Among the signaling pathways that regulate critical cell and tissue functions, the Hippo pathway is of great significance and under continuous investigation. The Hippo is a highly conversed pathway initially observed and decoded in Drosophila melanogaster. It consists of core molecules such as mammalian STE20-like kinase 1/2 (MST1/2), protein Salvador homologue 1 (SAV1) and MOBKL1A/B (MOB1A/B), and large tumour suppressor kinase 1/2 (LATS1/2), whereas the Yes-associated protein 1 (YAP), and WW-domain-containing transcription regulator 1 (TAZ) proteins act as transcriptional coactivators that bind to the transcriptional enhanced associated domain family 1(TEAD-1). The purpose of the current molecular review was to describe the main molecular and functional aspects of the Hippo pathway and its deregulation in specific eye lesions. A systematic review of the literature was carried out based on the international medical database PubMed. The year 2005 was set as a prominent time limit for the publication date of the majority of articles, whereas specific references of great importance and historical value in the field of the Hippo discovery and analysis were also included. The following keywords were used: Hippo, TAZ, LATS, YAP1, eye, signaling pathway. A pool of 70 important articles was selected for the present review describing the connections between the implicated in HIPPO pathway molecules, the corresponding normal biochemical and functional features of them, and the deregulation mechanisms that are involved in the pathogenesis of eye lesions. According to the selected publications, Hippo deregulation is observed in a broad spectrum of benign and neoplastic eye diseases. TAZ, LATS, and YAP1 proteins are negatively influenced by the deregulation of the core molecules in them. Hippo deregulation is critically involved in retina- and lens-related benign lesions as well as in conjunctival fibrosis and ocular surface squamous neoplasia. YAP/TEAD point mutations and abnormal expression of other molecules are the main genetic mechanisms in these lesions.

YAP/TAZ: An epitome of tumorigenesis.

Although stem/progenitor cell therapy shows potential for myocardial infarction repair, enhancing the therapeutic efficacy could be achieved through additional genetic modifications. HCLS1-associated protein X-1 (HAX1) has been identified as a versatile modulator responsible for cardio-protective signaling, while its role in regulating stem cell survival and functionality remains unknown. In this study, we investigated whether HAX1 can augment the protective potential of Sca1+ cardiac stromal cells (CSCs) for myocardial injury. The overexpression of HAX1 significantly increased cell proliferation and conferred enhanced resistance to hypoxia-induced cell death in CSCs. Mechanistically, HAX1 can interact with Mst1 (a prominent conductor of Hippo signal transduction) and inhibit its kinase activity for protein phosphorylation. This inhibition led to enhanced nuclear translocation of Yes-associated protein (YAP) and activation of downstream therapeutic-related genes. Notably, HAX1 overexpression significantly increased the pro-angiogenic potential of CSCs, as demonstrated by elevated expression of vascular endothelial growth factors. Importantly, implantation of HAX1-overexpressing CSCs promoted neovascularization, protected against functional deterioration, and ameliorated cardiac fibrosis in ischemic mouse hearts. In conclusion, HAX1 emerges as a valuable and efficient inducer for enhancing the effectiveness of cardiac stem or progenitor cell therapeutics.Graphical

Biomolecular condensates have been proposed to mediate cellular signaling transduction. However, the mechanism and functional consequences of signal condensates are not well understood. Here we report that LATS2, the core kinase of the Hippo pathway, responds to F-actin cytoskeleton reduction and forms condensates. The proline-rich motif (PRM) of LATS2 mediates its condensation. LATS2 partitions with the main components of the Hippo pathway to assemble a signalosome for LATS2 activation and for its stability by physically compartmentalizing from E3 ligase FBXL16 complex-dependent degradation, which in turn mediates yes-associated protein (YAP)-transcriptional coactivator with PDZ-binding motif (TAZ) recruitment and inactivation. This oncogenic FBXL16 complex blocks LATS2 condensation by binding to the PRM region to promote its degradation. Disruption of LATS2 condensation leads to tumor progression. Thus, our study uncovers that the signalosomes assembled by LATS2 condensation provide a compartmentalized and reversible platform for Hippo signaling transduction and protein stability, which have potential implications in cancer diagnosis and therapeutics.

