Transcriptional Enhanced Associate Domain Research Articles

The YAP/TAZ Signaling Pathway in the Tumor Microenvironment and Carcinogenesis: Current Knowledge and Therapeutic Promises.

The yes-associated protein (YAP) and the transcriptional coactivator with PDZ-binding motif (TAZ) are transcriptional coactivators, members of the Hippo signaling pathway, which play a critical role in cell growth regulation, embryonic development, regeneration, proliferation, and cancer origin and progression. The mechanism involves the nuclear binding of the un-phosphorylated YAP/TAZ complex to release the transcriptional enhanced associate domain (TEAD) from its repressors. The active ternary complex is responsible for the aforementioned biological effects. Overexpression of YAP/TAZ has been reported in cancer stem cells and tumor resistance. The resistance involves chemotherapy, targeted therapy, and immunotherapy. This review provides an overview of YAP/TAZ pathways’ role in carcinogenesis and tumor microenvironment. Potential therapeutic alternatives are also discussed.

Synthetic molecules targeting yes associated protein activity as chemotherapeutics against cancer.

The Hippo signaling pathway extorts several signals that concomitantly target the activity of transcriptional cofactor yes associated protein (YAP). YAP is a key regulator that elicits signature gene expression by coupling with transcriptional enhanced associate domain (TEAD) family of transcriptional factors. The YAP-TEAD complex via target gene expression gets associated with the development, proliferation, and progression of cancerous cells. Moreover, YAP adorns cells with several oncogenic traits such as inhibition of apoptosis, enhanced proliferation, drug resistance, and immune response suppression, which later became associated with various diseases, particularly cancer. Therefore, inhibition of the YAP activity is an appealing and viable therapeutic target for cancer treatment. This review highlights the recent advances in existing and novel synthetic therapeutics targeting YAP inhibition and regulation. The synthetically produced YAPD93A belonging to cyclic peptides and DC-TEADin02 and vinyl sulfonamide class of compounds are the most potent compounds to inhibit the YAP-TEAD expression by targeting protein-protein interaction (IC50 =25nM) and palmitate binding central pocket of TEAD (IC50 =197nM), respectively. On the other hand, Chlorpromazine belonging to phenothiazines class has the least potential to suppress YAP via proteasomal degradation (cell viability value of <20% at 40µM).

Significance of TEAD Family in Diagnosis, Prognosis and Immune Response for Ovarian Serous Carcinoma.

PurposeTo explore the molecular profiles of transcriptional enhanced associate domain (TEAD) family in ovarian serous carcinoma (OSC).MethodsIn this study, we use bioinformatics methods including GEPIA, GE-mini, Oncomine 3.0, Kaplan–Meier plotter, cBioPortal, WebGestalt, TIMER2.0 and DiseaseMeth2.0, and in vitro experimental RT-PCR to assess the expression profiles and prognostic significance of TEAD family in OSC.ResultsAccording to the bioinformatics analysis, TEAD family was abnormally expressed in OSC. In terms of prognosis, Kaplan–Meier plotter indicated that OSC patients with high level of TEAD4 showed poor overall survival (OS), progression-free survival (PFS) and post progression survival (PPS). TEAD family also had significantly diagnostic values for OSC patients. Tumor Immune Estimation Resource (TIMER) algorithm indicated that TEAD family was significantly associated with different types of infiltrating immune cells, including B cells, macrophages, dendritic cells, neutrophils, CD8+ T cells and CD4+ T cells. Gene set enrichment analysis of TEAD family-associated coexpression genes was further explored. In in vitro experiments, the RT-PCR results showed the upregulated TEAD2/4 in OSC tissues and cells (A2780 and TOV112D). Moreover, decreased expression of TEAD2 could induce the ferroptosis through increasing the ROS accumulation.ConclusionThus, TEAD family correlated with the diagnosis, prognosis and immune infiltration in OSC. These results could provide comprehensive understanding of TEAD family in the diagnosis and prognosis of OSC patients.

