The Hippo pathway: regulators and regulations.

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The Hippo pathway is a critical regulator of tissue size, and aberrations in pathway regulation lead to cancer. MST1/2 and LATS1/2 kinases comprise the core of the pathway that, in association with adaptor proteins SAV and MOB, functions in a sequential manner to phosphorylate and inhibit the transcription factors YAP and TAZ. Here we identify mammalian MARK family members as activators of YAP/TAZ. We show that depletion of MARK4 in MDA-MB-231 breast cancer cells results in the loss of nuclear YAP/TAZ and decreases the expression of YAP/TAZ targets. We demonstrate that MARK4 can bind to MST and SAV, leading to their phosphorylation, and that MARK4 expression attenuates the formation of a complex between MST/SAV and LATS, which depends on the kinase activity of MARK4. Abrogation of MARK4 expression using siRNAs and CRISPR/Cas9 gene editing attenuates the proliferation and migration of MDA-MB-231 cells. Our results show that MARK4 acts as a negative regulator of the Hippo kinase cassette to promote YAP/TAZ activity and that loss of MARK4 restrains the tumorigenic properties of breast cancer cells.

Article Tools UNDERSTANDING THE PATHWAY Article Tools OPTIONS & TOOLS Export Citation Track Citation Add To Favorites Rights & Permissions COMPANION ARTICLES No companion articles ARTICLE CITATION DOI: 10.1200/JCO.2015.61.2093 Journal of Clinical Oncology - published online before print June 8, 2015 PMID: 26056180 YAPing Hippo Forecasts a New Target for Lung Cancer Prevention and Treatment Duojia PanxDuojia PanSearch for articles by this author Show More Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD https://doi.org/10.1200/JCO.2015.61.2093 First Page Full Text PDF Figures and Tables © 2015 by American Society of Clinical Oncology

In multicellular organisms, regulation of growth control occurs because of the concerted action of multiple growth‐regulatory and patterning pathways. Among these, the Hippo (Hpo) pathway gained notoriety for its unique ability to simultaneously regulate cell proliferation and apoptosis to achieve the correct organ size. Initially discovered in Drosophila , this pathway is evolutionarily conserved, and acts as a universal regulator of organ size in metazoa. A complete pathway that integrates signals from the cell membrane to the nucleus has emerged, and multiple modes of Hpo pathway regulation have been elucidated. When the Hpo pathway is active, it acts as a brake on growth – cell proliferation is suppressed and apoptosis is promoted, whereas when the Hpo pathway is inactive, growth occurs – cell proliferation is promoted and apoptosis is suppressed. While the initial discovery of the pathway established its role in regulation of organ size and development, the focus has shifted considerably towards understanding its role in other biological processes such as maintaining tissue homeostasis, stem cell and tissue differentiation, cancer and regeneration. The Hpo signalling pathway functions in organ size control, and understanding its physiological functions will provide insights on its tissue‐ and cell‐type‐specific functions, and its involvement in diseases such as cancer. Key Concepts Cell proliferation and cell death must be regulated for balanced growth. The regulation of Hippo pathway activity is key to regulating cell number to achieve the correct organ size and growth during normal development. Hippo pathway activation promotes apoptosis and suppresses cell proliferation, whereas Hippo pathway inactivation promotes cell proliferation and suppresses apoptosis. The Hippo pathway is a network of tumour suppressor genes, and genes regulating cell‐ and planar‐polarity. Signalling interactions in the Hippo pathway ultimately converge on the regulation of the oncogene Yorkie pathway activity. The Hippo pathway is conserved across species. Hippo pathway is dysregulated in human cancer (e.g. loss of tumour suppressor gene neurofibromin 2 (NF2) is causal to schwannomas, and the YAP/TAZ oncoproteins are overexpressed in many human cancers [lung, breast, prostate and liver]). Hippo pathway regulates stem cell renewal, regeneration and differentiation in a tissue‐ and context‐specific manner.