Mechanism of Huatan Sanjie Fang in improving goiter in Graves' disease mice based on the Hippo signaling pathway

The survival of non-small cell lung cancer (NSCLC) patients has improved in the last decade as a result of introducing new therapeutics, such as immune checkpoint inhibitors, in the clinic. Still, some NSCLC patients do not benefit from these therapies due to intrinsic resistance or the development of acquired resistance and their malignant disease progresses. Further research on the molecular underpinnings of NSCLC pathobiology is required in order to discover clinically relevant molecular targets that regulate tumor immunity and to develop reasonable therapeutic combinations that will promote the efficacy of immune checkpoint inhibitors. Yes-associated Protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), the final effectors of the Hippo signaling transduction pathway, are emerging as key players in NSCLC development and progression. Herein, we overview studies that have investigated the oncogenic role of YAP/TAZ in NSCLC, focusing on immune evasion, and highlight the therapeutic potential of combining YAP/TAZ inhibitory agents with immune checkpoint inhibitors for the management of NSCLC patients.

The crumbs cell polarity complex plays a crucial role in apical-basal epithelial polarity, cellular adhesion, and morphogenesis. Homozygous variants in human CRB1 result in autosomal recessive Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP), with no established genotype-phenotype correlation. The associated protein complexes have key functions in developmental pathways; however, the underlying disease mechanism remains unclear. Using the oko meduzym289/m289 (crb2a-/- ) zebrafish, we performed integrative transcriptomic (RNA-seq data) and methylomic [reduced representation bisulphite sequencing (RRBS)] analysis of whole retina to identify dysregulated genes and pathways. Delayed retinal cell specification was identified in both the crb2a-/- zebrafish and CRB1 patient-derived retinal organoids, highlighting the dysfunction of cell cycle modulation and epigenetic transcriptional control. Differential DNA methylation analysis revealed novel hypermethylated pathways involving biological adhesion, Hippo, and transforming growth factor β (TGFβ) signalling. By integrating gene expression with DNA methylation using functional epigenetic modules (FEM), we identified six key modules involving cell cycle control and disturbance of TGFβ, bone morphogenetic protein (BMP), Hippo, and SMAD protein signal transduction pathways, revealing significant interactome hotspots relevant to crb2a function and confirming the epigenetic control of gene regulation in early retinal development, which points to a novel mechanism underlying CRB1-retinopathies. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract The Hippo pathway is critical to cancer progression, biogenesis, metastasis, and therapeutic resistance. Many cancer indications have a high frequency of mutations in the Hippo pathway. These pro-tumor mutations lead to constitutive TEAD transcription factor activation, which drives gene expression involved in cell growth and pro-survival signaling. To target cancers harboring mutations in the Hippo pathway, we have discovered and are developing IK-930, a novel small molecule that selectively inhibits TEAD-dependent transcription by directly blocking autopalmitoylation. IK-930 prevents this critical post-translational modification that is required for the functional interaction of TEAD with two transcriptional activators, YAP1 and TAZ (WWTR1). IK-930 inhibits in vitro proliferation of Hippo pathway-deficient cancer cell lines, but not Hippo pathway wild type cells. In human mesothelioma xenografts, IK-930 downregulates TEAD-dependent genes. Daily oral administration of IK-930 resulted in antitumor activity in Hippo-dysregulated mesothelioma xenograft models. In EGFR or KRAS mutated tumors, IK-930 enhanced apoptosis and in vivo antitumor activity in combination with EGFR and MEK inhibitors, respectively. IK-930 is inactive in a broad panel of kinases, receptors, and transporters, furthering evidence of selectivity. Multispecies pharmacokinetic analysis and additional in vitro ADME properties imply favorable pharmacokinetic properties, with a low potential for clinically significant drug-drug interactions. To identify indications that may be dependent on TEAD we evaluated tumor types for the incidence of gene alterations in the Hippo pathway, as well as for YAP1 and TAZ activation. YAP1 and TAZ activation was assessed by evaluating nuclear protein expression in tumor tissue microarrays. These analyses showed high YAP1 nuclear expression in tumors with frequent genetic alterations, that may help guide the development of IK-930. Mesothelioma ranks top among tumor types evaluated, due to high prevalence of YAP1 nuclear expression and Hippo pathway genetic alterations, which are predominantly mutations and copy number alterations in the tumor suppressor NF2. In summary, Hippo pathway dysregulation has been implicated in the etiology of multiple tumor types, including mesothelioma. IK-930 is a selective and potent TEAD inhibitor expected to enter the clinic in early 2022. Our analysis pointed to tumors with genetic alterations driving aberrant Hippo signaling. IK-930 demonstrates efficacy in tumor models representing these indications and beneficial combination activity with other targeted therapies. Taken together, these data have informed the clinical development plan for IK-930. Citation Format: Benjamin S. Amidon, Marta Sanchez-Martin, Wilmin Bartolini, Sakeena Syed, Karen McGovern, Lan Xu, Jeffrey Ecsedy, X. Michelle Zhang, Alex Constan, Alfredo C. Castro. IK-930 is a novel TEAD inhibitor for the treatment of cancers harboring mutations in the Hippo signal transduction pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2156.