Ophiopogonin B induces reactive oxygen species‑dependent apoptosis through the Hippo pathway in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a common malignant tumor in South China and is characterized by a high death rate. Ophiopogonin B (OP-B) is a bioactive component of Radix Ophiopogon japonicus, which is frequently used in traditional Chinese medicine to treat cancer. The present study aimed to examine the anti-cancer properties of OP-B on NPC cells. Cell viability and cell proliferation were measured using MTT and EdU assays. Flow cytometry was used to measure cell apoptosis, reactive oxygen species and mitochondrial membrane potential. Western blotting was used to investigate the expression of apoptosis and Hippo signaling pathway proteins. OP-B inhibited the proliferation of NPC cells by inducing apoptosis and disturbing the mitochondrial integrity. OP-B enhanced ROS accumulation. In addition, OP-B promoted the expression of mammalian STE20-like kinase 1, large tumor suppressor 1 and phosphorylated yes-associated protein (YAP) and suppressed the expression of YAP and transcriptional enhanced associate domain in NPC cells. OP-B increased the expression of forkhead box transcription factor O1 in the nuclear fraction. In conclusion, OP-B has therapeutic potential and feasibility in the development of novel YAP inhibitors for NPC.

Expression pattern of transcriptional enhanced associate domain family member 1 (Tead1) in developing mouse molar tooth

The Hippo pathway is essential for determining organ size by regulating cell proliferation. Previous reports have shown that impairing this pathway causes abnormal tooth development. However, the precise expression profile of the members of the transcriptional enhanced associate domain family (Tead), which are key transcription factors mediating Yap function, during tooth development is unclear. In this study, among the four isoforms of Tead (Tead1 - 4), only the expression of Tead1 mRNA was observed using semiquantitative RT- PCR in murine developing tooth germ at E16.5. The expression level of Tead1 mRNA in the excised murine mandibular molar tooth germ was significantly higher at E16.5 than at other developmental stages, as determined using quantitative PCR. We found that the mRNA expression of connective tissue growth factor (Ctgf), which is one of the Yap target genes directly controlling cell growth, changed consistently with that of Tead1 in developing molars. Fluorescent immunostaining revealed that Tead1 protein was expressed in both epithelial cells and mesenchymal cells of the dental lamina and dental epithelium, including the primary enamel knot during the cap stage. During the early bell stage (E16.5), Tead1 was expressed intensely in the inner and outer enamel epithelium, including the secondary enamel knot and the neighboring mesenchymal cells. Tead1 then specifically localized to the inner and outer enamel epithelium, which is responsible for enamel formation during the bell stage. These expression patterns were consistent with those of Yap, Taz, and Ctgf protein in developing molars. These results suggest that Tead1 acts as a mediator of the biological functions of Yap, such as the morphogenesis of cusp formation, during tooth development.

YTHDF1-regulated expression of TEAD1 contributes to the maintenance of intestinal stem cells

N6-methyladenosine (m6A) mRNA modification has been defined as a crucial regulator in various biological processes. Recent studies indicated an essential role of YTHDF1, an m6A reader, in the maintenance of intestinal stem cells (ISCs), while the detailed mechanism remains to be explored. By searching our m6A sequencing, RNA sequencing, and ribosome profiling data, we identified the transcriptional enhanced associate domain 1 (TEAD1) as a direct target of YTHDF1. We confirmed the presence of m6A modifications in TEAD1 mRNA and its binding with YTHDF1. Knockdown of either m6A methyltransferase METTL3 or YTHDF1 reduced the translation of TEAD1. TEAD1 was highly expressed in ISCs, while depletion of TEAD1 inhibited proliferation and induced differentiation of organoids. Overexpression of TEAD1 reversed the impaired stemness elicited by YTHDF1 depletion. These findings identify TEAD1 as a functional target of m6A-YTHDF1 in sustaining intestinal stemness.

