-regulation of downstream transcription, we dissected in detail the Tead4 or AP1-binding cis-regulatory elements identified in the ANKRD1, Dock9, and Tead4 loci (Figure 3A). Tead4 is a core Hippo component likely involved in pathway feedback regulation (Figure 1H), and ANKRD1 is a known YAP target gene (Dupont et al., 2011; Lei et al., 2015); however it was not clear whether Dock9 can be transcriptionally regulated by Tead or AP1. The analysis of histone modification status revealed that these Tead1 or JunD binding sites were located within the active promoter (ANKRD1 and Tead4) or active enhancer (Dock9) regions (Figure 3A). The lack of AP1 occupancy in the peak of the Tead4 locus suggested that its expression may be independent of AP1 (Figure 3A). Indeed, we found that YAP and TAZ shRNA knockdown, but not DN-JunD expression, decreased Tead4 expression in HCT116 cells (Figure 3C, D). In contrast, ANKRD1 and Dock9 expression was significantly decreased by inhibition of both Tead and AP1 activity (Figure 3B). Furthermore, we generated the luciferase reporter constructs driven by the promoter or enhancer peaks identified in the ANKRD1, Dock9, and Tead4 loci (ANK-Luc, Dock9-Luc and Tead4-Luc). We showed that, although Tead4-Luc was only responsive to YAP activation (Figure 3G), YAP and JunD co-expression synergistically induced reporter activation of ANK-Luc and Dock9-Luc (Figure 3E, F). Together, these data suggest that YAP/Tead-AP1 cooperation on the distal or proximal regulatory regions regulates a subset of target gene expression.

Figure 3. Tead and AP1 cooperation on downstream gene transcription.

Figure 3

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(A) Diagram showing the Tead4, JunD, H3K4me3, and H3K27ac peaks in the promoter or enhancer regions of the ANKRD1, Dock9 and Tead4 loci of the HCT116 genome. Scale bar = 2kb. (B) Immunoblot analysis of ANKRD1 and Dock9 expression in HCT116 cells with Tead1-4 knockdown/knockout (Teads KD/KO) or AP1 inhibition by DN-JunD. (C) Immunoblot analysis of Tead4 expression in HCT116 cells with shRNA knockdown against YAP and TAZ (shYAP/TAZ) or AP1 inhibition by DN-JunD. (D) Immunoblot analysis of YAP and TAZ expression in HCT116 cells expressing shRNAs against YAP and TAZ. (E–G) The luciferase reporters driven by the Tead4 peaks from the ANKRD1, Dock9 and Tead4 loci were generated, and the activity of ANK-Luc (E), Dock9-Luc (F) and Tead4-Luc (G) in 293T cells was measured with or without ectopic expression of JunD and YAP. (H, I) ChIP in HCT116 cells was performed with control IgG, Tead4, cJun, JunD, Fos, and Fra1 antibodies as indicated. The enrichment of the ANKRD1 promoter region was calculated based upon qPCR relative to the IgG control. (J) Sequential ChIP with antibody against Tead4 followed by antibody against JunD confirms the presence of both transcription factors on the ANKRD1 promoter. Enrichment is calculated based upon qPCR relative to the no antibody (Ab) or IgG control. (K) Diagrams of the wild type ANKRD1 luciferase reporter (ANK-Luc) and the luciferase reporters driven by the mutated peak lacking the Tead motif (ANK-mT-luc) or the AP1 motifs (ANK-mA-Luc). (L) Luciferase activity of the ANK-Luc reporter was measured in HCT116 cells with JunD, Fra1, or both. (M) Luciferase activity of ANK-Luc, ANK-mT-Luc and ANK-mA-Luc was measured with YAP/Tead4, JunD/Fra1, or both. (O,P) JunD binds both exogenous and endogenous Tead4 in cells. (O) Indicated plasmids were co-transfected into HEK293 cells and Tead4 was immunoprecipitated with anti-V5 antibody. Immunoblot analysis shows co-immunoprecipitation of JunD detected by anti-flag antibody. (P) Tead4 binds to endogenous JunD in HCT116 cells. Endogenous Tead4 was immunoprecipitated with anti-Tead4 antibody and co-immunoprecipitation of JunD and YAP was shown by anti-JunD and anti-YAP immunoblots. A control IgG was used as the negative control for immunoprecipitation. *P<0.01, error bars indicate mean ± s.d.