Abstract Polycystic ovary syndrome (PCOS) is a heterogeneous reproductive disease that can cause infertility. The Hippo signaling pathway, a network highly conserved throughout evolution, maintains the balance between follicle proliferation and dormancy. Dynamic changes in primordial follicles cannot occur without the participation of biological signals and mechanical force; however, little is known about the mechanism by which biomechanical signaling triggers PCOS, especially in the context of primordial follicle development. To investigate the contribution of mechanical stress and the Hippo signaling pathway to the onset of PCOS, we searched the literature via the PubMed database, and inclusion and exclusion criteria were established to ensure the rigor of this research. We eventually included 54 publications in which Hippo signaling and mechanical force were suggested to play a vital role in the development of primordial follicles as well as elucidate the pathogenesis of PCOS. The Hippo pathway modulating follicle growth can be perturbed via extracellular mechanical stress caused by the stiff ovarian cortical environment in PCOS. Clinical intervention targeting the Hippo pathway can alter the activity of core Hippo members, such as the Yes-associated protein/transcriptional co-activator PDZ-binding motif complex. In some patients with PCOS, follicle overactivation can be attributed to the dysfunction of Hippo signal transduction. PCOS, a condition with various patterns, cannot be accurately explained by a single, specific mechanism. The present review identifies potential targets and therapeutic strategies for PCOS.

The Hippo signal transduction network regulates transcription through Yap/Taz-Tead1-4 in many tissues including skeletal muscle. Whilst transgenic mice have been generated for many Hippo genes, the resultant skeletal muscle phenotypes were not always characterized. Here, we aimed to phenotype the hindlimb muscles of Hippo gene-mutated Lats1−/−, Mst2−/−, Vgll3−/−, and Vgll4+/− mice. This analysis revealed that Lats1−/− mice have 11% more slow type I fibers than age and sex-matched wild-type controls. Moreover, the mRNA expression of slow Myh7 increased by 50%, and the concentration of type I myosin heavy chain is 80% higher in Lats1−/− mice than in age and sex-matched wild-type controls. Second, to find out whether exercise-related stimuli affect Lats1, we stimulated C2C12 myotubes with the hypertrophy agent clenbuterol or the energy stress agent AICAR. We found that both stimulated Lats1 expression by 1.2 and 1.3 fold respectively. Third, we re-analyzed published datasets and found that Lats1 mRNA in muscle is 63% higher in muscular dystrophy, increases by 17–77% after cardiotoxin-induced muscle injury, by 41–71% in muscles during overload-induced hypertrophy, and by 19–21% after endurance exercise when compared to respective controls. To conclude, Lats1 contributes to the regulation of muscle fiber type proportions, and its expression is regulated by physiological and pathological situations in skeletal muscle.

