The Hippo signalling pathway is prominent and governs cell proliferation and stem cell activity, acting as a growth regulator and tumour suppressor. Defects in Hippo signalling and hyperactivation of its downstream effector's Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) play roles in cancer development, implying that pharmacological inhibition of YAP and TAZ activity could be an effective cancer treatment strategy. Conversely, YAP and TAZ can also have beneficial effects in promoting tissue repair and regeneration following damage, therefore their activation may be therapeutically effective in certain instances. Recently, a complex network of intracellular and extracellular signalling mechanisms that affect YAP and TAZ activity has been uncovered. The YAP/TAZ-TEAD interaction leads to tumour development and the protein structure of YAP/TAZ-TEAD includes three interfaces and one hydrophobic pocket. There are clinical and preclinical trial drugs available to inhibit the hippo signalling pathway, but these drugs have moderate to severe side effects, so researchers are in search of novel, potent, and selective hippo signalling pathway inhibitors. In this review, we have discussed the hippo pathway in detail, including its structure, activation, and role in cancer. We have also provided the various inhibitors under clinical and preclinical trials, and advancement of small molecules their detailed docking analysis, structure-activity relationship, and biological activity. We anticipate that the current study will be a helpful resource for researchers.

Hippo is a signaling pathway that is evolutionarily conserved and plays critical roles in wound healing and tissue regeneration. Disruption of the transcriptional activity of both Hippo-associated factors, the yes-associated protein (YAP), and the transcriptional co-activator with PDZ binding motif (TAZ) has been associated with cardiovascular diseases, fibrosis, and cancer. This makes the Hippo pathway an appealing target for therapeutic interventions.

Prior research has indicated that medical gas plasma promotes wound healing by delivering a combination of reactive species directly to the affected areas. However, the involvement of YAP/TAZ and other signaling pathways in diabetic wound healing remains unexplored.

To this extent, ear wounds were generated and treated with gas plasma in streptozotocin (STZ)-induced diabetic mice. Transcriptome profiling at two wound healing stages (days 9 and 20 post-wounding) was performed in female and male mice. Additionally, we employed gene and protein expression analyses, utilizing immunohistological and -chemical staining of various targets as well as quantitative PCR and Western blot analysis.

Gas plasma treatment accelerated healing by increasing re-epithelialization and modifying extracellular matrix components. Transcriptomic profiling charting the major alterations in gene expression following plasma treatment was followed by a validation of several targets using transcriptional and translational quantification as well as localization analyses.

Our study evaluated the cellular regulation of essential targets of the Hippo and related pathways such as YAP/TAZ, β-catenin, tumor growth factor β, and oxidative stress signaling after plasma treatment. The activation of genes, pathways, and their regulators is an attractive therapeutic aim for a therapeutic intervention in dermal skin repair in diabetic diseases using medical gas plasmas.

Inactivation of tumor-suppressive Hippo signaling pathway is frequently observed in mesothelioma, which leads to the activation of yes-associated protein (YAP) and TAZ (also known as WW domain-containing transcription regulator 1; YAP/TAZ) transcriptional coactivators. YAP/TAZ form complexes with TEAD family members, DNA-binding proteins, to activate transcription, which promotes cancer cell growth and proliferation. Recently developed TEAD inhibitors exhibit antitumor activity by inhibiting the formation of the transcription complex through binding to TEAD; however, the antitumor activity of TEAD inhibitors against mesothelioma remains to be fully elucidated. Here, we show that the TEAD inhibitor K-975 acts as a pan-TEAD inhibitor and selectively inhibits the binding of TEAD-binding proteins, especially YAP/TAZ, in mesothelioma cells. In studies using a panel of mesothelioma cell lines, K-975 showed a significant growth inhibitory effect on Hippo-inactivated mesothelioma cells, but some of these cell lines exhibited primary resistance to K-975. Differential gene expression analysis revealed that cells resistant to K-975 exhibited activation of MYC signaling in the presence of K-975, and cells overexpressed with MYC showed strong drug resistance, in vitro and in vivo. Our study revealed the features of a subset of mesothelioma cells that proliferate in a TEAD-independent manner and provides important insights for the successful development of therapeutic strategies for mesothelioma with Hippo pathway inactivation.

Dedifferentiated liposarcoma (DDLPS) accounts for 15-20% of liposarcoma (LPS) and has high rates of local recurrence and distant metastasis. Hyperactivation of yes-associated protein (YAP) has been implicated in DDLPS development. However, the mechanisms that drive aberrant YAP signaling remain largely unknown. Here, we show that tumor protein p53 inducible nuclear protein 2 (TP53INP2) is a potential negative modulator of the malignant progression of DDLPS. The TP53INP2 protein expression level in tumor tissues from 79 patients with DDLPS decreased progressively. Compared with primary tumors, recurrent tumors also exhibited reduced TP53INP2 expression. More importantly, low TP53INP2 expression is correlated with poor prognosis. TP53INP2 gain- or loss-of-function experiments in DDLPS cell lines showed profound inhibitory effects on processes and properties linked with cancer malignancy, such as proliferation, migration, stemness and dedifferentiation. Mechanistically, TP53INP2 is located mainly in mitochondria and promotes mitophagic degradation of YAP in a VDAC1-dependent manner. The WW domain in YAP and the PPTY motif in VDAC1 are required for their interaction. Taken together, these data demonstrate that TP53INP2 represses the malignant progression of DDLPS by inactivating YAP via a mitophagy-dependent mechanism and that TP53INP2 may constitute a novel prognostic biomarker for advanced DDLPS.