Impaired angiogenesis and wound healing carry significant morbidity and mortality in diabetic patients. Metabolic stress from hyperglycemia and elevated free fatty acids have been shown to inhibit endothelial angiogenesis. However, the underlying mechanisms remain poorly understood. In this study, we show that dysregulation of the Hippo-Yes-associated protein (YAP) pathway, an important signaling mechanism in regulating tissue repair and regeneration, underlies palmitic acid (PA)-induced inhibition of endothelial angiogenesis. PA inhibited endothelial cell proliferation, migration, and tube formation, which were associated with increased expression of mammalian Ste20-like kinases 1 (MST1), YAP phosphorylation/inactivation, and nuclear exclusion. Overexpression of YAP or knockdown of MST1 prevented PA-induced inhibition of angiogenesis. When searching upstream signaling mechanisms, we found that PA dysregulated the Hippo-YAP pathway by inducing mitochondrial damage. PA treatment induced mitochondrial DNA (mtDNA) release to cytosol, and activated cytosolic DNA sensor cGAS-STING-IRF3 signaling. Activated IRF3 bound to the MST1 gene promoter and induced MST1 expression, leading to MST1 up-regulation, YAP inactivation, and angiogenesis inhibition. Thus, mitochondrial damage and cytosolic DNA sensor cGAS-STING-IRF3 signaling are critically involved in PA-induced Hippo-YAP dysregulation and angiogenesis suppression. This mechanism may have implication in impairment of angiogenesis and wound healing in diabetes.

Yap1 Acts Downstream of α-Catenin to Control Epidermal Proliferation

Disease implications of the Hippo/YAP pathway

The Hippo pathway was initially identified in Drosophila melanogaster screens for tissue growth two decades ago and has been a subject extensively studied in both Drosophila and mammals in the last several years. The core of the Hippo pathway consists of a kinase cascade, transcription coactivators, and DNA-binding partners. Recent studies have expanded the Hippo pathway as a complex signaling network with >30 components. This pathway is regulated by intrinsic cell machineries, such as cell-cell contact, cell polarity, and actin cytoskeleton, as well as a wide range of signals, including cellular energy status, mechanical cues, and hormonal signals that act through G-protein-coupled receptors. The major functions of the Hippo pathway have been defined to restrict tissue growth in adults and modulate cell proliferation, differentiation, and migration in developing organs. Furthermore, dysregulation of the Hippo pathway leads to aberrant cell growth and neoplasia. In this review, we focus on recent developments in our understanding of the molecular actions of the core Hippo kinase cascade and discuss key open questions in the regulation and function of the Hippo pathway.

The Hippo tumor suppressor pathway plays an important role in tissue homeostasis that ensures development of functional organs at proper size. The YAP transcription coactivator is a major effector of the Hippo pathway and is phosphorylated and inactivated by the Hippo pathway kinases Lats1/2. It has recently been shown that YAP activity is regulated by G-protein-coupled receptor signaling. Here we demonstrate that cyclic adenosine monophosphate (cAMP), a second messenger downstream from Gαs-coupled receptors, acts through protein kinase A (PKA) and Rho GTPases to stimulate Lats kinases and YAP phosphorylation. We also show that inactivation of YAP is crucial for PKA-induced adipogenesis. In addition, PKA activation in Drosophila inhibits the expression of Yorki (Yki, a YAP ortholog) target genes involved in cell proliferation and death. Taken together, our study demonstrates that Hippo-YAP is a key signaling branch of cAMP and PKA and reveals new insight into mechanisms of PKA in regulating a broad range of cellular functions.

Department of Life Science, The University of Seoul, Seoul 130-743, Korea Balanced cell growth is crucial in animal development as well as tissue homeostasis. Concerted cross-regulation of multiple signaling pathways is essential for those purposes, and the dysregulation of signaling may lead to a variety of human diseases such as cancer. The time-honored Wnt/β-catenin and recently identified Hippo signaling pathways are evolutionarily conserved in both Drosophila and mammals, and are generally considered as having positive and negative roles in cell proliferation, respectively. While most mainstream regulators of the Wnt/β-catenin signaling pathway have been fairly well identified, the regulators of the Hippo pathway need to be more defined. The Hippo pathway controls organ size primarily by regulating cell contact inhibition. Recently, several crossregulations occurring between the Wnt/β-catenin and Hippo signaling pathways were determined through biochemical and genetic approaches. In the present mini-review, we mainly discuss the signal transduction mechanism of the Hippo signaling pathway, along with cross-talk between the regulators of the Wnt/β-catenin and Hippo signaling pathways. [BMB Reports 2014; 47(10): 540-545]