The Hippo pathway is an important regulator of organ growth and cell fate. In the R8 photoreceptor cells of the Drosophila melanogaster eye, the Hippo pathway controls the fate choice between one of two subtypes that express either the blue light-sensitive Rhodopsin 5 (Hippo inactive R8 subtype) or the green light-sensitive Rhodopsin 6 (Hippo active R8 subtype). The degree to which the mechanism of Hippo signal transduction and the proteins that mediate it are conserved in organ growth and R8 cell fate choice is currently unclear. Here, we identify Crumbs and the apical spectrin cytoskeleton as regulators of R8 cell fate. By contrast, other proteins that influence Hippo-dependent organ growth, such as the basolateral spectrin cytoskeleton and Ajuba, are dispensable for the R8 cell fate choice. Surprisingly, Crumbs promotes the Rhodopsin 5 cell fate, which is driven by Yorkie, rather than the Rhodopsin 6 cell fate, which is driven by Warts and the Hippo pathway, which contrasts with its impact on Hippo activity in organ growth. Furthermore, neither the apical spectrin cytoskeleton nor Crumbs appear to regulate the Hippo pathway through mechanisms that have been observed in growing organs. Together, these results show that only a subset of Hippo pathway proteins regulate the R8 binary cell fate decision and that aspects of Hippo signalling differ between growing organs and post-mitotic R8 cells.

The Hippo signal transduction pathway is an essential regulator of organ size during developmental growth by controlling multiple cellular processes such as cell proliferation, cell death, differentiation, and stemness. Dysfunctional Hippo signaling pathway leads to dramatic tissue overgrowth. Here, we will briefly introduce the Hippo tumor suppressor pathway before focusing on one of its members and the unexpected twists that followed our quest of its functions in its multifarious actions beside the Hippo pathway: the STK38 kinase. In this review, we will precisely discuss the newly identified role of STK38 on regulating the nuclear export machinery by phosphorylating and activating, the major nuclear export receptor XPO1. Finally, we will phrase STK38's role on regulating the subcellular distribution of crucial cellular regulators such as Beclin1 and YAP1 with its implication in cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide. The paralogous transcriptional cofactors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ, also called WWTR1), the main downstream effectors of the Hippo signal transduction pathway, are emerging as pivotal determinants of malignancy in lung cancer. Traditionally, studies have tended to consider YAP and TAZ as functionally redundant transcriptional cofactors with similar biological impact. However, there is growing evidence that each of them also possesses distinct attributes. Here we sought to systematically characterize the division of labor between YAP and TAZ in non-small cell lung cancer (NSCLC), the most common histological subtype of lung cancer. Representative NSCLC cell lines as well as patient-derived data showed that the two paralogs orchestrated nonoverlapping transcriptional programs in this cancer type. YAP preferentially regulated gene sets associated with cell division and cell-cycle progression, whereas TAZ preferentially regulated genes associated with extracellular matrix organization. Depletion of YAP resulted in growth arrest, whereas its overexpression promoted cell proliferation. Likewise, depletion of TAZ compromised cell migration, whereas its overexpression enhanced migration. The differential effects of YAP and TAZ on key cellular processes were also associated with differential response to anticancer therapies. Uncovering the different activities and downstream effects of YAP and TAZ may thus facilitate better stratification of patients with lung cancer for anticancer therapies. SIGNIFICANCE: Thease findings show that oncogenic paralogs YAP and TAZ have distinct roles in NSCLC and are associated with differential response to anticancer drugs, knowledge that may assist lung cancer therapy decisions.

Hippo signaling pathways are evolutionarily conserved signal transduction mechanisms mainly involved in organ size control, tissue regeneration, and tumor suppression. However, in mammals, the primary role of Hippo signaling seems to be regulation of immunity. As such, humans with null mutations in STK4 (mammalian homologue of Drosophila Hippo; also known as MST1) suffer from recurrent infections and autoimmune symptoms. Although dysregulated T cell homeostasis and functions have been identified in MST1-deficient human patients and mouse models, detailed cellular and molecular bases of the immune dysfunction remain to be elucidated. Although the canonical Hippo signaling pathway involves transcriptional co-activator Yes-associated protein (YAP) or transcriptional coactivator with PDZ motif (TAZ), the major Hippo downstream signaling pathways in T cells are YAP/TAZ-independent and they widely differ between T cell subsets. Here we will review Hippo signaling mechanisms in T cell immunity and describe their implications for immune defects found in MST1-deficient patients and animals. Further, we propose that mutual inhibition of Mst and Akt kinases and their opposing roles on the stability and function of forkhead box O and β-catenin may explain various immune defects discovered in mutant mice lacking Hippo signaling components. Understanding these diverse Hippo signaling pathways and their interplay with other evolutionarily-conserved signaling components in T cells may uncover molecular targets relevant to vaccination, autoimmune diseases, and cancer immunotherapies.