Contact inhibition of proliferation is a critical cell density control mechanism governed by the Hippo signalling pathway. The biochemical signalling underlying cell density-dependent cues regulating Hippo signalling and its downstream effectors, YAP, remains poorly understood. Here, we reveal that the tight junction protein ZO-2 is required for the contact-mediated inhibition of proliferation. We additionally determined that the well-established molecular players of this process, namely Hippo kinase LATS1 and YAP, are regulated by ZO-2 and that the scaffolding function of ZO-2 promotes the interaction with and phosphorylation of YAP by LATS1. Mechanistically, YAP is phosphorylated when ZO-2 brings LATS1 and YAP together via its SH3 and PDZ domains, respectively, subsequently leading to the cytoplasmic retention and inactivation of YAP. In conclusion, we demonstrate that ZO-2 maintains Hippo signalling pathway activation by promoting the stability of LATS1 to inactivate YAP.

Osteoarthritis (OA) is a prevalent joint disease characterized by pain, disability, and loss of physical function, posing a challenge to public health. However, molecular mechanisms of OA pathogenesis have not been fully described. We report that tripartite motif containing 15 (TRIM15) is a regulator in chondrocyte senescence and OA. Our study revealed heightened expression of TRIM15 in chondrocytes of senescent cartilage from patients with OA and in aged wild-type mice. Using gain- and loss-of-function studies, we found that TRIM15 facilitated human chondrocyte senescence. Conditional deletion of Trim15 in mouse chondrocytes severely impaired skeletal growth, partially because of impaired embryonic chondrocyte senescence. Compared with conditionally knocked out Col2a1-CreERT2/Trim15flox/flox mice, Trim15flox/flox control mice exhibited accelerated OA phenotypes, increased senescence markers, and senescence-associated secretory phenotype during aging. Mechanistically, TRIM15 bound with yes-associated protein (YAP) and mediated K48-linked YAP ubiquitination at K254, which interrupted the interaction between YAP and angiomotin, leading to enhanced YAP nuclear translocation. Dysregulation of TRIM15-YAP and transcriptional coactivator with PDZ-binding motif (TAZ) signaling promoted OA progression in both the surgery-induced and natural aging-induced mouse OA model. Intra-articular injection of adeno-associated virus 5 (AAV5)-Trim15 shRNA decelerated OA progression in mice. In particular, YAP and TAZ protein amounts were increased in chondrocytes of patients with OA. Our preclinical results demonstrated that the AAV5-TRIM15 shRNA treatment protected human OA explants against degeneration through inhibiting chondrocyte senescence. Together, our findings underscore the potential of targeting TRIM15 in reshaping the aging cartilage microenvironment and suggest a promising therapeutic avenue for OA.

The "Kick and Kill" strategy, which aims to reactivate latent HIV reservoirs and facilitate the clearance of reactivated HIV-infected cells, has yet to achieve a functional cure due to the limited efficacy of current latency reversal agents. This study evaluates the combination efficacy of histone deacetylase (HDAC) inhibitor with poly(ADP-ribose) polymerase (PARP) inhibitor in latency reversal and immune-mediated clearance. Latently infected J-Lat cells and dual-fluorescent HIV-infected primary CD4 T cells were treated with the HDAC inhibitor (vorinostat) and one of four PARP inhibitors (olaparib, rucaparib, niraparib, or talazoparib). PARP inhibitors, when administered alone, showed no latency reversal activity. However, when combined with vorinostat, their efficacy increased threefold compared to vorinostat alone. This effect was mediated by the inhibition of tankyrase, a PARP superfamily member, which modulates the Hippo signaling pathway. In HIVGR670-infected primary cells, the combination reduced the reservoir size by 67%. In addition, talazoparib alone significantly reduced actively infected cells by 50%. Talazoparib-treated peripheral blood mononuclear cells co-cultured with K562 cells demonstrated enhanced NK-cell-mediated cytotoxicity, with a 10% reduction in K562 cell viability. These findings demonstrate that combining HDAC and PARP inhibitors augments latency reversal and reservoir reduction. With both the HDAC inhibitors and PARP inhibitors used in this study approved by the FDA for cancer treatment, this combination therapy holds strong potential for rapid clinical integration, contingent upon the confirmation of efficacy and safety in ongoing in vivo studies.

All pancreatic lineages originate from a transitory structure known as the multipotent progenitor epithelium (MPE), which is an endodermal placode formed via epithelial stratification. Cells within the MPE undergo de novo lumenogenesis to give rise to an epithelial plexus, which serves as a progenitor niche for subsequent development of endocrine, ductal and acinar cell types. Recent evidence suggests that Hippo signaling is required for pancreatic cell differentiation, but little is known about the function of Hippo signaling in the development of the MPE. Here, we characterize the expression of YAP1, TAZ, and the Hippo regulators LATS1/2 kinases and MERLIN in early murine pancreatic epithelium, during epithelial stratification, plexus development and emergence of endocrine cells. We find that YAP1 expression is relatively low in the pancreas bud during stratification but increases by E11.5. Intriguingly, we find differing patterns of TAZ and YAP1 immunoreactivty throughout pancreatic development. We further find that MERLIN and LATS1/2 kinases are expressed during the period of rapid stratification and become markedly apical at nascent lumens. To gain a better understanding of how Hippo signaling and lumen formation are connected, we analyzed the subcellular localization of Hippo signaling components during varying stages of lumen formation and found that they are dynamically localized during lumenogenesis. Together, our results point to a previously unsuspected relationship between Hippo signaling and lumen formation during pancreatic development.