The Hippo pathway plays a crucial role in cell proliferation and differentiation during tumorigenesis, tissue homeostasis and early embryogenesis. Scaffold proteins from the ezrin-radixin-moesin (ERM) family, including neurofibromin 2 (NF2; Merlin), regulate the Hippo pathway through cell polarity. However, the mechanisms underlying Hippo pathway regulation via cell polarity in establishing outer cells remain unclear. In this study, we generated artificial Nf2 mutants in the N-terminal FERM domain (L64P) and examined Hippo pathway activity by assessing the subcellular localization of YAP1 in early embryos expressing these mutant mRNAs. The L64P-Nf2 mutant inhibited NF2 localization around the cell membrane, resulting in YAP1 cytoplasmic translocation in the polar cells. L64P-Nf2 expression also disrupted the apical centralization of both large tumor suppressor 2 (LATS2) and ezrin in the polar cells. Furthermore, Lats2 mutants in the FERM binding domain (L83K) inhibited YAP1 nuclear translocation. These findings demonstrate that NF2 subcellular localization mediates cell polarity establishment involving ezrin centralization. This study provides previously unreported insights into how the orchestration of the cell-surface components, including NF2, LATS2 and ezrin, modulates the Hippo pathway during cell polarization.

The success of stem cell therapies for acute myocardial infarction (AMI) has been hampered by poor cellular engraftment and survival, which may be partly due to the harmful microenvironment within ischemic myocardium. However, it may be possible to promote survival and cardiomyogenesis of transplanted adult mesenchymal cells by inducing the overexpression of myocardin (MYOCD), a promyogenic transcription factor with anti-apoptotic activity, and telomerase reverse transcriptase (TERT), an antisenescence protein. Objectives: We used a murine model of AMI to assess the efficacy of transplanted adipose tissuederived mesenchymal stromal cells (AT-MSCs) engineered to overexpress MYOCD and TERT. Methods: Twelve-month-old C57BL/6 mice underwent coronary artery ligation to induce AMI and were randomized into 3 treatment groups: phosphate-buffered saline (PBS) (20mL; n=7), mocktransduced AT-MSCs (2.5x105 cells in 20mL; n=5), or AT-MSCs overexpressing TERT and MYOCD (2.5x105 cells in 20mL; n=7). Sham-operated mice (n=7) were used as controls. The AT-MSCs were obtained from 12-month-old male green fluorescent protein-transgenic C57BL/6 mice and transduced with lentiviral vectors encoding TERT and MYOCD. Results: When transplanted into the infarcted hearts of C57BL/6 mice, AT-MSCs overexpressing TERT and MYOCD preserved myocardial fractional shortening (Figure A), increased arteriogenesis and cell engraftment and decreased fibrosis formation (Figure B), compared with PBS alone or mocktransduced AT-MSCs. These effects were accompanied by increased numbers of Ki-67+ cells and c-kit+ cells (Figure B) and enhanced expression of cardiac actin, GATA4, Nkx2.5, and myocardin A (Figure C). Conclusions: Delivering TERT and MYOCD genes into AT-MSCs before transplantation promotes activation of the cardiomyogenic pathway, vasculogenesis, and stem cell survival in a murine model of AMI.

G protein‐coupled receptors (GPCRs) regulate the Hippo signaling pathway, a developmental pathway involved in tumorigenesis and metastasis. Although GPCRs promote cancer progression and are highly druggable, there are currently no FDA‐approved drugs targeting GPCRs for cancer treatment. Protease‐activated receptor‐1 (PAR1) is a GPCR that both activates the Hippo pathway and promotes breast cancer progression. PAR1 is overexpressed in breast cancer patient tumor biopsies and in invasive breast carcinoma cell lines, and correlates with increased metastasis and poor prognosis. We found that PAR1 overexpression is due to defective lysosomal trafficking and results in persistent signaling observed in multiple triple‐negative breast carcinoma cell lines including the model invasive MDA‐MB‐231 cells. We recently showed that the α‐arrestin domain containing protein‐3 (ARRDC3), a tumor suppressor, is reduced or lost in highly aggressive triple‐negative breast carcinoma and this is responsible for aberrant PAR1 lysosomal trafficking and persistent signaling, leading to increased invasion. However, the link between ARRDC3 expression and PAR1‐stimulated Hippo pathway regulation in invasive breast cancer is not known and was examined. Here, we show that ARRDC3 regulates GPCR activation of the Hippo pathway in triple‐negative breast cancer, where the transcriptional co‐activators YAP and TAZ display distinct functions. ARRDC3 re‐expression in invasive breast carcinoma cells attenuates GPCR‐stimulated Hippo signaling and invasion that is mediated by activation of TAZ but not YAP. Furthermore, siRNA‐targeted depletion of TAZ, but not YAP inhibits GPCR‐induced Hippo signaling and invasion. An understanding of the mechanisms by which the Hippo pathway is regulated by GPCRs may lead to new potential therapeutic targets for the treatment or prevention of metastatic breast cancer.Support or Funding InformationFunding for this project was provided by the HHMI Gilliam Fellowship for Advanced Study, the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through NIH General Medical Sciences, T32 GM007752 and the UCSD Tribal Membership Initiative (Aleena Arakaki) and by the National Institute of Health NIGMS R01 GM090689 and R35 GM127121 (JoAnn Trejo).