Our ChIP-qPCR analysis showed that Tead4 and various AP1 proteins, including JunD, cJun, Fos and Fra1, were able to bind to the peak located in the ANKRD1 promoter (Figure 3H, I). The co-occupancy of Tead4 and AP1 was also confirmed by sequential re-ChIP experiments (Figure 3J). Further, we generated the ANK-Luc-based reporter constructs with mutated Tead or AP1 binding site (ANK-mT-Luc or ANK-mA-Luc) (Figure 3K). When JunD and Fra1 were co-expressed, the JunD/Fra1 heterodimer strongly induced ANK-Luc reporter luciferase activity (Figure 3L). However, expression of Tead4 alone with JunD/Fra1 did not further enhance reporter activity (data not shown), suggesting that, unlikely YAP, AP1 itself is not able to activate Tead proteins. In contrast, when Tead4 was activation by YAP, it acted synergistically with JunD/Fra1 to promote the wild type ANK-Luc reporter activity (Figure 3M). Furthermore, although YAP/Tead4 or JunD/Fra1 was capable of activating AP1 site-mutated or Tead site-mutated luciferase reporters respectively, they lost the ability to synergize with each other (Figure 3M), suggesting that YAP/Tead and AP1 proteins do not rely on each other to bind to the cis-regulatory region, but rather act synergistically to achieve maximum transcriptional output.

We also detected JunD and Tead4 protein-protein interactions exogenously in transfected 293T cells (Figure 3O) and endogenously in HCT116 cells (Figure 3P). Our co-immunoprecipitation (co-IP) assays detected Tead4 binding to endogenous YAP or AP1 proteins, including JunD, cJun, and to a lesser degree, Fos (Figure 3P, and data not shown). However we could not detect strong interaction between YAP and AP1 proteins by YAP and AP1 co-IP (data not shown), further suggesting the interaction occurs at the level of the Tead and AP1 transcription factors.

Tead and AP1 interaction is JNK independent

To explore the molecular mechanism underlying Tead-AP1 interaction, we first examined the possible involvement of JNK kinases. The activity of AP1 proteins can be regulated via N-terminal phosphorylation by upstream JNK kinases (Davis, 2000). Recent reports also showed that JNK can modulate Hippo/YAP signal transduction in certain contexts (Lee and Yonehara, 2012; Sun and Irvine, 2013; Tomlinson et al., 2010). Therefore, we sought to test whether Tead-AP1 cooperation depends on JNK activity in cancer cells. We utilized a recently developed highly-specific JNK inhibitor, JNK-IN-8 (Zhang et al., 2012), and showed that JNK-IN-8 treatment of HCT116 or A549 cells was able to effectively block cJun phosphorylation, but not Lats-mediated YAP phosphorylation (Figure 4A, and data not shown). JNK inhibition also did not alter YAP cellular localization or the expression of the YAP target genes, CTGF and ANKRD1 (Figure 4B; Figure S3). Furthermore, JNK inhibition or activation by overexpression of a MKK7-JNK construct did not affect Tead-AP1 interaction or transcriptional cooperation, measured by Tead/JunD co-IP and ANK-Luc activity (Figure 4C; Figure S3). In addition, we showed that, cJun4A, a mutant form of cJun with all four N-terminal JNK phosphorylation sites mutated, was equally efficient to induce ANK-Luc reporter activation as wild type cJun (Figure S3). These results indicated that JNK phosphorylation does not likely play a major role in regulating Tead-AP1 functional interaction, and either additional or different mechanism is involved.

Figure 4. JNK-independent AP1-Tead interaction engages SRC1-3 co-activators.