The Hippo signaling pathway is a highly conserved signaling network for controlling organ size, tissue homeostasis, and regeneration. It integrates a wide range of intracellular and extracellular signals, such as cellular energy status, cell density, hormonal signals, and mechanical cues, to modulate the activity of YAP/TAZ transcriptional coactivators. A key aspect of Hippo pathway regulation involves its spatial organization at the plasma membrane, where upstream regulators localize to specific membrane subdomains to regulate the assembly and activation of the pathway components. This spatial organization is critical for the precise control of Hippo signaling, as it dictates the dynamic interactions between pathway components and their regulators. Recent studies have also uncovered the role of biomolecular condensation in regulating Hippo signaling, adding complexity to its control mechanisms. Dysregulation of the Hippo pathway is implicated in various pathological conditions, particularly cancer, where alterations in YAP/TAZ activity contribute to tumorigenesis and drug resistance. Therapeutic strategies targeting the Hippo pathway have shown promise in both cancer treatment, by inhibiting YAP/TAZ signaling, and regenerative medicine, by enhancing YAP/TAZ activity to promote tissue repair. The development of small molecule inhibitors targeting the YAP-TEAD interaction and other upstream regulators offers new avenues for therapeutic intervention. SIGNIFICANCE STATEMENT: The Hippo signaling pathway is a key regulator of organ size, tissue homeostasis, and regeneration, with its dysregulation linked to diseases such as cancer. Understanding this pathway opens new possibilities for therapeutic approaches in regenerative medicine and oncology, with the potential to translate basic research into improved clinical outcomes.

Yes-associated protein (YAP), a focal point of current biological research, is involved in regulating various life processes. In this report, live-cell fluorescence resonance energy transfer (FRET) imaging was employed to unravel the YAP complexes in MCF-7 cells. Fluorescence imaging of living cells co-expressing CFP (cyan fluorescent protein)-YAP and YFP (yellow fluorescent protein)-LATS1 (large tumor suppressor 1) plasmids revealed that YAP promoted LATS1 oligomerization around mitochondria. Moreover, FRET two-hybrid assay showed that YAP directly interacted with LATS1 to form dimer. Similarly, we found that YAP directly interacted with large tumor suppressor 2 (LATS2) to form a heterotrimer with 1:2 in cytoplasm and around mitochondria. In addition, YAP directly interacted with angiomotin (AMOT) to form a heterodimer in cytoplasm. However, YAP did not interact with O-linked N-acetylglucosamine transferase (OGT). Furthermore, FRET assay also indicated that YAP exhibited a higher affinity with AMOT, followed by LATS1, and least with LATS2. In summary, YAP directly interacts with LATS1 and AMOT to form a heterodimer, with LATS2 to form a heterotrimer with 1:2, and shows a preference for binding to AMOT, followed by LATS1, and lastly LATS2, providing new insights into the Hippo-YAP signaling pathway.

Epithelial barriers serve as critical defense lines against microbial infiltration and maintain tissue homeostasis. E-cadherin, an essential component of adherens junctions, has emerged as a pivotal molecule that secures epithelial homeostasis. Lately, its pleiotropic role beyond barrier function, including its involvement in immune responses, has become more evident. Herein, we delve into the intricate relationship between (dys)regulation of epithelial homeostasis and the versatile functionality of E-cadherin, describing complex mechanisms that underlie barrier integrity and disruption in disease pathogenesis such as bacterial infection and inflammation, among others. Clinical implications of E-cadherin perturbations in host pathophysiology are emphasized; downregulation, proteolytic phenomena, abnormal localization/signaling and aberrant immune reactions are linked with a broad spectrum of pathology beyond infectious diseases. Finally, potential therapeutic interventions that may harness E-cadherin to mitigate barrier-associated tissue damage are explored. Overall, this review highlights the crucial role of E-cadherin in systemic health, offering insights that could pave the way for strategies to reinforce/restore barrier integrity and treat related diseases.

Elevated uptake of saturated fatty acid palmitic acid (PA) is associated with tumor metastasis; however, the precise mechanisms remain partially understood, hindering the development of therapy for PA-driven tumor metastasis. The Hippo-Yes-associated protein (Hippo/YAP) pathway is implicated in cancer progression. Here it is shown that a high-palm oil diet potentiates tumor metastasis in murine xenografts in part through YAP. It is found that the palmitoyltransferase ZDHHC15 is a YAP-regulated gene that forms a feedback loop with YAP. Notably, PA drives the ZDHHC15-YAP feedback loop, thus enforces YAP signaling, and hence promotes tumor metastasis in murine xenografts. In addition, it is shown that ZDHHC15 associates with Kidney and brain protein (KIBRA, also known as WW- and C2 domain-containing protein 1, WWC1), an upstream component of Hippo signaling, and mediates its palmitoylation. KIBRA palmitoylation leads to its degradation and regulates its subcellular localization and activity toward the Hippo/YAP pathway. Moreover, PA enhances KIBRA palmitoylation and degradation. It is further shown that combinatorial targeting of YAP and fatty acid synthesis exhibits augmented effects against metastasis formation in mice fed with a Palm diet. Collectively, these findings uncover a ZDHHC15-YAP feedback loop as a previously unrecognized mechanism underlying PA-promoted tumor metastasis and support targeting YAP and fatty acid synthesis as potential therapeutic targets in PA-driven tumor metastasis.

Genotype-informed anticancer therapies such as BRAF inhibitors can show remarkable clinical efficacy in BRAF-mutant melanoma; however, drug resistance poses a major hurdle to successful cancer treatment. Many resistance events to targeted therapies have been identified, suggesting a complex path to improve therapeutics. Here, we showed the utility of a piggyBac transposon activation mutagenesis screen for the efficient identification of genes that are resistant to BRAF inhibition in melanoma. Although several forward genetic screens performed in the same context have identified a broad range of resistance genes that poorly overlap, an integrative analysis revealed a much smaller functional diversity of resistance mechanisms, including reactivation of the MAPK pathway, PI3K-AKT pathway, and Hippo pathway, suggesting that a relatively small number of therapeutic strategies might overcome resistance manifested by a large gene set. Moreover, we illustrated the pivotal role of the Hippo pathway effector TAZ (encoded by the WWTR1 gene) in mediating BRAF inhibition resistance through transcriptional regulation of receptor tyrosine kinases and through interactions with the E3 ubiquitin ligase NEDD4L.

The aim of our study was to examine the association of Angiomotin (Amot-p130) and Yes-associated protein 1 (YAP1) expressions and their prognostic significance in epithelial ovarian cancer (EOC).

A total of 100 primary EOC samples were obtained for immunohistochemical analysis of Amot-p130 and YAP1 expressions. Correlation analysis was performed between Amot-p130 or YAP1 and clinical factors. The overall survival time was calculated.

