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Epithelioid hemangioendothelioma (EHE) is a poorly understood and devastating vascular cancer. Sequencing of EHE has revealed a unique gene fusion between the Hippo pathway nuclear effector TAZ (WWTR1) and the brain-enriched transcription factor CAMTA1 in ∼90% of cases. However, it remains unclear whether the TAZ-CAMTA1 gene fusion is a driver of EHE, and potential targeted therapies are unknown. Here, we show that TAZ-CAMTA1 expression in endothelial cells is sufficient to drive the formation of vascular tumors with the distinctive features of EHE, and inhibition of TAZ-CAMTA1 results in the regression of these vascular tumors. We further show that activated TAZ resembles TAZ-CAMTA1 in driving the formation of EHE-like vascular tumors, suggesting that constitutive activation of TAZ underlies the pathological features of EHE. We show that TAZ-CAMTA1 initiates an angiogenic and regenerative-like transcriptional program in endothelial cells, and disruption of the TAZ-CAMTA1-TEAD interaction or ectopic expression of a dominant negative TEAD in vivo inhibits TAZ-CAMTA1-mediated transformation. Our study provides the first genetic model of a TAZ fusion oncoprotein driving its associated human cancer, pinpointing TAZ-CAMTA1 as the key driver and a valid therapeutic target of EHE.

Studies of the regenerative capacity of the liver have converged on the Hippo pathway, a serine/threonine kinase cascade discovered in Drosophila and conserved from unicellular organisms to mammals. Genetic studies of mouse and rat livers have revealed that the Hippo pathway is a key regulator of liver size, regeneration, development, metabolism, and homeostasis and that perturbations in the Hippo pathway can lead to the development of common liver diseases, such as fatty liver disease and liver cancer. In turn, pharmacological targeting of the Hippo pathway may be utilized to boost regeneration and to prevent the development and progression of liver diseases. We review current insights provided by the Hippo pathway into liver pathophysiology. Furthermore, we present a path forward for future studies to understand how newly identified components of the Hippo pathway may control liver physiology and how the Hippo pathway is regulated in the liver.

Deng et al. report an essential role for the spectrin-based membrane skeleton in specifying cell shape by transmitting intracellular actomyosin force to cell membrane. Their findings uncover an essential mechanism that couples cell shape, cortical tension, and Hippo signaling in tissue morphogenesis., Although extracellular force has a profound effect on cell shape, cytoskeleton tension, and cell proliferation through the Hippo signaling effector Yki/YAP/TAZ, how intracellular force regulates these processes remains poorly understood. Here, we report an essential role for spectrin in specifying cell shape by transmitting intracellular actomyosin force to cell membrane. While activation of myosin II in Drosophila melanogaster pupal retina leads to increased cortical tension, apical constriction, and Yki-mediated hyperplasia, spectrin mutant cells, despite showing myosin II activation and Yki-mediated hyperplasia, paradoxically display decreased cortical tension and expanded apical area. Mechanistically, we show that spectrin is required for tethering cortical F-actin to cell membrane domains outside the adherens junctions (AJs). Thus, in the absence of spectrin, the weakened attachment of cortical F-actin to plasma membrane results in a failure to transmit actomyosin force to cell membrane, causing an expansion of apical surfaces. These results uncover an essential mechanism that couples cell shape, cortical tension, and Hippo signaling and highlight the importance of non–AJ membrane domains in dictating cell shape in tissue morphogenesis.

Sphingolipids (SLs) are structurally diverse lipids that are defined by the presence of a long-chain base (LCB) backbone. Typically, LCBs contain a single Δ4E double bond (DB) (mostly d18:1), whereas the dienic LCB sphingadienine (d18:2) contains a second DB at the Δ14Z position. The enzyme introducing the Δ14Z DB is unknown. We analyzed the LCB plasma profile in a gender-, age-, and BMI-matched subgroup of the CoLaus cohort (n = 658). Sphingadienine levels showed a significant association with gender, being on average ∼30% higher in females. A genome-wide association study (GWAS) revealed variants in the fatty acid desaturase 3 (FADS3) gene to be significantly associated with the plasma d18:2/d18:1 ratio (p = -log 7.9). Metabolic labeling assays, FADS3 overexpression and knockdown approaches, and plasma LCB profiling in FADS3-deficient mice confirmed that FADS3 is a bona fide LCB desaturase and required for the introduction of the Δ14Z double bond. Moreover, we showed that FADS3 is required for the conversion of the atypical cytotoxic 1-deoxysphinganine (1-deoxySA, m18:0) to 1-deoxysphingosine (1-deoxySO, m18:1). HEK293 cells overexpressing FADS3 were more resistant to m18:0 toxicity than WT cells. In summary, using a combination of metabolic profiling and GWAS, we identified FADS3 to be essential for forming Δ14Z DB containing LCBs, such as d18:2 and m18:1. Our results unravel FADS3 as a Δ14Z LCB desaturase, thereby disclosing the last missing enzyme of the SL de novo synthesis pathway.

Mutations in the Fused in Sarcoma (FUS) gene have been identified in familial ALS in human. Drosophila contains a single ortholog of human FUS called Cabeza (Caz). We previously established Drosophila models of ALS targeted to Caz, which developed the locomotive dysfunction and caused anatomical defects in presynaptic terminals of motoneurons. Accumulating evidence suggests that ALS and cancer share defects in many cellular processes. The Hippo pathway was originally discovered in Drosophila and plays a role as a tumor suppressor in mammals. We aimed to determine whether Hippo pathway genes modify the ALS phenotype using Caz knockdown flies. We found a genetic link between Caz and Hippo (hpo), the Drosophila ortholog of human Mammalian sterile 20-like kinase (MST) 1 and 2. Loss-of-function mutations of hpo rescued Caz knockdown-induced eye- and neuron-specific defects. The decreased Caz levels in nuclei induced by Caz knockdown were also rescued by loss of function mutations of hpo. Moreover, hpo mRNA level was dramatically increased in Caz knockdown larvae, indicating that Caz negatively regulated hpo. Our results demonstrate that hpo, Drosophila MST, is a novel modifier of Drosophila FUS. Therapeutic targets that inhibit the function of MST could modify the pathogenic processes of ALS.