Figure 4

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(A) Immunoblot analysis of cJun, phosphorylated cJun (p-cJun), YAP and phosphorylated YAP (p-YAP) in HCT116 cells with or without the JNK inhibitor, JNK-IN-8. (B) qPCR analysis of CTGF and ANKRD1 transcription in HCT116 cells with or without JNK-IN-8. (C) JNK inhibition or activation does not affect Tead/AP1 cooperation. Luciferase activity of the ANK-Luc reporter induced by JunD/Fra1 and YAP/Tead4 was measured with the presence of JNK-IN-8 or expression of MKK7-JNK. (D) Immunofluorecence staining of SRC1 in HCT116 cells. DAPI labels the nuclei of HCT116 cells. (E) Expression of SRC1 and SRC3 proteins in A549, HCT116, SK-N-SH, and ECC1 cells, assayed by immunoblotting. (F) SRC3 binds to endogenous JunD and Tead4 in HCT116 cells. Endogenous SRC3 was immunoprecipitated with anti-SRC3 antibody and co-immunoprecipitation of JunD and Tead4 was shown by anti-JunD and anti-Tead4 immunoblots. A control IgG was used as the negative control for immunoprecipitation. (G) Diagrams of wild type and truncated SRC3 constructs with a C-terminal V5 tag. Indicated are the bHLH-PAS domain, the nuclear receptor interacting domain (RID), and the activation domains 1 and 2 (AD1 and AD2, respectively) in SRC3, SRC3-N, SRC3-M and SRC3-C. (H) In HCT116 cells expressing wild type and truncated SRC3 proteins, immunoprecipitation was performed with anti-V5 antibody and co-immunoprecipitation of endogenous Tead4 and JunD was shown by anti-Tead4 and anti-JunD immunoblots. (I) ChIP from HCT116 cells was performed with control IgG or the SRC3 antibody as indicated. The enrichment of the ANKRD1 and Tead4 peak was calculated based upon qPCR relative to the IgG control. (J) Immunoblot analysis of SRC1, SRC2 and SRC3 in HCT116 cells with or without expression of Crispr knockout constructs against SRC1, SRC2 and SRC3 (SRC1-3 KO). (K) Immunoblot analysis of SRC1, SRC3 and Tead4 in HCT116 cells with or without the SRC1/3 inhibitor, Bufalin. (L) Luciferase activity of Tead-dependent luciferase reporter (Tead-Luc) induced by YAP5SA in HEK293 cells with or without SRC1-3 Crispr knockout or Bufalin treatment. (M) In HCT116 cells with SRC1-3 Crispr knockout or Bufalin treatment, endogenous JunD was immunoprecipitated with anti-JunD antibody and co-immunoprecipitation of endogenous Tead4 was shown by anti-Tead4 immunoblots. (N) In HCT116 cells expressing Crispr knockout constructs against SRC1-3 (SRC1-3 KO), luciferase activity of ANK-Luc was measured with YAP/Tead4, JunD/Fra1, or both. *P<0.05, **P<0.01, error bars indicate mean ± s.d. See also Figure S3.

Tead-AP1 cooperation engages SRC1-3 coactivators

The p160 family of steroid receptor coactivators, SRC1-3 (also known as NCOA1-3), were originally identified as nuclear hormone co-activators (Xu et al., 2009), although it was later discovered that they can interact with a broad range of other transcriptional factors, including AP1 proteins, to regulate gene transcription (Lee et al., 1998; Qin et al., 2014; Xu et al., 2009; Yan et al., 2006). Interestingly, a previous report identified all three SRCs as Tead binding partners through a yeast two-hybrid screen and that showed they can potentiate Tead-mediated transcription (Belandia and Parker, 2000). However, the cellular context and function of this interaction were not clear. Our immunofluoresence and immunoblot analyses revealed that SRC proteins were expressed in A549, HCT116, SK-N-SH and ECC1 cancer cells (Figure 4D, E and data not shown). More importantly, we demonstrated the binding of endogenous SRC3, JunD and Tead4 in HCT116 cells (Figure 4F). To further characterize the interactions among these proteins, we generated the expression constructs of three truncated mutants of SRC3 fused with a C-terminal V5 tag: SRC3-N, SRC3-M and SRC3-C (Figure 4G). We then examined their ability to bind to endogenous Tead4 and JunD proteins using the co-IP assay. Consistent with the previous report (Belandia and Parker, 2000), we showed that SRC3 interacted with Tead4 through its N-terminal bHLH-PAS domain (Figure 4H). Interestingly, we found that the SRC3 domain responsible for JunD binding was mainly located in its C-terminus (Figure 4H). These data raised an intriguing possibility that SRC factors bridge the interaction between Tead and AP1, thereby mediating their cooperation.