Low Amot-p130 and high YAP1 nuclear expression were identified in 34 and 56 of 100 EOC tissues, respectively. Both low Amot-p130 and high YAP1 nuclear expression were associated with advanced tumor stage, high-grade carcinoma, and non-response to chemotherapy (p<0.05). They were also associated with shorter overall survival time (p<0.05) by log-rank test. A marker of low Amot-p130 and high YAP1 expression was associated with high-grade ovarian carcinoma, late-stage disease, non-response to chemotherapy, and shorter overall survival time (p<0.05).

Low Amot-p130 and high YAP1 nuclear expression can provide additional prognostic information for patients with EOC. A marker of low Amot-p130 and high YAP1 expression may be a potent predictor of poor prognosis in patients with epithelial ovarian cancer.

The cornea, the anterior meniscus-shaped transparent and refractive structure of the eyeball, is the first mechanical barrier of the eye. Its functionality heavily relies on the health of its endothelium, its most posterior layer. The treatment of corneal endothelial cells (CECs) deficiency is allogeneic corneal graft using stored donor corneas. One of the main goals of eye banks is to maintain endothelial cell density (ECD) and endothelial barrier function, critical parameters influencing transplantation outcomes. Unlike in vivo, the stored cornea is not subjected to physiological mechanical stimuli, such as the hydrokinetic pressure of the aqueous humor and intraocular pressure (IOP). YAP (Yes-Associated Protein), a pivotal transcriptional coactivator, is recognized for its ability to sense diverse biomechanical cues and transduce them into specific biological signals, varying for each cell type and mechanical forces. The biomechanical cues that might regulate YAP in human corneal endothelium remain unidentified. Therefore, we investigated the expression and subcellular localization of YAP in the endothelium of corneas stored in organ culture (OC). Our findings demonstrated that CEC morphology, ECD and cell-cell interactions are distinctly and differentially associated with modifications in the expression, subcellular localization and phosphorylation of YAP. Notably, this phosphorylation occurs in the basal region of the primary cilium, which may play central cellular roles in sensing mechanical stimuli. The sustained recruitment of YAP in cellular junctions, nucleus, and cilium under long-term OC conditions strongly indicates its specific role in maintaining CEC homeostasis. Understanding these biophysical influences could aid in identifying molecules that promote homeostasis and enhance the functionality of CECs.

Decades of research into the Hippo signaling pathway have greatly advanced our understanding of its roles in organ growth, tissue regeneration, and tumorigenesis. The Hippo pathway is frequently dysregulated in human cancers and is recognized as a prominent cancer signaling pathway. Hence, the Hippo pathway represents an ideal molecular target for cancer therapies. This review will highlight recent advancements in targeting the Hippo pathway for cancer treatment and discuss the potential opportunities for developing new therapeutic modalities.

Stem cells regenerate differentiated cells to maintain and repair tissues and organs. They also replenish themselves, i.e. self-renew, to support a lifetime of regenerative capacity. Here we study the renewal of skeletal muscle stem cell (MuSC) during regeneration. The transcriptional co-factors TAZ/YAP (via the TEAD transcription factors) regulate cell cycle and growth while the transcription factor YY1 regulates metabolic programs for MuSC activation. We show that MPP7 and AMOT join TAZ and YY1 to regulate a selected number of common genes that harbor TEAD and YY1 binding sites. Among these common genes, Carm1 can direct MuSC renewal. We demonstrate that the L27 domain of MPP7 enhances the interaction as well as the transcriptional activity of TAZ and YY1, while AMOT acts as an intermediate to bridge them together. Furthermore, MPP7, TAZ and YY1 co-occupy the promoters of Carm1 and other common downstream genes. Our results define a renewal program comprised of two progenitor transcriptional programs, in which selected key genes are regulated by protein-protein interactions, dependent on promoter context.

The Hippo pathway is highly evolved to maintain tissue homeostasis in diverse species by regulating cell proliferation, differentiation, and apoptosis. In tumor biology, the Hippo pathway is a prime example of signaling molecules involved in cancer progression and metastasis. Hippo core elements LATS1, LATS2, MST1, YAP, and TAZ have critical roles in the maintenance of traditional tissue architecture and cell homeostasis. However, in cancer development, dysregulation of Hippo signaling results in tumor progression and the formation secondary cancers. Hippo components not only transmit biochemical signals but also act as mediators of mechanotransduction pathways during malignant neoplasm development and metastatic disease. This review confers knowledge of Hippo pathway core components and their role in cancer progression and metastasis and highlights the clinical role of Hippo pathway in cancer treatment. The Hippo signaling pathway and its unresolved mechanisms hold great promise as potential therapeutic targets in the emerging field of metastatic cancer research.

All pancreatic lineages originate from a transitory structure known as the multipotent progenitor epithelium (MPE), which is a placode formed via epithelial stratification. Cells within the MPE undergo de novo lumenogenesis to give rise to an epithelial plexus, which serves as a progenitor niche for subsequent development of endocrine, ductal and acinar cell types. Recent evidence suggests that Hippo signaling is required for pancreatic cell differentiation, but little is known about the function of Hippo signaling in the development of the MPE. Here, we characterize the expression of YAP1, TAZ, and the Hippo regulators LATS1/2 kinases and MERLIN in early murine pancreatic epithelium, during epithelial stratification, plexus development and emergence of endocrine cells. We find that YAP1 expression is relatively low in the pancreas bud during stratification, but increases by E11.5. Intriguingly, we find that TAZ, but not YAP1, is expressed in early endocrine cells. We further find that MERLIN and LATS1/2 kinases are robustly expressed during the period of rapid stratification and become markedly apical at nascent lumens. To gain a better understanding of how Hippo signaling and lumen formation are connected, we analyzed the expression of Hippo signaling components in an in vitro model of lumen formation and found that they are dynamically regulated during lumenogenesis. Together, our results point to a relationship between Hippo signaling and lumen formation during pancreatic development.