Regulatory T cells (Treg) are critical for maintaining self-tolerance and immune homeostasis, but their suppressive function can impede effective antitumor immune responses. FOXP3 is a transcription factor expressed in Tregs that is required for their function. However, the pathways and microenvironmental cues governing FOXP3 expression and Treg function are not completely understood. Herein, we report that YAP, a coactivator of the Hippo pathway, is highly expressed in Tregs and bolsters FOXP3 expression and Treg function in vitro and in vivo. This potentiation stemmed from YAP-dependent upregulation of activin signaling, which amplifies TGFβ/SMAD activation in Tregs. YAP deficiency resulted in dysfunctional Tregs unable to suppress antitumor immunity or promote tumor growth in mice. Chemical YAP antagonism and knockout or blockade of the YAP-regulated activin receptor similarly improved antitumor immunity. Thus, we identify YAP as an unexpected amplifier of a Treg-reinforcing pathway with significant potential as an anticancer immunotherapeutic target. Significance: Tregs suppress antitumor immunity, and pathways supporting their function can be novel immunotherapy targets. Here, the selective expression of YAP by Tregs, its importance for their function, and its unexpected enhancement of pro-Treg Activin/SMAD signaling are reported, as are validations of potential cancer-fighting antagonists of YAP and its regulatory targets. Cancer Discov; 8(8); 1026–43. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 899

Rationale: Microvascular inflammation and endothelial dysfunction secondary to unchecked activation of endothelium play a critical role in the pathophysiology of sepsis and organ failure. The intrinsic signaling mechanisms responsible for dampening excessive activation of endothelial cells are not completely understood. Objective: To determine the central role of YAP (Yes-associated protein), the major transcriptional coactivator of the Hippo pathway, in modulating the strength and magnitude of endothelial activation and vascular inflammation. Methods and Results: Endothelial-specific YAP knockout mice showed increased basal expression of E-selectin and ICAM (intercellular adhesion molecule)-1 in endothelial cells, a greater number of adherent neutrophils in postcapillary venules and increased neutrophil counts in bronchoalveolar lavage fluid. Lipopolysaccharide challenge of these mice augmented NF-κB (nuclear factor-κB) activation, expression of endothelial adhesion proteins, neutrophil and monocyte adhesion to cremaster muscle venules, transendothelial neutrophil migration, and lung inflammatory injury. Deletion of YAP in endothelial cells also markedly augmented the inflammatory response and cardiovascular dysfunction in a polymicrobial sepsis model induced by cecal ligation and puncture. YAP functioned by interacting with the E3 ubiquitin-protein ligase TLR (Toll-like receptor) signaling adaptor TRAF6 (tumor necrosis factor receptor-associated factor 6) to ubiquitinate TRAF6, and thus promoted TRAF6 degradation and modification resulting in inhibition of NF-κB activation. TRAF6 depletion in endothelial cells rescued the augmented inflammatory phenotype in mice with endothelial cell–specific deletion of YAP. Conclusions: YAP modulates the activation of endothelial cells and suppresses vascular inflammation through preventing TRAF6-mediated NF-κB activation and is hence essential for limiting the severity of sepsis-induced inflammation and organ failure.

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disorder caused by mutations in PKD1 or PKD2 and affects one in 500–1000 humans. Limited treatment is currently available for ADPKD. Here we identify the Hippo signaling effector YAP and its transcriptional target, c-Myc, as promoters of cystic kidney pathogenesis. While transgenic overexpression of YAP promotes proliferation and tubule dilation in mouse kidneys, loss of YAP/TAZ or c-Myc suppresses cystogenesis in a mouse ADPKD model resulting from Pkd1 deficiency. Through a comprehensive kinase inhibitor screen based on a novel three-dimensional (3D) culture of Pkd1 mutant mouse kidney cells, we identified a signaling pathway involving the RhoGEF (guanine nucleotide exchange factor) LARG, the small GTPase RhoA, and the RhoA effector Rho-associated kinase (ROCK) as a critical signaling module between PKD1 and YAP. Further corroborating its physiological importance, inhibition of RhoA signaling suppresses cystogenesis in 3D culture of Pkd1 mutant kidney cells as well as Pkd1 mutant mouse kidneys in vivo. Taken together, our findings implicate the RhoA–YAP–c-Myc signaling axis as a critical mediator and potential drug target in ADPKD.

Summary: The Hippo pathway controls organ size and tissue homeostasis through a kinase cascade leading from the Ste20-like kinase Hpo (MST1/2 in mammals) to the transcriptional coactivator Yki (YAP/TAZ in mammals). Whereas previous studies have uncovered positive and negative regulators of Hpo/MST, how they are integrated to maintain signaling homeostasis remains poorly understood. Here, we identify a self-restricting mechanism whereby autophosphorylation of an unstructured linker in Hpo/MST creates docking sites for the STRIPAK PP2A phosphatase complex to inactivate Hpo/MST. Mutation of the phospho-dependent docking sites in Hpo/MST or deletion of Slmap, the STRIPAK subunit recognizing these docking sites, results in constitutive activation of Hpo/MST in both Drosophila and mammalian cells. In contrast, autophosphorylation of the Hpo/MST linker at distinct sites is known to recruit Mats/MOB1 to facilitate Hippo signaling. Thus, multisite autophosphorylation of Hpo/MST linker provides an evolutionarily conserved built-in molecular platform to maintain signaling homeostasis by coupling antagonistic signaling activities. : The Hippo pathway was named after the Ste20-like kinase Hpo/MST, but how its activity is regulated remains unclear. Zheng et al. identify a self-restricting mechanism whereby autophosphorylation of an unstructured linker in Hpo/MST creates docking sites for the STRIPAK PP2A phosphatase complex to inactivate Hpo/MST in both Drosophila and mammals. Keywords: Hippo signaling, Hpo, MST1/2, STRIPAK, PP2A, SLMAP, autophosphorylation