Consistent with this idea, our SRC3 ChIP-qPCR analysis also revealed that SRC3 was significantly more enriched at the ANKRD1 peak with Tead and AP1 co-occupancy than the Tead4 peak with only Tead occupancy (Figure 4I). To further examine the importance of SRC proteins in Tead-AP1 cooperation, we generated the Crispr-based constructs to knock out all three SRC proteins in HCT116 cells (Figure 4J). In addition, we utilized a recently identified SRC1/3 specific inhibitor, Bufalin (Wang et al., 2014) (Figure 4K). We found that inhibition of SRC function by SRC1/2/3 crispr knockout or Bufalin treatment did not affect Tead4 protein expression (Figur 4K) or the Tead-dependent luciferase reporter (Tead-Luc) activity induced by YAP5SA, a constitutively active form of YAP (Zhao et al., 2008) (Figure 4L). However, we found that SRC inhibition significantly blocked the endogenous Tead-JunD interaction (Figure 4M) and inhibited the synergistic effect on ANK-Luc reporter activation by YAP/Tead-JunD/Fra1 co-expression (Figure 4N). Taken together, these studies suggested that Tead-AP1 cooperation is mediated at least in part by the SRC1-3 co-activators.

Tead-AP1 drives a core set of target genes to regulate migration and invasion

Our analysis of Tead4 ChIPseq in a broad range of cancer cells identified the previously known YAP/Tead targets, including CTGF, Cyr61, Axl, Birc5 and AREG that are involved in regulation of proliferation and apoptosis (Dong et al., 2007; Lai et al., 2011; Xu et al., 2011; Zhang et al., 2009b; Zhao et al., 2008). However, it was not clear whether the cooperation between YAP/Tead and AP1 drives additional function or distinct targets in cancer cells.

To explore potential downstream transcriptional programs controlled by Tead and AP1, we performed functional clustering analysis of the overlapping genes with Tead4, JunD, and Fra1/2 peaks from A549, HCT116 and SK-N-SH cells (Figure 5A). By comparing the functional clustering data between the genes with the Tead4 peaks and the genes with Tead4/AP1 co-occupied peaks, we found that, interestingly, a significant enrichment of the clusters associated with cell adhesion, motility and migration within the genes carrying the Tead/AP1 co-occupied peaks (Table 1), suggesting a potential regulation of cell migration and invasion by Tead-AP1. In addition, we noted that Tead4, JunD and Fra1/2 often bound to the distal or proximal regulatory regions of the genes encoding the Dock proteins (Table S1–4). Dock is a family of eleven related proteins functioning as the specific guanine nucleotide exchange factors for small G proteins Rac and Cdc42 that play a pivotal role in orchestrating cell adhesion and movement (Laurin and Cote, 2014). qPCR and immunoblot analyses in HCT116 cells showed that transcription and expression of many Dock factors, including Dock4, Dock5 and Dock9, were down-regulated by inhibition of both AP1 and Tead activation (Figure 3B, 5C, 5E; Figure S4). Moreover, we found that the activity of Rac1 and CDC42, but not the protein levels, was significantly decreased following Tead and AP1 inhibition (Figure 5D, E). In contrast, the activity and expression of RhoA, the other member of the Rho small G protein family, were largely unaffected (Figure 5D, E). These data suggested that Tead and AP1 act as important upstream regulators for the Dock-Rac/CDC42 functional module in cancer cells.

