From birth to adulthood, the mammalian heart grows primarily through increasing cardiomyocyte (CM) size, which is known as maturational hypertrophic growth. The Hippo-YAP signaling pathway is well known for regulating heart development and regeneration, but its roles in CM maturational hypertrophy have not been clearly addressed. Vestigial-like 4 (VGLL4) is a crucial component of the Hippo-YAP pathway, and it functions as a suppressor of YAP/TAZ, the terminal transcriptional effectors of this signaling pathway. To develop an in vitro model for studying CM maturational hypertrophy, we compared the biological effects of T3 (triiodothyronine), Dex (dexamethasone), and T3/Dex in cultured neonatal rat ventricular myocytes (NRVMs). The T3/Dex combination treatment stimulated greater maturational hypertrophy than either the T3 or Dex single treatment. Using T3/Dex treatment of NRVMs as an in vitro model, we found that activation of VGLL4 suppressed CM maturational hypertrophy. In the postnatal heart, activation of VGLL4 suppressed heart growth, impaired heart function, and decreased CM size. On the molecular level, activation of VGLL4 inhibited the PI3K-AKT pathway, and disrupting VGLL4 and TEAD interaction abolished this inhibition. In conclusion, our data suggest that VGLL4 suppresses CM maturational hypertrophy by inhibiting the YAP/TAZ-TEAD complex and its downstream activation of the PI3K-AKT pathway.

Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, is widely prescribed for hyperlipidemia management. Recent studies also showed that it has therapeutic potential in various liver diseases. However, its effects on hepatomegaly and liver regeneration and the involved mechanisms remain unclear. Here, the study showed that fenofibrate significantly promoted liver enlargement and regeneration post-partial hepatectomy in mice, which was dependent on hepatocyte-expressed PPARα. Yes-associated protein (YAP) is pivotal in manipulating liver growth and regeneration. We further identified that fenofibrate activated YAP signaling by suppressing its K48-linked ubiquitination, promoting its K63-linked ubiquitination, and enhancing the interaction and transcriptional activity of the YAP-TEAD complex. Pharmacological inhibition of YAP-TEAD interaction using verteporfin or suppression of YAP using AAV Yap shRNA in mice significantly attenuated fenofibrate-induced hepatomegaly. Other factors, such as MYC, KRT23, RAS, and RHOA, might also participate in fenofibrate-promoted hepatomegaly and liver regeneration. These studies demonstrate that fenofibrate-promoted liver enlargement and regeneration are PPARα-dependent and partially through activating the YAP signaling, with clinical implications of fenofibrate as a novel therapeutic agent for promoting liver regeneration.

Hippo pathway functions as a tumor suppressor pathway by inhibiting the oncogenic potential of pathway effectors YAP/TAZ. However, YAP can also function as a context-dependent tumor suppressor in several types of cancer including clear cell renal cell carcinomas (ccRCC). Here we show that YAP blocks NF-κB signaling in ccRCC to inhibit cancer cell growth. Mechanistically, YAP inhibits the expression of ZHX2, a critical p65 co-factor in ccRCC. Furthermore, YAP competes with ZHX2 for binding to p65. Consequently, elevated nuclear YAP blocks the cooperativity between ZHX2 and p65, leading to diminished NF-κB target gene expression. Pharmacological inhibition of Hippo/MST1/2 blocked NF-κB transcriptional program and suppressed ccRCC cancer cell growth, which can be rescued by ZHX2/p65 overexpression. Our study uncovers a novel crosstalk between the Hippo and NF-κB pathways and its involvement in ccRCC growth inhibition, suggesting that targeting the Hippo pathway may provide a therapeutical opportunity for ccRCC treatment.

The major histocompatibility complex class I (MHC class I)-mediated tumor antigen processing and presentation (APP) pathway is essential for the recruitment and activation of cytotoxic CD8+ T lymphocytes (CD8+ CTLs). However, this pathway is frequently dysregulated in many cancers, thus leading to a failure of immunotherapy. Here, we report that activation of the tumor-intrinsic Hippo pathway positively correlates with the expression of MHC class I APP genes and the abundance of CD8+ CTLs in mouse tumors and patients. Blocking the Hippo pathway effector Yes-associated protein/transcriptional enhanced associate domain (YAP/TEAD) potently improves antitumor immunity. Mechanistically, the YAP/TEAD complex cooperates with the nucleosome remodeling and deacetylase complex to repress NLRC5 transcription. The upregulation of NLRC5 by YAP/TEAD depletion or pharmacological inhibition increases the expression of MHC class I APP genes and enhances CD8+ CTL-mediated killing of cancer cells. Collectively, our results suggest a crucial tumor-promoting function of YAP depending on NLRC5 to impair the MHC class I APP pathway and provide a rationale for inhibiting YAP activity in immunotherapy for cancer.