Although mutations in the phosphoinositide 3‐kinase catalytic subunit (PIK3CA) are common in breast cancer, PI3K inhibitors alone have shown modest efficacy. We sought to identify additional pathways altered in PIK3CA‐mutant tumors that might be targeted in combination with PI3K inhibitors. We generated two transgenic mouse models expressing the human PIK3CA‐H1047R‐ and the ‐E545K hotspot‐mutant genes in the mammary gland and evaluated their effects on development and tumor formation. Molecular analysis identified pathways altered in these mutant tumors, which were also targeted in multiple cell lines derived from the PIK3CA tumors. Finally, public databases were analyzed to determine whether novel pathways identified in the mouse tumors were altered in human tumors harboring mutant PIK3CA. Mutant mice showed increased branching and delayed involution of the mammary gland compared to parental FVB/N mice. Mammary tumors arose in 30% of the MMTV‐PIK3CA‐H1047R and in 13% of ‐E545K mice. Compared to MMTV‐Her‐2 transgenic mouse mammary tumors, H1047R tumors showed increased upregulation of Wnt/β‐catenin/Axin2, hepatocyte growth factor (Hgf)/Stat3, insulin‐like growth factor 2 (Igf‐2), and Igf‐1R pathways. Inhibitors of STAT3, β‐catenin, and IGF‐1R sensitized H1047R‐derived mouse tumor cells and PIK3CA‐H1047R overexpressing human HS578T breast cancer cells to the cytotoxic effects of PI3K inhibitors. Analysis of The Cancer Genome Atlas database showed that, unlike primary PIK3CA‐wild‐type and HER‐2+ breast carcinomas, PIK3CA‐mutant tumors display increased expression of AXIN2, HGF, STAT3, IGF‐1, and IGF‐2 mRNA and activation of AKT, IGF1‐MTOR, and WNT canonical signaling pathways. Drugs targeting additional pathways that are altered in PIK3CA‐mutant tumors may improve treatment regimens using PI3K inhibitors alone.

The regenerative capacity of Schwann cells in the PNS underlies functional repair after injury. In this study, Mindos et al. show a new function for the tumor suppressor Merlin and Hippo/YAP signaling in the generation of repair-competent Schwann cells after injury., Loss of the Merlin tumor suppressor and activation of the Hippo signaling pathway play major roles in the control of cell proliferation and tumorigenesis. We have identified completely novel roles for Merlin and the Hippo pathway effector Yes-associated protein (YAP) in the control of Schwann cell (SC) plasticity and peripheral nerve repair after injury. Injury to the peripheral nervous system (PNS) causes a dramatic shift in SC molecular phenotype and the generation of repair-competent SCs, which direct functional repair. We find that loss of Merlin in these cells causes a catastrophic failure of axonal regeneration and remyelination in the PNS. This effect is mediated by activation of YAP expression in Merlin-null SCs, and loss of YAP restores axonal regrowth and functional repair. This work identifies new mechanisms that control the regenerative potential of SCs and gives new insight into understanding the correct control of functional nerve repair in the PNS.

Objective: Yap1 oncogene is essential for KRAS-induced pancreatic ductal adenocarcinoma (PDAC) initiation. We recently demonstrated that YAP1 is capable of bypassing KRAS-dependency. However, genetic alterations of YAP1 pathway are rare in human PDAC and its role in tumour maintenance remains unclear. This study investigates YAP1 function in transcriptionally distinct subsets of PDAC and explores the molecular mechanisms for YAP1 activation. Design: Conditional Yap1 allele was crossed into LSL-KrasG12D/+; Trp53R172H/+; Mist1-CreERT2+ mice to study the requirement of Yap1 for PDAC development in adult mice. YAP1 function in advanced PDAC was analyzed in human tissues, PDAC cell lines, patient-derived xenograft samples and mouse PDAC cells, using in vitro and in vivo assays and gene expression analyses. WNT5A expression and its effect on YAP1 activation as well as tumorigenic activity was studied in aforementioned human and mouse systems using genetic and pharmacological approaches. The bypass of KRAS-dependency by WNT5A was evaluated in KrasG12D-driven mouse PDAC cells. Results: Yap1 deletion blocked KrasG12D/Trp53R172H/+ induced PDAC development in mice. YAP1 activation correlated with squamous subtype of human PDAC and poor prognosis. YAP1 activation enhanced malignant phenotype and YAP1 depletion suppressed the tumorigenic activity specifically in squamous subtype tumours. WNT5A was significantly overexpressed in squamous subtype tumours and positively correlated with YAP1 activity. Conversely, WNT5A depletion resulted in YAP1 inactivation and inhibition of tumorigenic activity. WNT5A expression enabled cell survival and tumour growth in the absence of oncogenic Kras. Conclusions: YAP1 oncogene is a major driver for squamous subtype PDAC whose activation is mediated by WNT5A overexpression.