YAP1 expression in the early mouse pancreatic anlagen is low until approximately E11.5, when it becomes localized to cell nuclei in multipotent progenitor cells. At E14.5, we find nuclear YAP1 in ductal cells.YAP1 is not expressed in early and midgestation endocrine cells. By contrast, TAZ is expressed in first transition endocrine cells.Hippo regulators MERLIN and LATS1/2 kinases are robustly expressed in the early pancreatic bud by E10.5. Both MERLIN and LATS1/2 exhibit strong apical localization in epithelial cells at nascent microlumens. Using in vitro models of de novo pancreas lumen formation, we show that YAP1 nuclear localization is high in early phases of lumen formation and gradually decreases as lumens matures.

Human pluripotent stem cells (hPSCs) have the potential to differentiate into various cell types, including pancreatic insulin-producing β cells, which are crucial for developing therapies for diabetes. However, current methods for directing hPSC differentiation towards pancreatic β-like cells are often inefficient and produce cells that do not fully resemble the native counterparts. Here, we report that highly selective tankyrase inhibitors, such as WIKI4, significantly enhances pancreatic differentiation from hPSCs. Our results show that WIKI4 promotes the formation of pancreatic progenitors that give rise to islet-like cells with improved β-like cell frequencies and glucose responsiveness compared to our standard cultures. These findings not only advance our understanding of pancreatic development, but also provide a promising new tool for generating pancreatic cells for research and potential therapeutic applications.

Angiomiotin (AMOT) family comprises three members: AMOT, AMOT-like protein 1 (AMOTL1), and AMOT-like protein 2 (AMOTL2). AMOTL2 is widely expressed in endothelial cells, epithelial cells, and various cancer cells. Specifically, AMOTL2 predominantly localizes in the cytoplasm and nucleus in human normal cells, whereas associates with cell-cell junctions and actin cytoskeleton in non-human cells, and locates at cell junctions or within the recycling endosomes in cancer cells. AMOTL2 is implicated in regulation of tube formation, cell polarity, and shape, although the specific impact on tumorigenesis remains to be conclusively determined. It has been shown that AMOTL2 enhances tumor growth and metastasis in pancreatic, breast, and colon cancer, however inhibits cell proliferation and migration in lung, hepatocellular cancer, and glioblastoma. In addition to its role in cell shape and cytoskeletal dynamics through co-localization with F-actin, AMOTL2 modulates the transcription of Yes-associated protein (YAP) by binding to it, thereby affecting its phosphorylation and cellular sequestration. Furthermore, the stability and cellular localization of AMOTL2, influenced by its phosphorylation and ubiquitination mediated by specific proteins, affects its cellular function. Additionally, we observe that AMOTL2 is predominantly downregulated in some tumors, but significantly elevated in colorectal adenocarcinoma (COAD). Moreover, overall analysis, GSEA and ROC curve analysis indicate that AMOTL2 exerts as an oncogenic protein in COAD by modulating Wnt pathway, participating in synthesis of collagen formation, and interacting with extracellular matrix receptor. In addition, AMOTL2 potentially regulates the distribution of immune cells infiltration in COAD. In summary, AMOTL2 probably functions as an oncogene in COAD. Consequently, further in-depth mechanistic research is required to elucidate the precise roles of AMOTL2 in various cancers.

Circular RNAs (circRNAs) and eukaryotic translation initiation factor 4A3 (EIF4A3) have been reported to participate in the pathogenesis of nasopharyngeal carcinoma (NPC), but their mechanism has not been fully understood. This research aimed to confirm the role and regulatory mechanism of hsa_circ_0049396 interacting with EIF4A3 in NPC tumorigenesis. Quantitative real time polymerase chain reaction (qRT-PCR) was executed to detect the levels of hsa_circ_0049396 and EIF4A3. Cell function experiments and nude mice xenograft assay were used to confirm the role of hsa_circ_0049396 in NPC. The regulatory effect of EIA4A3 on hsa_circ_0049396 was determined by circInteractome prediction, RNA binding protein immunoprecipitation (RIP) assay, and qRT-PCR. In addition, the Hippo-YAP pathway-related proteins and EIF4A3 protein were detected by western blotting. hsa_circ_0049396 was proved to be downregulated in NPC samples, and its low expression indicated the poor prognosis of NPC. After upregulating hsa_circ_0049396 in NPC cells, the proliferation, migration, invasion, and tumor growth in vivo were suppressed by inhibiting the Hippo-YAP pathway. Moreover, EIF4A3 bound to the flanking regions of the hsa_circ_0049396 to enhance hsa_circ_0049396 expression in NPC cells. hsa_circ_0049396 mediated by EIF4A3 in NPC can attenuate NPC tumorigenesis by inhibiting the Hippo-YAP pathway. This finding may provide a potential early diagnostic biomarker or drug target to improve the precision medicine approaches of NPC.

Our understanding of the transitions of human embryonic stem cells (hESCs) between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of hESCs as they transition from primed to naïve pluripotency which includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for formation of the actin ring, to establish uniform cell mechanics within naïve colonies, to promote nuclear translocation of the Hippo pathway effectors YAP and TAZ, and for effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. Moreover, expression of YAP-S127A partially restores the actin filament fence with Arp2/3 complex inhibition, suggesting that actin filament remodeling is both upstream and downstream of YAP activity. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.