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.

The Hippo pathway is recognized as an important regulator of tissue growth and cell fate [1-3]. Originally indentified in Drosophila, the Hippo pathway, also known as the Salvador–Warts–Hippo pathway, contains core kinases cascade, Hippo and Warts(Wts) coupled by the scaffold protein Salvador (Sav), as well as Mats. The activation of the Hippo pathway kinases results in phosphorylation and inactivation of the downstream transcriptional co-activator Yorkie which binds to the sequence-specific DNA-binding protein Scalloped and enhances the expression of proliferative and pro-survival genes. In general, the primary function of the Hippo signaling pathway is to inhibit the activation of Yorkie, inasmuch as deletion of Yki reverses the overgrowth phenotypes resulted from loss of Hippo, Warts, Salvador or Mats. Components of the Hippo pathway are highly conserved throughout evolution. The counterparts for the Hippo pathway in Drosophila can all be found in mammals, although they are more diverse and complex [4]. The Hippo orthologs Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 to regulate the Warts orthologs Lats1/Lats2. Activated Lats kinases phosphorylate the transcriptional regulators TAZ/YAP (Yorkie orthologs) which promotes 14-3-3 binding to YAP, causing YAP nuclear exit, hereby inhibiting its function. In recent years, increasing numbers of mammalian studies have expanded the large proteins network of the Hippo signaling pathway that controls tissues growth during development and regeneration, as well as in pathological states such as cancer [5]. The upstream regulator of the Hippo pathway and the downstream of Mst1/Mst2 have been diversified considerably in mammals compared with the Drosophila Hippo pathway. Multiple cellular stresses can trigger an adaptive response by activating the Hippo signaling pathway, which may, in turn, maintain the cellular homeostasis. The Hippo/Warts/Mats/Yorkie pathway predicated in Drosophila is not universal in all mammalian tissues in which their regulation and function are different in selected cell types. For examples, Mst1/2 negatively regulates YAP1 in mammalian liver, however, Mst1/2 is not required for YAP1 phosporylation and nuclear exclusion resulted from the cell-cell contact in mouse embryonic fibroblasts(MEFs); Mst1/2 is dispensable for Lats1/2 signaling in MEFs, but not in HeLa cells [6]. Independent of YAP, Mst1 negatively regulates naive T cell proliferation upon the T cell receptor stimulation, as well as regulates peripheral naive T cell trafficking and thymus egress [7]. Furthermore, patients with Mst1 deficiency are reported to have a primary immunodeficiency phenotype [8,9]. During the tissues regeneration and tumorigenesis, the Hippo signaling pathway has been shown to cross talk with other signaling players such as Notch and Wnt [10]. Thus, not just for the organ size control, the Hippo pathway receives inputs from multiple extracellular or intracellular signals and interacts with other essential signaling pathways to play critical roles in many aspects for cell fate decisions. In this issue of the Cell & Bioscience, we have provided some updates on the regulations beyond the canonical Hippo signaling, and their implications in pathological states. Qin et al. will review the recent updates of the roles of Mst1/2 on the cellular redox state regulation, the effects of Mst1/2 deficiency on the development process and tumorigenesis in multiple organs, and their involvement in the immune regulation. The review by Hergovich will summarize the current understanding of mammalian Lats1/2 kinases together with their closest relatives, the NDR1/2 kinases. He will focus on discussion about the regulation of the LATS/NDR family of kinases and their currently known substrates, as well as the biological roles of LATS/NDR kinases. Guo and Zhao follows with a discussion of the function of YAP and TAZ as effectors of cell responses to several extracellular signals including mechanical stress, GPCR signaling, and the Wnt signaling pathway, emphasizing that YAP and TAZ might have different role with cell-type specificity in the promotion of specific cancers. Collectively, these reviews have provided additional information to address the complexity of the hippo signaling pathway in response to physiological signals for regulating cellular and tissues homeostasis.