The Hippo tumor suppressor pathway regulates tissue growth in Drosophila by restricting the activity of the transcriptional coactivator Yorkie (Yki), which normally complexes with the TEF/TEAD family DNA-binding transcription factor Scalloped (Sd) to drive the expression of growth-promoting genes. Given its pivotal role as a central hub in mediating the transcriptional output of Hippo signaling, there is great interest in understanding the molecular regulation of the Sd-Yki complex. In this study, we identify Nerfin-1 as a transcriptional repressor that antagonizes the activity of the Sd-Yki complex by binding to the TEA DNA-binding domain of Sd. Consistent with its biochemical function, ectopic expression of Nerfin-1 results in tissue undergrowth in an Sd-dependent manner. Conversely, loss of Nerfin-1 enhances the ability of winner cells to eliminate loser cells in multiple scenarios of cell competition. We further show that INSM1, the mammalian ortholog of Nerfin-1, plays a conserved role in repressing the activity of the TEAD-YAP complex. These findings reveal a novel regulatory mode converging on the transcriptional output of the Hippo pathway that may be exploited for modulating the YAP oncoprotein in cancer and regenerative medicine., eLife digest Animals uses a range of mechanisms to stop their organs from growing once they have reached the right shape and size. One of these processes, a set of chemical messages called the Hippo pathway, controls the balance of cell death and cell division. In fruit flies, Hippo works by repressing a complex formed of two proteins, Yorkie and Scalloped, which normally switch genes on to encourage cells to grow. Yorkie is also involved in cell competition, a process in which cells in a tissue compare themselves to each other. Healthier ‘winner’ cells then kill neighboring ‘loser’ cells that are weaker or damaged. This ensures that the tissue keeps working properly. Despite Yorkie and Scalloped being key to control the growth and health of tissues, how the activity of these proteins is regulated was not well understood. To investigate, Guo et al. conducted a series experiments on fruit flies and found that a protein called Nerfin-1 can bind onto Scalloped to stop the Scalloped-Yorkie complex from switching on genes. As a result, flies with too much Nerfin-1 had stunted tissue growth. In addition, Guo et al. confirmed that the Nerfin-1 equivalent in mammals acts in the same way. Further work revealed that Nerfin-1 also plays a role in cell competition: without this protein, ‘winner’ cells became 'super winners', eliminating even more of the loser cells. Besides regulating the size of organs, the Hippo pathway is also involved in stopping cells from dividing uncontrollably and becoming cancerous. Further research may therefore focus on Nerfin-1 and its equivalent in mammals to understand how this protein could contribute to the emergence of cancer.

The Hippo signaling pathway regulates diverse physiological processes, and its dysfunction has been implicated in an increasing number of human diseases, including cancer. Here, we provide an updated review of the Hippo pathway; discuss its roles in development, homeostasis, regeneration, and diseases; and highlight outstanding questions for future investigation and opportunities for Hippo-targeted therapies.

The Hippo pathway effector Yes-associated protein (YAP) regulates liver size by promoting cell proliferation and inhibiting apoptosis. However, recent in vivo studies suggest that YAP has important cellular functions other than controlling proliferation and apoptosis. Transgenic YAP expression in mouse hepatocytes results in severe jaundice. A possible explanation for the jaundice could be defects in adherens junctions that prevent bile from leaking into the blood stream. Indeed, immunostaining of E-cadherin and electron microscopic examination of bile canaliculi of Yap transgenic livers revealed abnormal adherens junction structures. Using primary hepatocytes from Yap transgenic livers and Yap knockout livers, we found that YAP antagonizes E-cadherin-mediated cell-cell junction assembly by regulating the cellular actin architecture, including its mechanical properties (elasticity and cortical tension). Mechanistically, we found that YAP promoted contractile actin structure formation by upregulating nonmuscle myosin light chain expression and cellular ATP generation. Thus, by modulating actomyosin organization, YAP may influence many actomyosin-dependent cellular characteristics, including adhesion, membrane protrusion, spreading, morphology, and cortical tension and elasticity, which in turn determine cell differentiation and tissue morphogenesis.

Rationale: NF2 (neurofibromin 2) is an established tumor suppressor that promotes apoptosis and inhibits growth in a variety of cell types, yet its function in cardiomyocytes remains largely unknown. Objective: We sought to determine the role of NF2 in cardiomyocyte apoptosis and ischemia/reperfusion (I/R) injury in the heart. Methods and Results: We investigated the function of NF2 in isolated cardiomyocytes and mouse myocardium at baseline and in response to oxidative stress. NF2 was activated in cardiomyocytes subjected to H 2 O 2 and in murine hearts subjected to I/R. Increased NF2 expression promoted the activation of Mst1 (mammalian sterile 20–like kinase 1) and the inhibition of Yap (Yes-associated protein), whereas knockdown of NF2 attenuated these responses after oxidative stress. NF2 increased the apoptosis of cardiomyocytes that appeared dependent on Mst1 activity. Mice deficient for NF2 in cardiomyocytes, NF2 cardiomyocyte-specific knockout (CKO), were protected against global I/R ex vivo and showed improved cardiac functional recovery. Moreover, NF2 cardiomyocyte-specific knockout mice were protected against I/R injury in vivo and showed the upregulation of Yap target gene expression. Mechanistically, we observed nuclear association between NF2 and its activator MYPT-1 (myosin phosphatase target subunit 1) in cardiomyocytes, and a subpopulation of stress-induced nuclear Mst1 was diminished in NF2 CKO hearts. Finally, mice deficient for both NF2 and Yap failed to show protection against I/R indicating that Yap is an important target of NF2 in the adult heart. Conclusions: NF2 is activated by oxidative stress in cardiomyocytes and mouse myocardium and facilitates apoptosis. NF2 promotes I/R injury through the activation of Mst1 and inhibition of Yap, thereby regulating Hippo signaling in the adult heart.