Structure-based derivation and optimization of YAP-like coactivator-derived peptides to selectively target TEAD family transcription factors by hydrocarbon stapling and cyclization.

Human transcriptional enhanced associate domain (TEAD) family consists of four paralogous transcription factors that function to modulate gene expression by interacting with YAP-like coactivators and have been recognized as potential therapeutic targets of diverse diseases including lung cancer and gastric tumor. Here, we attempt to explore the systematic interaction profile between the 4 TEAD proteins and the peptides derived from the binding sites of 8 known YAP-like coactivators, in order to analyze the binding affinity and recognition specificity of these peptides toward the TEAD family, and to design hydrocarbon-stapled/cyclized peptides that can target the specific interaction profile for each coactivator. Structural, energetic, and dynamic investigations of TEAD-coactivator interactions reveal that the coactivators adopt three independent secondary structure regions (β-strand, α-helix, and Ω-loop) to surround on the surface of TEAD proteins, in which the α-helical and Ω-loop regions are primarily responsible for the interactions. Five α-helical peptides and four Ω-loop peptides are derived from the 8 YAP-like coactivators, and their systematic binding profile toward the 4 TEAD proteins is created, and hydrocarbon stapling and cyclization strategies are employed to constrain the free α-helical and Ω-loop peptides into their native conformations, respectively, thus effectively promoting peptide binding to TEADs. The all-hydrocarbon and disulfide bridges are designed to point out the TEAD-peptide complex interface, which would not disrupt the direct intermolecular interaction between the TEAD and peptide. Therefore, the stapling and cyclization only improve peptide binding affinity to these TEADs, but do not alter peptide recognition specificity over different TEADs.

RTEF-1 Inhibits Vascular Smooth Muscle Cell Calcification through Regulating Wnt/\u03b2-Catenin Signaling Pathway

Vascular calcification (VC) is highly prevailing in cardiovascular disease, diabetes mellitus, and chronic kidney disease and, when present, is associated with cardiovascular events and mortality. The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is regarded as the foundation for mediating VC. Related transcriptional enhancer factor (RTEF-1), also named as transcriptional enhanced associate domain (TEAD) 4 or transcriptional enhancer factor-3 (TEF-3), is a nuclear transcriptional factor with a potent effect on cardiovascular diseases, apart from its oncogenic role in the canonical Hippo pathway. However, the role and mechanism of RTEF-1 in VC, particularly in calcification of VSMCs, are poorly understood. Our results showed that RTEF-1 was reduced in calcified VSMCs. RTEF-1 significantly ameliorated β-glycerophosphate (β-GP)-induced VSMCs calcification, as detected by alizarin red staining and calcium content assay. Also, RTEF-1 reduced alkaline phosphatase (ALP) activity and decreased expressions of osteoblast markers such as Osteocalcin and Runt-related transcription factor-2 (Runx2), but increased expression of contractile protein, including SM α-actin (α-SMA). Additionally, RTEF-1 inhibited β-GP-activated Wnt/β-catenin pathway which plays a critical role in calcification and osteogenic differentiation of VSMCs. Specifically, RTEF-1 reduced the levels of Wnt3a, p-β-catenin (Ser675), glycogen synthase kinase-3β (GSK-3β), and p-GSK-3β (Ser9), but increased the levels of p-β-catenin (Ser33/37). Also, RTEF-1 increased the ratio of p-β-catenin (Ser33/37) to β-catenin proteins and decreased the ratio of p-GSK-3β (Ser9) to GSK-3β protein. LiCl, a Wnt/β-catenin signaling activator, was observed to reverse the protective effect of RTEF-1 overexpression on VSMCs calcification induced by β-GP. Accordingly, Dickkopf-1 (Dkk1), a Wnt antagonist, attenuated the role of RTEF-1 deficiency in β-GP-induced VSMCs calcification. Taken together, we concluded that RTEF-1 ameliorated β-GP-induced calcification and osteoblastic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling pathway.