Figure 5. Tead and AP1 drive a core set of downstream targets to regulate cell migration and invasion.

Figure 5

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(A) Venn diagram showing the overlapping Tead4, JunD and Fra1/2 peaks identified by ChIP-seq in A549, HCT116, and SK-N-SH genomes. (B) qPCR analysis of transcription of selected target genes involved in regulation of cell migration and invasion in HCT116 cells with Tead1-4 knockdown/knockout (Teads KD/KO) and AP1 inhibition by DN-JunD. (C) qPCR analysis of transcription of Dock4, Dock5, and Dock9 in HCT116 cells with Tead1-4 knockdown/knockout (Teads KD/KO) or AP1 inhibition by DN-JunD. (D) Measurement of the activity of the Rho family small G proteins, RhoA, RAC1, and CDC42, in HCT116 cells with Tead1-4 knockdown/knockout (Teads KD/KO) or AP1 inhibition by DN-JunD. (E) Immunoblot analysis of DOCK4, RAC1 and CDC42 in HCT116 cells expressing DN-JunD, Teads KD/KO, or both. (F) Representative images of transwell migration of HT29 cells with ectopic expression of JunD, YAP5SA, or both. (G) Relative migration activity of HT29 cells expressing YAP5SA or TAZ4SA with or without DN-JunD. (H) Immunoblot analysis of Dock4 and Dock9 in HCT116 cells expressing Crispr knockout constructs against Dock4 and Dock9 (Dock4/9 KO). (I) Representative images of transwell migration of YAP5SA/JunD-expressing HT29 cells with knockout of SRC1-3 or Dock4/9. (J) Quantification of transwell migration assay shown in (I). (K) Representative images of cell scratch assay of HCT116 cells expressing DN-JunD or Teads KD/KO at 0 hour or 48 hours. (L) Representative images of matrigel invasion assay in control (i) or HCT116 cells expressing DN-JunD (ii), Teads KD/KO (iii), or both (iv). (M) Quantification of matrigel invasion assay shown in (L). (N–Q) Co-expression of JunD and Tead4 proteins in human lung adenocarcinomas (n=30) and matched lymph node metastases (n=30) by tissue microarray (TMA) assays. (N) Representative IHC images of nuclear expression of JunD and Tead4 in lymph node metastases of lung adenocarcinoma. (O, P) Higher expression of JunD (O) and Tead4 (P) in lymph node metastases in comparison to matched primary lung adenocarcinoma samples. (Q) The percentile of co-expression of high level JunD and Tead proteins in primary lung adenocarcinoma and matched lymph node metastases. P: primary lung adenocarcinoma; M: matched lymph node metastases. *P<0.05, **P<0.01, error bars indicate mean ± s.d. See also Figure S4 and Table S4.

Table 1.

Functional clustering of the overlapping genes carrying Tead4 peaks or Tead/AP1-cooccupied peaks from A549, HCT116 and SK-N-SH cells.

TEAD4 Peaks TEAD4-AP1 Peaks
blood vessel morphogenesis blood vessel morphogenesis
transcription activator activity focal adhesion
regulation of transcription regulation of cell adhesion
regulation of kinase activity cell-cell adhesion
regulation of cell motion cell migration
pattern specification process regulation of kinase activity
lung development regulation of protein modification process
focal adhesion cytoskeletal protein binding
chordate embryonic development regulation of cell motion
regulation of protein modification process regulation of cell growth
regulation of RNA metabolic process contractile fiber
protein amino acid phosphorylation protein amino acid phosphorylation
TGFb receptor binding actin cytoskeleton organization
growth factor binding GTPase activation
regulation of cell growth embryonic morphogenesis
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In addition to the Dock-Rac/CDC42 axis, our functional annotation revealed that more than 50 out of the 300 genes with Tead and AP1 co-occupied peaks were directly implicated in regulation of cell migration and invasion (Table S4). Among them, we found that transcription of ABL2, CDH2, CNN3, DAAM1, GRP126, ITBG5, MACF1, MKLN1, NRP1, PARD3, PHLDB2 and TNS3 was inhibited by blocking Tead and AP1 activity in HCT116 cells (Figure 5B, Figure S5), suggesting that these genes represent a core set of new direct targets of Tead and AP1 in cancer cells. Moreover, these results support the idea that a key functional output of YAP/Tead-AP1 cooperation is to regulate cancer cell migration and invasion.