Efforts to induce the differentiation of cancer stem cells through treatment with all-trans retinoic acid (ATRA) have yielded limited success, partially due to the epigenetic silencing of the retinoic acid receptor (RAR)-β. The histone deacetylase inhibitor entinostat is emerging as a promising antitumor agent when added to the standard-of-care treatment for breast cancer. However, the combination of epigenetic, cellular differentiation, and chemotherapeutic approaches against triple-negative breast cancer (TNBC) has not been investigated. In this study, we found that combined treatment of TNBC xenografts with entinostat, ATRA, and doxorubicin (EAD) resulted in significant tumor regression and restoration of epigenetically silenced RAR-β expression. Entinostat and doxorubicin treatment inhibited topoisomerase II-β (TopoII-β) and relieved TopoII-β-mediated transcriptional silencing of RAR-β. Notably, EAD was the most effective combination in inducing differentiation of breast tumor–initiating cells in vivo. Furthermore, gene expression analysis revealed that the epithelium-specific ETS transcription factor-1 (ESE-1 or ELF3), known to regulate proliferation and differentiation, enhanced cell differentiation in response to EAD triple therapy. Finally, we demonstrate that patient-derived metastatic cells also responded to treatment with EAD. Collectively, our findings strongly suggest that entinostat potentiates doxorubicin-mediated cytotoxicity and retinoid-driven differentiation to achieve significant tumor regression in TNBC. Cancer Res; 76(7); 2013–24. ©2016 AACR.

Cell-cell contact inhibition and the mechanical environment of cells have both been shown to regulate YAP nuclear localization to modulate cell proliferation. Changes in cellular contractility by genetic, pharmacological, and matrix stiffness perturbations regulate YAP nuclear localization. However, because contractility and F-actin organization are interconnected cytoskeletal properties, it remains unclear which of these distinctly regulates YAP localization. Here we show that in the absence of cell-cell contact, actomyosin contractility suppresses YAP phosphorylation at Ser(112), however, neither loss of contractility nor increase in YAP phosphorylation is sufficient for its nuclear exclusion. We find that actin cytoskeletal integrity is essential for YAP nuclear localization, and can override phosphoregulation or contractility-mediated regulation of YAP nuclear localization. This actin-mediated regulation is conserved during mechanotransduction, as substrate compliance increased YAP phosphorylation and reduced cytoskeletal integrity leading to nuclear exclusion of both YAP and Ser(P)(112)-YAP. These data provide evidence for two actin-mediated pathways for YAP regulation; one in which actomyosin contractility regulates YAP phosphorylation, and a second that involves cytoskeletal integrity-mediated regulation of YAP nuclear localization independent of contractility. We suggest that in non-contact inhibited cells, this latter mechanism may be important in low stiffness regimes, such as may be encountered in physiological environments.

TEA domain (TEAD) transcription factors bind to the coactivators YAP and TAZ and regulate the transcriptional output of the Hippo pathway, playing critical roles in organ size control and tumorigenesis. Protein S-palmitoylation attaches a fatty acid, palmitate, to cysteine residues and regulates protein trafficking, membrane localization and signaling activities. Using activity-based chemical probes, we discovered that human TEADs possess intrinsic palmitoylating enzyme-like activities and undergo autopalmitoylation at evolutionarily conserved cysteine residues under physiological conditions. We determined the crystal structures of lipid-bound TEADs and found that the lipid chain of palmitate inserts into a conserved deep hydrophobic pocket. Strikingly, palmitoylation did not alter TEAD's localization, but it was required for TEAD's binding to YAP and TAZ and was dispensable for its binding to the Vgll4 tumor suppressor. Moreover, palmitoylation-deficient TEAD mutants impaired TAZ-mediated muscle differentiation in vitro and tissue overgrowth mediated by the Drosophila YAP homolog Yorkie in vivo. Our study directly links autopalmitoylation to the transcriptional regulation of the Hippo pathway.

The Hippo signaling pathway functions through Yorkie to control tissue growth and homeostasis. How this pathway regulates non-developmental processes remains largely unexplored. Here, we report an essential role for Hippo signaling in innate immunity whereby Yorkie directly regulates the transcription of the Drosophila IκB homolog, Cactus, in Toll receptor-mediated antimicrobial response. Loss of Hippo pathway tumor suppressors or activation of Yorkie in fat bodies, the Drosophila immune organ, leads to elevated cactus mRNA levels, decreased expression of antimicrobial peptides, and vulnerability to infection by Gram-positive bacteria. Furthermore, Gram-positive bacteria acutely activate Hippo-Yorkie signaling in fat bodies via the Toll-Myd88-Pelle cascade through Pelle-mediated phosphorylation and degradation of the Cka subunit of the Hippo-inhibitory STRIPAK PP2A complex. Our studies elucidate a Toll-mediated Hippo signaling pathway in antimicrobial response, highlight the importance of regulating IκB/Cactus transcription in innate immunity, and identify Gram-positive bacteria as extracellular stimuli of Hippo signaling under physiological settings.

Cdc14 is an evolutionarily conserved serine/threoninephosphatase. Originally identified in S. cerevisiae as a cell cycle regulator, its role in other eukaryotic organisms remains unclear. In Drosophila melanogaster, Cdc14 is encoded by a single gene, thus facilitating its study. We found that Cdc14 expression is highest in the testis of adult flies and that cdc14 null flies are viable. cdc14 null female and male flies do not display altered fertility. cdc14 null males, however, exhibit decreased sperm competitiveness. Previous studies have shown that Cdc14 plays a role in ciliogenesis during zebrafish development. In Drosophila, sensory neurons are ciliated. We found that the Drosophila cdc14 null mutants have defects in chemosensation and mechanosensation as indicated by decreased avoidance of repellant substances and decreased response to touch. In addition, we show that cdc14 null mutants have defects in lipid metabolism and resistance to starvation. These studies highlight the diversity of Cdc14 function in eukaryotes despite its structural conservation.