Hippo signaling regulates the balance between cell proliferation and apoptosis to control the size of organs during development. Appropriate Hippo signaling is associated with stem cell differentiation, and inappropriate signaling can result in tumorigenesis and cancer. Hippo signaling activity is influenced not only by biochemical signals but also by mechanical force and the cytoskeleton transmitted through cell-cell junctions and cell-matrix adhesions. In this review, we describe the evidence for the regulation of Hippo signaling by the spatial reorganization of signaling components, mechanical force, and the cytoskeleton. Although our understanding of the relationship between Hippo signal transduction and mechanical force and the cytoskeleton is developing rapidly, many unresolved questions remain.

Background: Human neonatal cardiac progenitor cells (ckit+/CD45-/Lin-, CPCs) improve cardiac function and attenuate adverse left ventricular remodeling after myocardial infarction (MI) through their exosomes (nEXOs). Success of cell therapy to treat injured myocardium depends mainly on reactivation of endogenous quiescent cardiomyocytes to proliferate and recover the lost myocardium. Here, we investigated the mechanism of reactivating quiescent cardiomyocyte proliferation by exosomes. Hypothesis: RNAseq analysis of nEXOs identified miRs which may be responsible for modulation of Hippo signaling, thereby promoting cardiomyocytes proliferation. nEXOs effectively stimulate endogenous cardiomyocyte (CM) proliferation by targeting the Hippo pathway to restore cardiac function in an injured heart. Methods and Results: nCPCs were conditioned for 48 hours in serum free nutrition mix (Ham’sF12) and nEXOs were purified from supernatant using size exclusion chromatography (SEC; CL2B) coupled with ultracentrifugation. nEXOs were quantified by Nanosight (NS300) and characterized by transmission electron microscopy and flow cytometry for the presence of CD63 and CD9. Our results show recovery of cardiac function (ejection fraction = 63.4% vs 40.5%, n=10, p<0.001)) and generation of myocardial mass in a rodent MI model following nEXOs’ intra-myocardial injection. We further show that nEXOs are preferentially acquired by CMs in the border zone of the infarction (m-cherry-Alix labeled EXOs). Our In vitro experiments show that miR-582-3p and miR-7641 (25nM) are the most effective miRs to induce proliferation of quiescent cardiomyocytes (40.1% and 48.8%, respectively, n=3). Increased miR-7641 expression led to a profound increase in quiescent CMs proliferation, in part through repression of the Hippo signal transduction pathway (increased YAP/pYAP ratio). By immunoblotting we show that LATs1/2 (a protein in the Hippo pathway) is directly targeted by miR-7641. nEXOs enriched with miR 7641 promote CM proliferation by 3 folds as compared to non-enriched nEXOs. Conclusion: Our data for the first time demonstrates the ability of nEXOs derived miRNA based therapeutic approaches to activate cardiomyocyte proliferation.

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are two homologous transcriptional coactivators and the final effectors of the Hippo signaling transduction pathway. The transcriptional activity of YAP/TAZ is dependent on their recruitment to the nucleus, which promotes binding to the transcription factor of TEA domain family members 1–4 (TEAD1-4). In Hippo-signaling pathway, YAP/TAZ is inactivated and its translocation to the nucleus is blocked via a core kinase cascade stimulated by a variety of upstream signals, such as G-protein-coupled receptor signaling, mechanical pressure, and adherens junction signaling. This pathway plays a very important role in regulating organ size, tissue homeostasis, and tumor development. In recent years, many studies have reported upregulation or nuclear localization of YAP/TAZ in a number of human malignancies, such as breast cancer, melanoma, lung cancer, especially squamous cell carcinoma in different organs. A large number of experiments demonstrate that YAP/TAZ activation promotes cancer formation, progression, and metastasis. Therefore, in this review, we summarize the evidence of regulation and function of YAP/TAZ and discuss its role in squamous cell carcinoma. Collectively, this summary strongly suggests that targeting aberrant YAP/TAZ activation is a promising strategy for the suppression of squamous cell carcinoma.