Opposite expression and pro- or anti-cancer function of YAP and its paralog TAZ/WWTR1 stratify cancers into binary YAPon and YAPoff classes. These transcriptional coactivators are oncogenic in YAPon cancers. In contrast, YAP/TAZ are silenced epigenetically along with their integrin and extracellular matrix adhesion target genes in neural and neuroendocrine YAPoff cancers (e.g., small cell lung cancer, retinoblastoma). Forced YAP/TAZ expression induces these targets, causing cytostasis in part through Integrin-αV/β5, independent of the integrin-binding RGD ligand. Other effectors of this anticancer YAP function are unknown. Here, using clustered regularly interspaced short palindromic repeats (CRISPR) screens, we link the Netrin receptor UNC5B to YAP-induced cytostasis in YAPoff cancers. Forced YAP expression induces UNC5B through TEAD DNA-binding partners, as either TEAD1/4-loss or a YAP mutation that disrupts TEAD-binding (S94A) blocks, whereas a TEAD-activator fusion (TEAD(DBD)-VP64) promotes UNC5B induction. Ectopic YAP expression also upregulates UNC5B relatives and their netrin ligands in YAPoff cancers. Netrins are considered protumorigenic, but knockout and peptide/decoy receptor blocking assays reveal that in YAPoff cancers, UNC5B and Netrin-1 can cooperate with integrin-αV/β5 to mediate YAP-induced cytostasis. These data pinpoint an unsuspected Netrin-1/UNC5B/integrin-αV/β5 axis as a critical effector of YAP tumor suppressor activity.

Netrins are widely perceived as procancer proteins; however, we uncover an anticancer function for Netrin-1 and its receptor UNC5B.

Angiomotin-like 2 (AMOTL2) is related to numerous physiological and pathological conditions by affecting signal transduction. However, whether AMOTL2 is linked to pulmonary arterial hypertension (PAH) has not been addressed. This work aimed to investigate the potential role of AMOTL2 in PAH. A decrease in AMOTL2 abundance was observed in the lungs of PAH rats. The upregulation of AMOTL2 significantly decreased right ventricle systolic pressure and right ventricular hypertrophy in PAH rats. Overexpression of AMOTL2 also led to a noteworthy decrease in vascular wall thickness, pulmonary artery area, and collagen deposition in rats with PAH. AMOTL2 was downregulated in hypoxia-stimulated pulmonary arterial smooth muscle cells (PASMCs). Moreover, AMOTL2 overexpression impeded hypoxia-evoked proliferation, migration, and phenotypic transformation in rat PASMCs. Mechanistic investigation revealed that Yes-associated protein 1 (YAP1) activation in PAH rats or hypoxia-stimulated PASMCs was markedly inhibited by AMOTL2 overexpression, which was associated with increased large tumor suppressor 1/2 phosphorylation. The inhibition of large tumor suppressor 1/2 reversed the AMOTL2-mediated inactivation of YAP1. Restoring the activity of YAP1 reversed the inhibitory effect of AMOTL2 on hypoxia-evoked proliferation, migration, and phenotypic transformation of PASMCs. Collectively, these results suggest that AMOTL2 can ameliorate PAH in a rat model by interfering with pulmonary arterial remodeling via the inactivation of YAP1 signaling. Our work indicates that AMOTL2 may be a candidate target for novel drug development for the treatment of PAH.

Epithelia consist of proliferating and differentiating cells that often display patterned arrangements. However, the mechanism regulating these spatial arrangements remains unclear. Here, we show that cell-cell adhesion dictates multicellular patterning in stratified epithelia. When cultured keratinocytes, a type of epithelial cell in the skin, are subjected to starvation, they spontaneously develop a pattern characterized by areas of high and low cell density. Pharmacological and knockout experiments show that adherens junctions are essential for patterning, whereas the mathematical model that only considers local cell-cell adhesion as a source of attractive interactions can form regions with high/low cell density. This phenomenon, called cell-cell adhesion-induced patterning (CAIP), influences cell differentiation and proliferation through Yes-associated protein modulation. Starvation, which induces CAIP, enhances the stratification of the epithelia. These findings highlight the intrinsic self-organizing property of epithelial cells.

Increased extracellular matrix (ECM) and matrix stiffness promote solid tumor progression. However, mechanotransduction in cancers arising in mechanically active tissues remains underexplored. Here, we report upregulation of multiple ECM components accompanied by tissue stiffening in vocal fold cancer (VFC). We compare non-cancerous (NC) and patient-derived VFC cells - from early (mobile, T1) to advanced-stage (immobile, T3) cancers - revealing an association between VFC progression and cell-surface receptor heterogeneity, reduced laminin-binding integrin cell-cell junction localization and a flocking mode of collective cell motility. Mimicking physiological movement of healthy vocal fold tissue (stretching/vibration), decreases oncogenic nuclear β-catenin and YAP levels in VFC. Multiplex immunohistochemistry of VFC tumors uncovered a correlation between ECM content, nuclear YAP and patient survival, concordant with VFC sensitivity to YAP-TEAD inhibitors in vitro. Our findings present evidence that VFC is a mechanically sensitive malignancy and restoration of tumor mechanophenotype or YAP/TAZ targeting, represents a tractable anti-oncogenic therapeutic avenue for VFC.

RhoA (ras homolog family member A) is a small G-protein that transduces intracellular signaling to regulate a broad range of cellular functions such as cell growth, proliferation, migration, and survival. RhoA serves as a proximal downstream effector of numerous G protein-coupled receptors (GPCRs) and is also responsive to various stresses in the heart. Upon its activation, RhoA engages multiple downstream signaling pathways. Rho-associated coiled-coil-containing protein kinase (ROCK) is the first discovered and best characterized effector or RhoA, playing a major role in cytoskeletal arrangement. Many other RhoA effectors have been identified, including myocardin-related transcription factor A (MRTF-A), Yes-associated Protein (YAP) and phospholipase Cε (PLCε) to regulate transcriptional and post-transcriptional processes. The role of RhoA signaling in the heart has been increasingly studied in last decades. It was initially suggested that RhoA signaling pathway is maladaptive in the heart, but more recent studies using cardiac-specific expression or deletion of RhoA have revealed that RhoA activation provides cardioprotection against stress through various mechanisms including the novel role of RhoA in mitochondrial quality control. This review summarizes recent advances in understanding the role of RhoA in the heart and its signaling pathways to prevent progression of heart disease.