Genetic studies in Drosophila have been instrumental in characterizing the Hippo pathway, which converges on the co-activator Yorkie to regulate target gene transcription. A routinely used strategy to interrogate upstream regulators of Yorkie involves the examination of selected Hippo target genes upon loss or gain of function of a suspected pathway regulator. A caveat with this strategy is that aberrant expression of a given Hippo target per se does not distinguish whether it is caused by changes in Yorkie or Yorkie-independent inputs converging on the same target gene. Building on previous findings that the DNA-binding transcription factor Scalloped mediates both Yorkie overexpression and loss-of-function phenotypes yet is itself dispensable for normal eye development, we describe a simple strategy to distinguish these possibilities by analyzing double-mutant clones of scalloped and a suspected Yorkie regulator. We provide proof of principle that this strategy can be used effectively to validate canonical Yorkie regulators and to exclude proteins that impact target expression independent of Yorkie. The described methodology and reagents should facilitate efforts to assess the expanding repertoire of proteins implicated in regulation of Yorkie activity.

The Hippo signaling pathway and the Notch signaling pathway are evolutionary conserved signaling cascades that have important roles in embryonic development of many organs. In murine liver, disruption of either pathway impairs intrahepatic bile duct development. Recent studies suggested that the Notch signaling receptor Notch2 is a direct transcriptional target of the Hippo signaling pathway effector YAP, and the Notch signaling is a major mediator of the Hippo signaling in maintaining biliary cell characteristics in adult mice. However, it remains to be determined whether the Hippo signaling pathway functions through the Notch signaling in intrahepatic bile duct development. We found that loss of the Hippo signaling pathway tumor suppressor Nf2 resulted in increased expression levels of the Notch signaling pathway receptor Notch2 in cholangiocytes but not in hepatocytes. When knocking down Notch2 on the background of Nf2 deficiency in mouse livers, the excessive bile duct development induced by Nf2 deficiency was suppressed by heterozygous and homozygous deletion of Notch2 in a dose-dependent manner. These results implicated that Notch signaling is one of the downstream effectors of the Hippo signaling pathway in regulating intrahepatic bile duct development.

During development, progenitor expansion, lineage allocation, and implementation of differentiation programs need to be tightly coordinated so that different cell types are generated in the correct numbers for appropriate tissue size and function. Pancreatic dysfunction results in some of the most debilitating and fatal diseases, including pancreatic cancer and diabetes. Several transcription factors regulating pancreas lineage specification have been identified, and Notch signalling has been implicated in lineage allocation, but it remains unclear how these processes are coordinated. Using a combination of genetic approaches, organotypic cultures of embryonic pancreata, and genomics, we found that sphingosine-1-phosphate (S1p), signalling through the G protein coupled receptor (GPCR) S1pr2, plays a key role in pancreas development linking lineage allocation and specification. S1pr2 signalling promotes progenitor survival as well as acinar and endocrine specification. S1pr2-mediated stabilisation of the yes-associated protein (YAP) is essential for endocrine specification, thus linking a regulator of progenitor growth with specification. YAP stabilisation and endocrine cell specification rely on Gαi subunits, revealing an unexpected specificity of selected GPCR intracellular signalling components. Finally, we found that S1pr2 signalling posttranscriptionally attenuates Notch signalling levels, thus regulating lineage allocation. Both S1pr2-mediated YAP stabilisation and Notch attenuation are necessary for the specification of the endocrine lineage. These findings identify S1p signalling as a novel key pathway coordinating cell survival, lineage allocation, and specification and linking these processes by regulating YAP levels and Notch signalling. Understanding lineage allocation and specification in the pancreas will shed light in the origins of pancreatic diseases and may suggest novel therapeutic approaches., Author summary The pancreas develops from a field of progenitor cells localised in a restricted region of the embryonic endoderm. These progenitor cells proliferate and eventually differentiate to generate the three distinct lineages comprising the endocrine (which include insulin-producing β cells), acinar, and ductal cells. The molecular pathways implicated in the early generation of pancreas progenitors and their proliferation are well understood. It is also known that the Notch signalling pathway is implicated in sequential binary cell fate decisions that generate the three lineages; however, other signals that may regulate this process remain unknown. Here, we show that a phospholipid, sphingosine-1-phosphate (S1p), generated by the progenitor cells themselves, acts as a signal necessary to define the acinar and endocrine lineage. We observe that in the absence of S1p only duct cells are generated and the survival of pancreas progenitors is compromised. The function of this signalling pathway in the generation of the endocrine cells is two-fold. Firstly, it stabilises yes-associated protein (YAP), a transcriptional gene coactivator known to regulate pancreatic progenitor proliferation, and we show that this stability is necessary for the activation of the endocrine specification program. Secondly, it attenuates Notch signalling, allowing the generation of endocrine and acinar cells. Notch attenuation is necessary for the stabilisation of the transcription factor Ngn3, which is required for the generation of endocrine cells. We conclude that S1p acts as an autocrine signal regulating YAP stabilisation and Notch attenuation to mediate pancreas specification. Understanding lineage allocation and specification in the pancreas will shed light on the origins of pancreatic diseases and may suggest novel therapeutic approaches.