Yes-associated protein (YAP)-a major effector protein of the Hippo pathway- regulates cell proliferation, differentiation, apoptosis, and senescence. Amp-activated protein kinase (AMPK) is a key sensor that monitors cellular nutrient supply and energy status. Although YAP and AMPK are considered to regulate cellular senescence, it is still unclear whether AMPK is involved in YAP-regulated cellular senescence. Here, we found that YAP promoted AMPKα1 aggregation and localization around mitochondria by co-transfecting CFP-YAP and YFP-AMPKα1 plasmids. Subsequent live cell fluorescence resonance energy transfer (FRET) assay did not exhibit direct interaction between YAP and AMPKα1. FRET, Co-immunoprecipitation, and western blot experiments revealed that YAP directly bound to TEAD, enhancing the expression of AMPKα1 and p-AMPKα. Treatment with verteporfin inhibited YAP\'s binding to TEAD and reversed the elevated expression of AMPKα1 in the cells overexpressing CFP-YAP. Verteporfin also reduced the proportion of AMPKα1 puncta in the cells co-expressing CFP-YAP and YFP-AMPKα1. In addition, the AMPKα1 puncta were demonstrated to inhibit cell viability, autophagy, and proliferation, and ultimately promote cell senescence. In conclusion, YAP binds to TEAD to upregulate AMPKα1 and promotes the formation of AMPKα1 puncta around mitochondria under the condition of co-expression of CFP-YAP and YFP-AMPKα1, in which AMPKα1 puncta lead to cellular senescence.

Evolutionarily conserved, the Hippo signaling pathway is critical in regulating organ size and tissue homeostasis. The activity of this pathway is tightly regulated under normal circumstances, since its physical function is precisely maintained to control the rate of cell proliferation. Failure of maintenance leads to a variety of tumors. Our understanding of the mechanism of Hippo dysregulation and tumorigenesis is becoming increasingly precise, relying on the emergence of upstream inhibitor or activator and the connection linking Hippo target genes, mutations, and related signaling pathways with phenotypes. In this review, we summarize recent reports on the signaling network of the Hippo pathway in tumorigenesis and progression by exploring its critical mechanisms in cancer biology and potential targeting in cancer therapy.

Transcriptional enhanced associate domain family members 1 to 4 (TEADs) are a family of four transcription factors and the major transcriptional effectors of the Hippo pathway. In order to activate transcription, TEADs rely on interactions with other proteins, such as the transcriptional effectors Yes-associated protein and transcriptional co-activator with PDZ-binding motif. Nuclear protein interactions involving TEADs influence the transcriptional regulation of genes involved in cell growth, tissue homeostasis, and tumorigenesis. Clearly, protein interactions for TEADs are functionally important, but the full repertoire of TEAD interaction partners remains unknown. Here, we employed an affinity purification mass spectrometry approach to identify nuclear interacting partners of TEADs. We performed affinity purification mass spectrometry experiment in parallel in two different cell types and compared a wildtype TEAD bait protein to a nuclear localization sequence mutant that does not localize to the nucleus. We quantified the results using SAINT analysis and found a significant enrichment of proteins linked to DNA damage including X-ray repair cross-complementing protein 5 (XRCC5), X-ray repair cross-complementing protein 6 (XRCC6), poly(ADP-ribose) polymerase 1 (PARP1), and Rap1-interacting factor 1 (RIF1). In cellular assays, we found that TEADs co-localize with DNA damage-induced nuclear foci marked by histone H2AX phosphorylated on S139 (γH2AX) and Rap1-interacting factor 1. We also found that depletion of TEAD proteins makes cells more susceptible to DNA damage by various agents and that depletion of TEADs promotes genomic instability. Additionally, depleting TEADs dampens the efficiency of DNA double-stranded break repair in reporter assays. Our results connect TEADs to DNA damage response processes, positioning DNA damage as an important avenue for further research of TEAD proteins.

Hippo signaling restricts tumor growth by inhibiting the oncogenic potential of YAP/TAZ-TEAD transcriptional complex. Here, we uncover a context-dependent tumor suppressor function of YAP in androgen receptor (AR) positive prostate cancer (PCa) and show that YAP impedes AR+ PCa growth by antagonizing TEAD-mediated AR signaling. TEAD forms a complex with AR to enhance its promoter/enhancer occupancy and transcriptional activity. YAP and AR compete for TEAD binding and consequently, elevated YAP in the nucleus disrupts AR-TEAD interaction and prevents TEAD from promoting AR signaling. Pharmacological inhibition of MST1/2 or LATS1/2, or transgenic activation of YAP suppressed the growth of PCa expressing therapy resistant AR splicing variants. Our study uncovers an unanticipated crosstalk between Hippo and AR signaling pathways, reveals an antagonistic relationship between YAP and TEAD in AR+ PCa, and suggests that targeting the Hippo signaling pathway may provide a therapeutical opportunity to treat PCa driven by therapy resistant AR variants.

Transcriptional enhanced associate domain (TEAD) transcription factors play important roles in embryonic stem cell (ESC) renewal and differentiation. Four TEAD transcription factors (Tead1, Tead2, Tead3 and Tead4) and their various splice variants have been discovered in mice, but the expression pattern of them during pluripotency state transition is unclear. Here, we investigated the expression of TEADs and their splice variants in mouse ESCs at different pluripotent/differentiating states and adult mouse tissues. Our results preliminarily revealed the diversity and heterogeneity of TEAD family, which is helpful for understanding their overlapping and distinctive functions. Furthermore, a novel splice variant of Tead1 was identified and named Tead1 isoform 4.

The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.