Decades of work in developmental genetics has given us a deep mechanistic understanding of the fundamental signaling pathways underlying animal development. However, little is known about how these pathways emerged and changed over evolutionary time. Here, we review our current understanding of the evolutionary emergence of the Hippo pathway, a conserved signaling pathway that regulates tissue size in animals. This pathway has deep evolutionary roots, emerging piece by piece in the unicellular ancestors of animals, with a complete core pathway predating the origin of animals. Recent functional studies in close unicellular relatives of animals and early-branching animals suggest an ancestral function of the Hippo pathway in cytoskeletal regulation, which was subsequently co-opted to regulate proliferation and animal tissue size.

The Motin family proteins (Motins) are a class of scaffolding proteins consisting of Angiomotin (AMOT), AMOT-like protein 1 (AMOTL1), and AMOT-like protein 2 (AMOTL2). Motins play a pivotal role in angiogenesis, tumorigenesis, and neurogenesis by modulating multiple cellular signaling pathways. Recent findings indicate that Motins are components of the Hippo pathway, a signaling cascade involved in development and cancer. This review discusses how Motins are integrated into the Hippo signaling network, as either upstream regulators or downstream effectors, to modulate cell proliferation and migration. The repression of YAP/TAZ by Motins contributes to growth inhibition, whereas subcellular localization of Motins and their interactions with actin fibers are critical in regulating cell migration. The net effect of Motins on cell proliferation and migration may contribute to their diverse biological functions.

Metastasis is the major culprit of treatment failure in nasopharyngeal carcinoma (NPC). Aryl hydrocarbon receptor nuclear translocator like 2 (ARNTL2), a core circadian gene, plays a crucial role in the development of various tumors. Nevertheless, the biological role and mechanism of ARNTL2 are not fully elucidated in NPC. In this study, ARNTL2 expression was significantly upregulated in NPC tissues and cells. Overexpression of ARNTL2 facilitated NPC cell migration and invasion abilities, while inhibition of ARNTL2 in similarly treated cells blunted migration and invasion abilities in vitro. Consistently, in vivo xenograft tumor models revealed that ARNTL2 silencing reduced nude mice inguinal lymph node and lung metastases, as well as tumor growth. Mechanistically, ARNTL2 negatively regulated the transcription expression of AMOTL2 by directly binding to the AMOTL2 promoter, thus reducing the recruitment and stabilization of AMOTL2 to LATS1/2 kinases, which strengthened YAP nuclear translocation by suppressing LATS-dependent YAP phosphorylation. Inhibition of AMOTL2 counteracted the effects of ARNTL2 knockdown on NPC cell migration and invasion abilities. These findings suggest that ARNTL2 may be a promising therapeutic target to combat NPC metastasis and further supports the crucial roles of circadian genes in cancer development.

Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear.

We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays.

Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation.

These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.

Angiomotins (Amots) are a family of adaptor proteins with important roles in cell growth, migration, and proliferation. The Amot coiled-coil homology (ACCH) domain has a high affinity for non-phosphorylated and mono-phosphorylated phosphatidylinositol which provides specificity in the membrane association. The membrane specificity is linked with targeting and recycling of the membrane protein to maintain normal cell phenotypes and function. Therefore, we endeavored to find the minimal amount of DMSO to stabilize the Amot lipid binding domain to eventually understand the protein function by studying its atomic structure. Our laboratory looked to determine the structure using nuclear magnetic resonance (NMR), which requires higher protein concentrations than those possible in our current buffered solutions. Based on literature reported on other proteins, DMSO can be used as a stabilizing agent up to 33-70%. Therefore, this work shows our preliminary findings for the minimal amount of dimethyl sulfoxide (DMSO) needed to stabilize the domain at higher concentrations without disrupting its native structure. To that end, we determined DMSO related changes in protein structure by analyzing shifts in the melting point determined by dynamic scanning fluorescence measurements. As a result, we found that the ACCH domain is denatured in solutions >10% DMSO.

Angiomotin (Amot) family members, including Amot, Amot-like protein 1 (Amotl1) and Amot-like protein 2 (Amotl2), have been found to interact with angiostatins. In addition, Amot family members are involved in various physiological and pathological functions such as embryonic development, angiogenesis and tumorigenesis. Some studies have also demonstrated its regulation in signaling pathways such as the Hippo signaling pathway, AMPK signaling pathway and mTOR signaling pathways. Amot family members play an important role in neural stem cell differentiation, dendritic formation and synaptic maturation. In addition, an increasing number of studies have focused on their function in promoting and/or suppressing cancer, but the underlying mechanisms remain to be elucidated. The present review integrated relevant studies on upstream regulation and downstream signals of Amot family members, as well as the latest progress in physiological and pathological functions and clinical applications, hoping to offer important ideas for further research.

Maintenance of the intestinal epithelium requires constant self-renewal and regeneration. Tight regulation of proliferation and differentiation of intestinal stem cells within the crypt region is critical to maintaining homeostasis. The transcriptional co-factors β-catenin and YAP are required for proliferation during normal homeostasis as well as intestinal regeneration after injury: aberrant signaling activity results in over proliferation and tumorigenesis. Although both YAP and β-catenin activity are controlled along canonical pathways, it is becoming increasingly clear that non-canonical regulation of these transcriptional regulators plays a role in fine tuning their activity. We have shown previously that MAMDC4 (Endotubin, AEGP), an integral membrane protein present in endosomes, regulates both YAP and β-catenin activity in kidney epithelial cells and in the developing intestinal epithelium. Here we show that MAMDC4 interacts with members of the signalosome and mediates cross-talk between YAP and β-catenin. Interestingly, this cross-talk occurs through a non-canonical pathway involving interactions between AMOT:YAP and AMOT:β-catenin.