Structure-Based Optimization of Conformationally Constrained Peptides to Target Esophageal Cancer TEAD Transcription Factor

The transcriptional enhanced associate domain (TEAD) transcription factor is a core regulator of Hippo signaling pathway and has been recognized as a promising therapeutic target against gastrointestinal tumors. The intermolecular interaction of TEAD with its transcription coactivator vestigial-like protein (VGLL) is involved in the tumorigenesis of esophageal cancer, which possesses an additional double-stranded β-sheet (DSβS) motif in its TEAD-recognition domain as compared to the classic coactivators YAP and TAZ. Here, the DSβS motif is examined systematically based on the complex crystal structure of TEAD with VGL. It is found that the DSβS is one of the most important recognition sites at TEAD–VGLL complex interface, which can confer stability and specificity to the complex recognition and elicit the transcription activation event. Protein segments with different lengths are derived from the DSβS motif of VGLL to define a number of linear peptides; their binding modes and affinities to TEAD are computationally analyzed to reveal that these linear peptides cannot maintain in native double-strand conformation in free solution state, thus unfavorable for their rebinding to TEAD–VGLL interface. A head-to-tail cyclization strategy is described to constrain the conformation of linear peptides, which can considerably reduce the peptide flexibility and help the peptide folding to an approximately native conformation in solution. We also demonstrate that the binding affinity and biological activity of cyclic peptides are improved effectively relative to their linear counterparts at molecular and cellular levels.

Multiscale Analysis of Extracellular Matrix Remodeling in the Failing Heart.

Cardiac ECM (extracellular matrix) comprises a dynamic molecular network providing structural support to heart tissue function. Understanding the impact of ECM remodeling on cardiac cells during heart failure (HF) is essential to prevent adverse ventricular remodeling and restore organ functionality in affected patients. We aimed to (1) identify consistent modifications to cardiac ECM structure and mechanics that contribute to HF and (2) determine the underlying molecular mechanisms. We first performed decellularization of human and murine ECM (decellularized ECM) and then analyzed the pathological changes occurring in decellularized ECM during HF by atomic force microscopy, 2-photon microscopy, high-resolution 3-dimensional image analysis, and computational fluid dynamics simulation. We then performed molecular and functional assays in patient-derived cardiac fibroblasts based on YAP (yes-associated protein)-transcriptional enhanced associate domain (TEAD) mechanosensing activity and collagen contraction assays. The analysis of HF decellularized ECM resulting from ischemic or dilated cardiomyopathy, as well as from mouse infarcted tissue, identified a common pattern of modifications in their 3-dimensional topography. As compared with healthy heart, HF ECM exhibited aligned, flat, and compact fiber bundles, with reduced elasticity and organizational complexity. At the molecular level, RNA sequencing of HF cardiac fibroblasts highlighted the overrepresentation of dysregulated genes involved in ECM organization, or being connected to TGFβ1 (transforming growth factor β1), interleukin-1, TNF-α, and BDNF signaling pathways. Functional tests performed on HF cardiac fibroblasts pointed at mechanosensor YAP as a key player in ECM remodeling in the diseased heart via transcriptional activation of focal adhesion assembly. Finally, in vitro experiments clarified pathological cardiac ECM prevents cell homing, thus providing further hints to identify a possible window of action for cell therapy in cardiac diseases. Our multiparametric approach has highlighted repercussions of ECM remodeling on cell homing, cardiac fibroblast activation, and focal adhesion protein expression via hyperactivated YAP signaling during HF.