SummaryAlthough Merlin/NF2 was discovered two decades ago as a tumor suppressor underlying Neurofibromatosis type II, its precise molecular mechanism remains poorly understood. Recent studies in Drosophila revealed a potential link between Merlin and the Hippo pathway by placing Merlin genetically upstream of the kinase Hpo/Mst. In contrast to the commonly depicted linear model of Merlin functioning through Hpo/Mst, here we show that in both Drosophila and mammals, Merlin promotes downstream Hippo signaling without activating the intrinsic kinase activity of Hpo/Mst. Instead, Merlin directly binds and recruits the effector kinase Wts/Lats to the plasma membrane. Membrane recruitment, in turn, promotes Wts phosphorylation by the Hpo-Sav kinase complex. We further show that disruption of the actin cytoskeleton promotes Merlin-Wts interactions, which implicates Merlin in actin-mediated regulation of Hippo signaling. Our findings elucidate an important molecular function of Merlin and highlight the plasma membrane as a critical subcellular compartment for Hippo signal transduction.

Summary The Hippo tumor suppressor pathway restricts tissue growth by inactivating the transcriptional coactivator Yki. Although Sd has been implicated as a DNA-binding transcription factor partner for Yki and can genetically account for gain-of-function Yki phenotypes, how Yki regulates normal tissue growth remains a long-standing puzzle because Sd, unlike Yki, is dispensable for normal growth in most Drosophila tissues. Here we show that the yki mutant phenotypes in multiple developmental contexts are rescued by inactivation of Sd, suggesting that Sd functions as a default repressor and that Yki promotes normal tissue growth by relieving Sd-mediated default repression. We further identify Tgi as a cofactor involved in Sd's default repressor function and demonstrate that the mammalian ortholog of Tgi potently suppresses the YAP oncoprotein in transgenic mice. These findings fill a major gap in Hippo-mediated transcriptional regulation and open up possibilities for modulating the YAP oncoprotein in cancer and regenerative medicine.

Nonaggregative multicellularity requires strict control of cell number. The Hippo signaling pathway coordinates cell proliferation and apoptosis and is a central regulator of organ size in animals. Recent studies have shown the presence of key members of the Hippo pathway in nonbilaterian animals, but failed to identify this pathway outside Metazoa. Through comparative analyses of recently sequenced holozoan genomes, we show that Hippo pathway components, such as the kinases Hippo and Warts, the coactivator Yorkie, and the transcription factor Scalloped, were already present in the unicellular ancestors of animals. Remarkably, functional analysis of Hippo components of the amoeboid holozoan Capsaspora owczarzaki, performed in Drosophila melanogaster, demonstrate that the growth-regulatory activity of the Hippo pathway is conserved in this unicellular lineage. Our findings show that the Hippo pathway evolved well before the origin of Metazoa and highlight the importance of Hippo signaling as a key developmental mechanism predating the origin of Metazoa. © 2012 The Authors., This work was supported by an Institució Catalana per a la Recerca i Estudis Avançats contract, a European Research Council Starting Grant (ERC-2007-StG-206883), a grant (BFU2008-02839/BMC) from Ministerio de Ciencia e Innovación (MICINN) to I. R.-T., and a National Institutes of Health grant (R01 EY015708) to D.P. A.S-P. was supported by a pregraduate Formacion Profesorado Universitario grant from MICINN. D.P. is an investigator of the Howard Hughes Medical Institute.

SummaryThe Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. Central to this pathway is a kinase cascade wherein Hippo (Hpo), in complex with Salvador (Sav), phosphorylates and activates Warts (Wts), which in turn phosphorylates and inactivates the Yorkie (Yki) oncoprotein, known as the YAP coactivator in mammalian cells. The FERM domain proteins Merlin (Mer) and Expanded (Ex) are upstream components that regulate Hpo activity through unknown mechanisms. Here we identify Kibra as another upstream component of the Hippo signaling pathway. We show that Kibra functions together with Mer and Ex in a protein complex localized to the apical domain of epithelial cells, and that this protein complex regulates the Hippo kinase cascade via direct binding to Hpo and Sav. These results shed light on the mechanism of Ex and Mer function and implicate Kibra as a potential tumor suppressor with relevance to neurofibromatosis.

SummaryTGF-β and BMP receptor kinases activate Smad transcription factors by C-terminal phosphorylation. We have identified a subsequent agonist-induced phosphorylation that plays a central dual role in Smad transcriptional activation and turnover. As receptor-activated Smads form transcriptional complexes, they are phosphorylated at an interdomain linker region by CDK8 and CDK9, which are components of transcriptional mediator and elongation complexes. These phosphorylations promote Smad transcriptional action, which in the case of Smad1 is mediated by the recruitment of YAP to the phosphorylated linker sites. An effector of the highly conserved Hippo organ size control pathway, YAP supports Smad1-dependent transcription and is required for BMP suppression of neural differentiation of mouse embryonic stem cells. The phosphorylated linker is ultimately recognized by specific ubiquitin ligases, leading to proteasome-mediated turnover of activated Smad proteins. Thus, nuclear CDK8/9 drive a cycle of Smad utilization and disposal that is an integral part of canonical BMP and TGF-β pathways.

SummaryCoordination of cell proliferation and cell death is essential to attain proper organ size during development and for maintaining tissue homeostasis throughout postnatal life. In Drosophila, these two processes are orchestrated by the Hippo kinase cascade, a growth-suppressive pathway that ultimately antagonizes the transcriptional coactivator Yorkie (Yki). Here we demonstrate that a single phosphorylation site in Yki mediates the growth-suppressive output of the Hippo pathway. Hippo-mediated phosphorylation inactivates Yki by excluding it from the nucleus, whereas loss of Hippo signaling leads to nuclear accumulation and therefore increased Yki activity. We further delineate a mammalian Hippo signaling pathway that culminates in the phosphorylation of YAP, the mammalian homolog of Yki. Using a conditional YAP transgenic mouse model, we demonstrate that the mammalian Hippo pathway is a potent regulator of organ size, and that its dysregulation leads to tumorigenesis. These results uncover a universal size-control mechanism in metazoan.