Microcystins (MCs) are toxic to the central nervous system of mammals. However, the direct toxicity of MCs on mammalian brain cells and the involved molecular mechanisms are not fully elucidated. Here, we incubated primary astrocytes, the major glial cell-type in the brain, with 0-12.5 μM concentrations of MC-LR for 48 h, and the impairment was evaluated. We found that MC-LR caused significant increases in the cell viability at the range of 0.05-1 μM concentrations with the highest density at 0.1 μM concentration. Treatment with 0.1 μM MC-LR induced YAP nuclear translocation and decreased the ratio of p-YAP to YAP. It also decreased mRNA levels of the upstream regulator (AMOT), and enhanced expressions of YAP interacted genes (Egfr, Tead1, and Ctgf) in primary astrocytes. Overexpression of AMOT significantly attenuated the increase of MC-LR-induced astrocyte proliferation and the expression of YAP downstream genes. These results indicate that Hippo signaling contributed to MC-LR-caused astrocyte proliferation. Further, reactive astrogliosis was observed in the mice brain after MC-LR exposure to environmentally relevant concentrations (20 or 100 μg/L) through drinking water for 16 weeks. Pathological observations revealed that 100 μg/L MC-LR exposure caused neuronal damages with characteristics of shrunken or vacuolation in the region of the cerebral cortex, striatum and cerebellum. These results were accompanied with increased oxidative stress and inflammatory response. Our data reveal the potential astrocytic mechanisms in MC-induced neurotoxicity and raise an alarm for neurodegenerative disease risk following daily exposure to MC-LR.

Cells have an incredible ability to physically interact with neighboring cells and their environment. They can detect and respond to mechanical forces by converting mechanical stimuli into biochemical signals in a process known as mechanotransduction. This is a key process for the adaption of vascular smooth muscle and endothelial cells to altered flow and pressure conditions. Mechanical stimuli, referring to a physical force exerted on cells, are primarily sensed by transmembrane proteins and the actin cytoskeleton, which initiate a cascade of intracellular events, including the activation of signaling pathways, ion channels, and transcriptional regulators. Recent work has highlighted an important role of the transcriptional coactivators YAP/TAZ for mechanotransduction in vascular cells. Interestingly, the activity of YAP/TAZ decreases with age, providing a potential mechanism for the detrimental effects of aging in the vascular wall. In this review, we summarize the current knowledge on the functional role of YAP and TAZ in vascular endothelial and smooth muscle cells for mechanotransduction in homeostasis and disease. In particular, the review is focused on in vivo observations from conditional knockout (KO) models of YAP/TAZ and the potential implications these studies may have for our understanding of vascular disease development.

CHO cells are extensively employed in biological drug industry to manufacture therapeutic proteins. Nevertheless, production of biopharmaceuticals faces obstacles such as limited growth and inadequate productivity. Employing host cell engineering techniques for CHO cells serves as a valuable approach to address the constraints encountered in biologics manufacturing. Despite advancements, most techniques focus on specific genes to address individual cellular challenges. The significance of YAP, transcriptional co-activator, cannot be overstated due to its involvement in regulating organ size and tumor formation. YAP's influence extends to various cellular processes and is regulated by kinase cascade in the Hippo pathway, which phosphorylates serine residues in specific LATS recognition motifs. Activation of YAP has been observed to impact both the size and quantity of cells. This research investigates the effects of YAP5SA on proliferation, apoptosis, and productivity in CHO-K1 cells. YAP5SA, with mutations in all five LATS-target sites, is selected for its heightened activity and resistance to repression through the Hippo-LATS1/2 kinase signaling pathway. Plasmid harboring YAP5SA was transfected into EPO-CHO and the influence of YAP5SA overexpression was investigated. According to our findings, transfection of EPO-CHO cells with YAP5SA exhibited a substantial enhancement in CHO cell productivity, resulting in a 3-fold increase in total protein and EPO, as well as a 1.5-fold increase in specific productivity. Additionally, it significantly contributes in augmenting viability, size, and proliferation. Overall, the findings of this study exemplify the potential of utilizing YAP5SA to impact particular cellular mechanisms, thereby presenting an avenue for customizing cells to fulfill production demands. KEY POINTS: • YAP5SA in CHO cells boosts growth, reduces apoptosis, and significantly improves productivity. • YAP5SA regulates genes involved in proliferation, survival, and mTOR activation. • YAP5SA increases productivity by improving cell cycle, c-MYC expression, and mTOR pathway.

The Hippo signaling pathway is a central growth control mechanism in multicellular organisms. By integrating diverse mechanical, biochemical, and stress cues, the Hippo pathway orchestrates proliferation, survival, differentiation, and mechanics of cells, which in turn regulate organ development, homeostasis, and regeneration. A deep understanding of the regulation and function of the Hippo pathway therefore holds great promise for developing novel therapeutics in regenerative medicine. Here, we provide updates on the molecular organization of the mammalian Hippo signaling network, review the regulatory signals and functional outputs of the pathway, and discuss the roles of Hippo signaling in development and regeneration.

Activation of the Hippo pathway by angiomotins to limit colorectal cancer progression is prevalent, whereas the regulation of angiomotins remains elusive. In this study, we uncover the involvement of an upregulated E3 ubiquitin ligase called RNF166, which destabilizes angiomotins, activates YAP, and is associated with a poor prognosis in colorectal cancer patients. Mechanistically, RNF166 specifically recognizes PARsylated angiomotin, a modification mediated by tankyrase at specific amino acid residues (D506, E513, E516, and E528). The tankyrase inhibitor XAV939, effectively prevents RNF166-dependent destabilization of angiomotins and subsequent activation of YAP. Additionally, YAP-5SA, a constitutively active form of YAP, rescues colorectal cancer progression following knockdown of RNF166. Importantly, the C-terminus of RNF66, particularly the Di19-ZF domain, is the crucial region responsible for recognizing ADP-ribosylated angiomotins. Together, this work not only sheds light on the regulation of the Hippo pathway in colorectal cancer but also uncovers a novel poly(ADP-ribose)-binding domain, which may serve as a potential therapeutic target for intervention.