VGLL1 phosphorylation and activation promotes gastric cancer malignancy via TGF-β/ERK/RSK2 signaling

We previously reported that vestigial-like 1 (VGLL1), a cofactor of transcriptional enhanced associate domain 4 (TEAD4), is transcriptionally regulated by PI3K and β-catenin signaling and is involved in gastric cancer malignancy. However, the precise mechanism underlying the regulation of VGLL1 activation remains unknown. Therefore, we aimed to investigate the molecular mechanism underlying the transforming growth factor-β (TGF-β)–mediated activation of VGLL1 and the VGLL1-TEAD4 interaction in gastric cancer cells. We showed that TGF-β enhanced VGLL1 phosphorylation and that this phosphorylated VGLL1 functioned as a transcription cofactor of TEAD4 in NUGC3 cells. TGF-β also increased the phosphorylation of ERK and ribosomal S6 kinase 2 (RSK2) in NUGC3 cells, thereby triggering the translocation of phosphorylated RSK2 to the nucleus. Site-directed mutagenesis and immunoprecipitation experiments revealed that RSK2 phosphorylated VGLL1 at S84 in the presence of TGF-β. Mutation of VGLL1 at S84 suppressed VGLL1-TEAD4 binding and the subsequent transcriptional activation of matrix metalloprotease 9 (MMP9). Moreover, VGLL1 peptide containing S84 suppressed the TGF-β-induced MMP9 expression and reduced the invasion and proliferation of gastric cancer cells, whereas VGLL1 peptide containing S84A did not. Furthermore, suppression of expression or activation of VGLL1 enhances the therapeutic effects of lapatinib. Collectively, these results indicate that VGLL1 phosphorylation via TGF-β/ERK/RSK2 signaling plays a crucial role in MMP9-mediated malignancy of gastric cancer. In addition, our study highlights the therapeutic potential of the peptide containing VGLL1 S84 for the treatment of gastric cancer.

Discovery of Covalent Inhibitors Targeting the Transcriptional Enhanced Associate Domain Central Pocket

Transcriptional enhanced associate domain (TEAD) transcription factors together with coactivators and corepressors modulate the expression of genes that regulate fundamental processes, such as organogenesis and cell growth, and elevated TEAD activity is associated with tumorigenesis. Hence, novel modulators of TEAD and methods for their identification are in high demand. We describe the development of a new “thiol conjugation assay” for identification of novel small molecules that bind to the TEAD central pocket. The assay monitors prevention of covalent binding of a fluorescence turn-on probe to a cysteine in the central pocket by small molecules. Screening of a collection of compounds revealed kojic acid analogues as TEAD inhibitors, which covalently target the cysteine in the central pocket, block the interaction with coactivator yes-associated protein with nanomolar apparent IC50 values, and reduce TEAD target gene expression. This methodology promises to enable new medicinal chemistry programs aimed at the modulation of TEAD activity.

Infarct Collagen Topography Regulates Fibroblast Fate via p38-Yes-Associated Protein Transcriptional Enhanced Associate Domain Signals.

Myocardial infarction causes spatial variation in collagen organization and phenotypic diversity in fibroblasts, which regulate the heart's ECM (extracellular matrix). The relationship between collagen structure and fibroblast phenotype is poorly understood but could provide insights regarding the mechanistic basis for myofibroblast heterogeneity in the injured heart. To investigate the role of collagen organization in cardiac fibroblast fate determination. Biomimetic topographies were nanofabricated to recapitulate differential collagen organization in the infarcted mouse heart. Here, adult cardiac fibroblasts were freshly isolated and cultured on ECM topographical mimetics for 72 hours. Aligned mimetics caused cardiac fibroblasts to elongate while randomly organized topographies induced circular morphology similar to the disparate myofibroblast morphologies measured in vivo. Alignment cues also induced myofibroblast differentiation, as >60% of fibroblasts formed αSMA (α-smooth muscle actin) stress fibers and expressed myofibroblast-specific ECM genes like Postn (periostin). By contrast, random organization caused 38% of cardiac fibroblasts to express αSMA albeit with downregulated myofibroblast-specific ECM genes. Coupling topographical cues with the profibrotic agonist, TGFβ (transforming growth factor beta), additively upregulated myofibroblast-specific ECM genes independent of topography, but only fibroblasts on flat and randomly oriented mimetics had increased percentages of fibroblasts with αSMA stress fibers. Increased tension sensation at focal adhesions induced myofibroblast differentiation on aligned mimetics. These signals were transduced by p38-YAP (yes-associated protein)-TEAD (transcriptional enhanced associate domain) interactions, in which both p38 and YAP-TEAD (yes-associated protein transcriptional enhanced associate domain) binding were required for myofibroblast differentiation. By contrast, randomly oriented mimetics did not change focal adhesion tension sensation or enrich for p38-YAP-TEAD interactions, which explains the topography-dependent diversity in fibroblast phenotypes observed here. Spatial variations in collagen organization regulate cardiac fibroblast phenotype through mechanical activation of p38-YAP-TEAD signaling, which likely contribute to myofibroblast heterogeneity in the infarcted myocardium.