To test this hypothesis, we ectopically expressed YAP5SA and JunD in the HT29 cells, a relatively less invasive colon cancer cell line (de Both et al., 1999), and found that YAP5SA and JunD co-expression significantly promoted migration of HT29 cells, measured by transwell migration assay (Figure 5F). In addition, we showed that YAP5SA or TAZ4SA (an active form of TAZ) induced migration was partially inhibited by DN-JunD (Figure 5G). Furthermore, we found that Crispr-mediated SRC1/2/3 or Dock4/9 knockout inhibited YAP5SA/JunD-dependent migration in HT29 cells (Figure 5H–J), highlighting the functional importance of these proteins in YAP/Tead-AP1 cooperation. Next, we demonstrated that the combined inhibition of Tead and AP1 activity in the highly invasive HCT116 colon cancer cells (de Both et al., 1999) led to effective inhibition of cell migration and invasion, measured by cell scratch and matrigel invasion assays (Figure 5K–M). Moreover, we analyzed the expression of JunD and Tead4 in sixty two human lung adenocarcinomas and their matched lymph node metastases, and found that JunD and Tead4 proteins were expressed significantly higher in the lymph node metastases than in the matched primary cancers (Figure 5N–Q), suggesting that the potential involvement of Tead-AP1 cooperation in promoting tumor invasion and metastasis.

DISCUSSION

By intersecting the transcriptional networks employed by both Tead4 and AP1 proteins in diverse cancer cells, we uncovered a critical mechanism underlying Tead and AP1-mediated oncogenic regulation. Our data support a model that Tead and AP1 interaction at the active cis-regulatory genomic regions promotes or maximizes transcription output of target genes (Figure 6). The Tead-AP1 cooperation we described here is distinct from the recently reported Tead-independent Fos regulation by Kras and YAP in pancreatic cancer cells (Shao et al., 2014). However, our data are consistent with a recent report of Tead and AP1 co-occupancy on cis-regulatory regions in the invasive melanoma genome (Verfaillie et al., 2015). Further, during the revision of this manuscript, a recent study described the genomic association between YAP/TAZ/Tead and AP1 in breast cancer cells that drives oncogenic growth (Zanconato et al., 2015). Our analyses of both AP1 and Tead4 genome-wide ChIPseq data in a broad range of tumor cells, including neuroblastoma, colon, lung and endometrial cancers, suggest that Tead-AP1 cooperation on transcription is a general mechanism utilized by both families of transcription factors during tumorigenesis.

Figure 6.

Figure 6

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A schematic model showing YAP/Tead-AP1 cooperation on cis-regulatory regions engages SRC1-3 co-activators and drives downstream gene expression to regulate cancer cell migration and invasion.

Recent reports showed the possible JNK regulation of Hippo signaling via upstream Lats kinases or direct phosphorylation of YAP in fly and mammalian cells during apoptosis (Lee and Yonehara, 2012; Sun and Irvine, 2013; Tomlinson et al., 2010). However, our data indicate that Tead-AP1 cooperation in cancer cells does not appear to require JNK phosphorylation, highlighting the complex crosstalkes between these pathways in different functional and cellular contexts. More importantly, our study here revealed a key molecular mechanism underlying Tead-AP1 interaction that is mediated by the SRC1-3 transcriptional cofactors (Figure 6). The SRC1-3 (NCOA1-3) family transcriptional co-activators, originally identified as nuclear receptor co-regulators, have been shown to interact with a variety of transcription factors. Our study suggest a model that Tead and AP1 co-occupancy at the promoter and active enhancer regions engages SRC factors, which bind to Tead and AP1 through distinct N-terminal and C-terminal domains to bridge the Tead-AP1 interaction, thereby mediating at least in part their cooperation on downstream transcription in cancer cells (Figure 6).