ABSTRACTVGLL proteins are transcriptional co-factors that bind TEAD family transcription factors to regulate events ranging from wing development in fly, to muscle fibre composition and immune function in mice. Here, we characterise Vgll3 in skeletal muscle. We found that mouse Vgll3 was expressed at low levels in healthy muscle but that its levels increased during hypertrophy or regeneration; in humans, VGLL3 was highly expressed in tissues from patients with various muscle diseases, such as in dystrophic muscle and alveolar rhabdomyosarcoma. Interaction proteomics revealed that VGLL3 bound TEAD1, TEAD3 and TEAD4 in myoblasts and/or myotubes. However, there was no interaction with proteins from major regulatory systems such as the Hippo kinase cascade, unlike what is found for the TEAD co-factors YAP (encoded by YAP1) and TAZ (encoded by WWTR1). Vgll3 overexpression reduced the activity of the Hippo negative-feedback loop, affecting expression of muscle-regulating genes including Myf5, Pitx2 and Pitx3, and genes encoding certain Wnts and IGFBPs. VGLL3 mainly repressed gene expression, regulating similar genes to those regulated by YAP and TAZ. siRNA-mediated Vgll3 knockdown suppressed myoblast proliferation, whereas Vgll3 overexpression strongly promoted myogenic differentiation. However, skeletal muscle was overtly normal in Vgll3-null mice, presumably due to feedback signalling and/or redundancy. This work identifies VGLL3 as a transcriptional co-factor operating with the Hippo signal transduction network to control myogenesis.

The Hippo signal transduction pathway is highly conserved in mammals. It plays a critical role in tissue and organ size by regulating the balance between cell proliferation and apoptosis. However, there have been few reports concerning Yes-activated protein-1 (YAP-1) elevation in papillary thyroid cancer (PTC). The objective of this study was to determine whether YAP-1 expression is a biomarker and high-risk clinicopathologic prognosticator in PTC. A large series of patients of PTC with a long follow-up were investigated for YAP-1 expression. Our study was carried out in the laboratory of breast and thyroid and Department of pathology. Immunohistochemical staining was performed on 240 patient-derived PTC specimens to analyze the correlation of YAP-1 expression with clinicopathologic features and prognosis in patients with PTC. The 240 PTC patients were immunohistochemically assessed for YAP-1 expression. Kaplan-Meier analysis was conducted to assess recurrence-free survival (RFS). Univariate and multivariate analyses were conducted to determine prognosticators of RFS. YAP-1 expression was observed in 62.1% of PTC tumors. There were significant positive correlations between YAP-1 expression and tumor size, lymph node metastases, extrathyroidal extension, and tissue infiltration. YAP-1 expression was significantly associated with RFS. Univariate analysis revealed that YAP-1 expression significantly affects RFS. YAP-1 and extrathyroidal extension were significant independent prognosticators for RFS. YAP-1 expression was significantly correlated with high-risk clinicopathologic features and inferior RFS in patients with PTC.

In the study of the Hippo signal transduction pathway, protein-protein interactions are often explored, because various proteins such as MOB1, NDR1/NDR2, and LATS1/LATS2 are very important members in this complicated signaling pathway. The transduction of signals from upstream to downstream is largely dependent on the mutual recognition of proteins and the formation of specific non-covalent complexes between them. In general, protein-protein associations, protein-DNA associations, or protein-small molecule associations cause the release or absorption of heat during the association reaction. The isothermal titration calorimetry (ITC) assay is a convenient and widely used approach to directly measure the amount of heat released or absorbed during association processes of biomolecules (such as protein-protein, protein-DNA, or protein-small molecules) in solution and to quantitatively estimate the interaction affinity.