&lt;p&gt;Rab14 Overexpression Promotes Proliferation and Invasion Through YAP Signaling in Non-Small Cell Lung Cancers&lt;/p&gt;

IntroductionSeveral reports have shown that Rab14 is dysregulated in human cancers suggesting that it is an oncogenic protein closely related to tumorigenesis. However, whether Rab14 plays a role in the development and progression of human non-small cell lung cancer (NSCLC) remains unclear.MethodsRab14 protein levels were examined in 115 cases of NSCLC tissues and 6 cancer cell lines. Rab14 knockdown was performed in H1299 and A549 cell lines. Rab14 plasmid transfection was performed in the LK2 cell line. The biological roles and mechanisms of Rab14 were examined using MTT, colony formation, Matrigel invasion assay, migration assay, cell cycle analysis, Western blotting, and RT-qPCR.ResultsWe found that Rab14 was upregulated in 65 of 115 lung cancer tissues. Rab14 high expression was significantly correlated with advanced TNM stage and nodal metastasis. Rab14 protein levels were higher in lung cancer cell lines than in normal bronchial cell line. Functionally, Rab14 overexpression increased growth rate, colony formation, invasion/migration ability and cell cycle progression, while Rab14 siRNA decreased the cell proliferation rate, colony numbers and inhibited invasion/migration ability and cell cycle progression. Rab14 upregulated cyclin D1, cyclin E, connective tissue growth factor (CTGF) and downregulated p27 protein and mRNA levels in both A549 and H1299 cell lines, while Rab14 siRNA produced the opposite effects. Further study showed that Rab14 overexpression increased luciferase reporter activity from transcriptional enhanced associate domain (TEAD) protein. Accordingly, Rab14 increased total Yes-associated protein (YAP) and nuclear YAP protein while decreased phosphorylated (p)-YAP and cytoplasmic YAP protein expression. Cycloheximide treatment showed that Rab14 downregulated the level of YAP degradation. Depletion of YAP using siRNA abolished the influence of Rab14 on cyclin D1, cyclin E, and CTGF. YAP knockdown also partly abolished the effects of Rab14 on cell proliferation and invasion.ConclusionIn summary, our data showed that Rab14 is overexpressed in human NSCLC. Rab14 facilitated proliferation and invasion, possibly through regulation of YAP signaling.

Abstract A45: In silico drug discovery targeting Hippo pathway and YAP-TEAD protein-protein interactions for small-molecule anticancer agent