Current knowledge of the YAP/Tead downstream targets in cancers has been largely focused on the genes such as CTGF, Cyr61, Birc5, AXL and AREG that are involved in regulation of proliferation, apoptosis, and oncogenic growth (Dong et al., 2007; Lai et al., 2011; Xu et al., 2011; Zanconato et al., 2015; Zhang et al., 2009b; Zhao et al., 2008). Our results here not only identified a core subset of downstream YAP/Tead target genes, but uncovered a critical functional output of Tead-AP1 in coordination of cell migration and invasion in diverse types of cancer cells (Figure 6). These Tead and AP1 downstream targets include the members of the Dock family proteins and other genes such as CDH2, MACF1, ABL2 and TNS3, which all have been directly implicated in controlling different aspects of cell motility, including cytoskeleton organization, cell adhesion and migration (Bradley and Koleske, 2009; Laurin and Cote, 2014; Qian et al., 2009; Wu et al., 2008b). Interestingly, recent studies have reported that cell attachment, cell-matrix interaction and mechanical forces can influence upstream Hippo signaling converging on YAP/TAZ regulation (Chang et al., 2015; Dupont et al., 2011; Halder et al., 2012; Zhao et al., 2012). Our study suggests the importance of the interplay and possible feedback regulation between Tead-AP1 activation and cell adhesion, migration and invasion during tumorigenesis and metastasis.

EXPERIMENTAL PROCEDURES

Cell culture, transfection and lentiviral infection

HCT116, SK-N-SH, ECC1, FET, DLD1, LS174T, HEK293T cells were cultured in DMEM supplemented with 10% FBS. A549 cells were cultured in F-12K Medium supplemented with 10% FBS. HT29 cells were cultured in McCoy 5A medium supplemented with 10% FBS. Caco2 and RKO cells were cultured in EMEM medium supplemented with 20% FBS. SW48 cells were cultured in L15 medium supplemented with 10% FBS. Detailed information of lentiviral expression vectors, Crispr knockout/shRNA knockdown constructs and luciferase reporter constructs are described in Supplemental Experimental Procedures.

Cell and biochemical assays

For immunohistochemistry, human cancer tissue microarray samples were purchased from Biomax, Genvelop or UMass Cancer Center Tissue Bank. Antibody information for immunohistochemistry, immunofluorescence, and immunoblot analyses are shown in Supplemental Materials. Detailed protocols for assaying cell migration and invasion, anchorage-independent colony formation, chromatin immunoeprecipitation, protein immunoprecipitation, and small G protein activation are described in Supplemental Experimental Procedures.

ChIP-seq, de novo motif discovery, and functional clustering analyses

Tead4, JunD, Fra1, Fra2, H3K4me1, H3K4me3, and H3K27ac ChIP-seq data were obtained from the ENCODE project (http://genome.ucsc.edu/ENCODE/downloads.html), and data analysis and de novo motif discovery were performed using Homer software (http://homer.salk.edu/homer/). Functional clustering analysis was done using DAVID v6.7 (http://david.abcc.ncifcrf.gov/). Additional details are provided in the Supplemental Experimental Procedures.

Supplementary Material

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Acknowledgments

National Institutes of Health grant R01DK099510 (to J.M.) and American Cancer Society grant RSG-11-040-01-DDC (to J.M.) were the major support for this work. X.L. was also supported in part by a grant from national nature science foundation of China (81201913). R.J.D. is an investigator of the Howard Hughes Medical Institute. We also thank members of Mao lab for helpful discussion.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AUTHOR CONTRIBUTIONS

X.L., H.L., M.R., Q.L., J.L.C., and H.L.G. conducted the experiments and analyzed the data. J.O., L.J.Z., and J-S.P. performed bioinformatics analyses. A.M.M., R.J.D., and J.M. designed the experiment. J.M. supervised the project and wrote the manuscript.

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Supplementary Materials

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2
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3
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