Abstract The Hippo pathway is one of the important pathways regulating tissue growth and proliferation. Dysregulation of this pathway can result in overgrowth of phenotypes because of a malfunction of stem cell proliferation, differentiation, and apoptosis, which are directly involved with components to cancer cell developments. Protein-protein interaction (PPI) in YAP (Yes-associated protein) and TEAD (transcriptional enhancer associate domain) is a key interaction that regulates cancer cell growth in the Hippo pathway. Regulating YAP-TEAD protein-protein interaction by small molecules could play an important role in cancer therapy. In this study, we identified small molecules inhibiting YAP-TEAD interactions by an in silico approach. Molecular hits were identified by pharmacophore-based virtual screening. The hit compounds from virtual screening were tested by surface plasmon resonance (SPR), Luciferase activity test. As a result, several hit compounds were identified to inhibit YAP-TEAD protein-protein interactions. These selected hit compounds were also evaluated by molecular docking study and FMO (fragment molecular orbital) binding energy calculations. In our overall results, we identified hit compounds to inhibit YAP-TEAD PPIs and also analyzed hit compounds’ binding interaction energy to TEAD protein. Citation Format: Kim Jongwan, Hocheol Lim, K.T. No. In silico drug discovery targeting Hippo pathway and YAP-TEAD protein-protein interactions for small-molecule anticancer agent [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A45.

Small Molecule Dysregulation of TEAD Lipidation Induces a Dominant-Negative Inhibition of Hippo Pathway Signaling

The transcriptional enhanced associate domain (TEAD) family of transcription factors serves as the receptors for the downstream effectors of the Hippo pathway, YAP and TAZ, to upregulate the expression of multiple genes involved in cellular proliferation and survival. Recent work identified TEAD S-palmitoylation as critical for protein stability and activity as the lipid tail extends into a hydrophobic core of the protein. Here, we report the identification and characterization of a potent small molecule that binds the TEAD lipid pocket (LP) and disrupts TEAD S-palmitoylation. Using a variety of biochemical, structural, and cellular methods, we uncover that TEAD S-palmitoylation functions as a TEAD homeostatic protein level checkpoint and that dysregulation of this lipidation affects TEAD transcriptional activity in a dominant-negative manner. Furthermore, we demonstrate that targeting the TEAD LP is a promising therapeutic strategy for modulating the Hippo pathway, showing tumor stasis in a mouse xenograft model.

Targeting Human Hippo TEAD Binding Interface with YAP/TAZ-Derived, Flexibility-Reduced Peptides in Gastric Cancer

Human Hippo signaling pathway plays an important role in the tumorigenesis of diverse cancers and has been recognized as an attractive therapeutic target of gastric cancer. The transcriptional coactivators Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) are the major downstream effectors of Hippo, which interact primarily with transcriptional enhanced associate domain (TEAD) protein through their TEAD-binding domain (TBD). Competitive disruption of the TEAD–YAP/TAZ interaction using peptide inhibitors has been exploited as a potential strategy to treat gastric cancer by regulating Hippo signaling. Here, the crystal structures of TEAD complex with YAP/TAZ TBD domain are investigated systematically at structural, energetic and dynamic levels, from which two binding hotspots are identified; they separately correspond to an α-helix and a Ω-loop of TBD domain, and contribute essentially to the complex interaction. Several linear peptide segments derived from the hotspot regions are highly flexibility and exhibit moderate or modest affinity for TEAD. The Ω-loop-derived peptides are found to have a higher affinity, which are cyclized by introducing a disulfide bridge across their two termini. Affinity assay confirms that the cyclization can considerably improve peptide affinity by 3.7–6.6-fold. Computational analysis reveals that the designed cyclic peptides exhibit a decreased flexibility and intrinsic disorder; they can roughly maintain in native active conformation out of TBD protein context, with a reduced entropy cost upon binding to TEAD.

The Hippo Effector Transcriptional Coactivator with PDZ-Binding Motif Cooperates with Oncogenic β-Catenin to Induce Hepatoblastoma Development in Mice and Humans

Hepatoblastoma (HB) is the most common pediatric liver tumor. Though Wnt/β-catenin and Hippo cascades are implicated in HB development, studies on crosstalk between β-catenin and Hippo downstream effector transcriptional coactivator with PDZ-binding motif (TAZ) in HB are lacking. Expression levels of TAZ and β-catenin in human HB specimens were assessed by immunohistochemistry. Functional interplay between TAZ and β-catenin was determined by overexpression of an activated form of TAZ (TAZS89A), either alone or combined with an oncogenic form of β-catenin (ΔN90-β-catenin), in mouse liver via hydrodynamic transfection. Activation of TAZ often co-occurred with that of β-catenin in clinical specimens. Although the overexpression of TAZS89A alone did not induce hepatocarcinogenesis, concomitant overexpression of TAZS89A and ΔN90-β-catenin triggered the development of HB lesions exhibiting both epithelial and mesenchymal features. Mechanistically, TAZ/β-catenin-driven HB development required TAZ interaction with transcriptional enhanced associate domain factors. Blockade of the Notch cascade did not inhibit TAZ/β-catenin-dependent HB formation in mice but suppressed the mesenchymal phenotype. Neither Yes-associated protein nor heat shock factor 1 depletion affected HB development in TAZ/β-catenin mice. In human HB cell lines, silencing of TAZ resulted in decreased cell growth, which was further reduced when TAZ knockdown was associated with suppression of either β-catenin or Yes-associated protein. Overall, our study identified TAZ as a crucial oncogene in HB development and progression.

Lats1/2 Sustain Intestinal Stem Cells and Wnt Activation through TEAD-Dependent and Independent Transcription.

Intestinal homeostasis is tightly regulated by complex yet poorly understood signaling networks. Here, we demonstrate that Lats1/2, the core Hippo kinases, are essential to maintain Wnt pathway activity and intestinal stem cells. Lats1/2 deletion leads to loss of intestinal stem cells but drives Wnt-uncoupled crypt expansion. To explore the function of downstream transcriptional enhanced associate domain (TEAD) transcription factors, we identified a selective small-molecule reversible inhibitor of TEAD auto-palmitoylation that directly occupies its lipid-binding site and inhibits TEAD-mediated transcription invivo. Combining this chemical tool with genetic and proteomics approaches, we show that intestinal Wnt inhibition by Lats deletion is Yes-associated protein (YAP)/transcriptional activator with PDZ-binding domain (TAZ) dependent but TEAD independent. Mechanistically, nuclear YAP/TAZ interact with Groucho/Transducin-Like Enhancer of Split (TLE) to block Wnt/T-cell factor (TCF)-mediated transcription, and dual inhibition of TEAD and Lats suppresses Wnt-uncoupled Myc upregulation and epithelial over-proliferation in Adenomatous polyposis coli (APC)-mutated intestine. Our studies highlight a pharmacological approach to inhibit TEAD palmitoylation and have important implications for targeting Wnt and Hippo signaling in human malignancies.

The YAP1-NMU Axis Is Associated with Pancreatic Cancer Progression and Poor Outcome: Identification of a Novel Diagnostic Biomarker and Therapeutic Target.

Yes-associated protein (YAP)-1 is highly upregulated in pancreatic cancer and associated with tumor progression. However, little is known about the role of YAP1 and related genes in pancreatic cancer. Here, we identified target genes regulated by YAP1 and explored their role in pancreatic cancer progression and the related clinical implications. Analysis of different pancreatic cancer databases showed that Neuromedin U (NMU) expression was positively correlated with YAP1 expression in the tumor group. The Cancer Genome Atlas data indicated that high YAP1 and NMU expression levels were associated with poor mean and overall survival. YAP1 overexpression induced NMU expression and transcription and promoted cell motility in vitro and tumor metastasis in vivo via upregulation of epithelial–mesenchymal transition (EMT), whereas specific inhibition of NMU in cells stably expressing YAP1 had the opposite effect in vitro and in vivo. To define this functional association, we identified a transcriptional enhanced associate domain (TEAD) binding site in the NMU promoter and demonstrated that YAP1–TEAD binding upstream of the NMU gene regulated its transcription. These results indicate that the identified positive correlation between YAP1 and NMU is a potential novel drug target and biomarker in metastatic pancreatic cancer.