The Hippo pathway controls tissue growth through a core kinase cascade that impinges on the transcription of growth-regulatory genes. Understanding how this pathway is regulated in development remains a major challenge. Recent studies suggested that Hippo signaling can be modulated by cytoskeletal tension through a Rok-myosin II pathway. How cytoskeletal tension is regulated or its relationship to the other known upstream regulators of the Hippo pathway remains poorly defined. In this study, we identify spectrin, a contractile protein at the cytoskeleton-membrane interface, as an upstream regulator of the Hippo signaling pathway. We show that, in contrast to canonical upstream regulators such as Crumbs, Kibra, Expanded, and Merlin, spectrin regulates Hippo signaling in a distinct way by modulating cortical actomyosin activity through non-muscle myosin II. These results uncover an essential mediator of Hippo signaling by cytoskeleton tension, providing a new entry point to dissecting how mechanical signals regulate Hippo signaling in living tissues. DOI: http://dx.doi.org/10.7554/eLife.06567.001, eLife digest Organs including the liver, eyes, and lungs are made up of millions of cells, and how these organs stop growing once they reach their final size has fascinated scientists for decades. The cells in developing organs must communicate with each other and respond appropriately to the signals that they receive from other cells. This requires so-called “signaling pathways”. One such pathway that involves a protein called Hippo is known to control when cells should grow and divide and when they should stop. If this pathway does not work correctly, it can cause too many cells to be formed, which may result in cancer. The Hippo signaling pathway can also be regulated by an extensive network of protein filaments found within cells, called the cytoskeleton. This network can exert forces on the cells, which can have a major impact on cell growth. However, the mechanism behind the interaction between the cytoskeleton and the Hippo signaling pathway is poorly understood. Now, Deng et al. have engineered fruit flies in which the expression of individual genes had been artificially reduced, and looked for flies that had enlarged wings. Three genes identified in these experiments encode different subunits of a large spring-like protein, called spectrin, which is part of the cytoskeleton. This suggests that normally spectrin limits wing size. Furthermore, spectrin was also found to control the size of other organs in the fruit flies, such as the eyes and ovaries. In all of these organs, the Hippo signaling pathway failed to work properly in the absence of spectrin. Deng et al. then further explored the relationship between spectrin and Hippo signaling and found that cells without spectrin show abnormally high levels of tension in their cytoskeleton. When flies that lacked spectrin were engineered to reduce this tension, these flies developed normal sized organs. These findings reveal the importance of cytoskeleton tension in controlling tissue growth, and provide a new entry point to understand how normal tissues grow to their characteristic size and how such process goes awry in cancer. DOI: http://dx.doi.org/10.7554/eLife.06567.002

The Hippo signaling pathway regulates tissue growth in Drosophila through the transcriptional coactivator Yorkie (Yki). How Yki activates target gene transcription is poorly understood. Here, we identify Nuclear receptor coactivator 6 (Ncoa6), a subunit of the Trithorax-related (Trr) histone H3 lysine 4 (H3K4) methyltransferase complex, as a Yki-binding protein. Like Yki, Ncoa6 and Trr are functionally required for Hippo-mediated growth control and target gene expression. Strikingly, artificial tethering of Ncoa6 to Sd is sufficient to promote tissue growth and Yki target expression even in the absence of Yki, underscoring the importance of Yki-mediated recruitment of Ncoa6 in transcriptional activation. Consistent with the established role for the Trr complex in histone methylation, we show that Yki, Ncoa6, and Trr are required for normal H3K4 methylation at Hippo target genes. These findings shed light on Yki-mediated transcriptional regulation and uncover a potential link between chromatin modification and tissue growth. DOI: http://dx.doi.org/10.7554/eLife.02564.001, eLife digest Cells need to work together for a multi-celled organism, such as a plant or an animal, to thrive. Many complicated signaling pathways therefore exist that allow cells to communicate with one another and to control their own activity in response to the signals that they receive. One such pathway, called the Hippo signaling pathway, regulates when cells grow and divide, which allows organs and tissues to develop correctly and helps to prevent cancerous tumors from forming. Signaling pathways often control the activity of cells by affecting how particular genes are expressed from DNA. One way of doing this is to activate or inactivate proteins called transcription factors, which bind to sections of DNA to alter the expression of nearby genes. In fruit flies, the Hippo signaling pathway stops cells from dividing by inactivating Yorkie, a protein that binds to and activates certain transcription factors. However, exactly how Yorkie is able to activate these transcription factors was unclear. For transcription factors to function correctly, they must be able to reach the stretch of DNA where they bind. Therefore, another way to alter gene expression is to change how the DNA is packaged in a cell. This can be done by modifying the proteins that the DNA is wrapped around, which are called histones, for example by using enzymes called methyltransferases to add methyl groups to these proteins. Qing, Yin et al. looked at the Hippo signaling pathway in the fruit fly Drosophila, and found that the Yorkie protein only activates transcription factors when another protein called Ncoa6—which is part of a methyltransferase—binds to it. Furthermore, when the Ncoa6 protein was bound directly to the transcription factor, the tissue grew normally even when the Yorkie protein was not present. These findings reveal the importance of histone modifications in controlling tissue growth, and could provide a new direction in the search for cancer treatments. DOI: http://dx.doi.org/10.7554/eLife.02564.002

SummaryThe tumor-suppressive Hippo pathway controls tissue homeostasis through balancing cell proliferation and apoptosis. Activation of the kinases Mst1 and Mst2 (Mst1/2) is a key upstream event in this pathway and remains poorly understood. Mst1/2 and their critical regulators RASSFs contain Salvador/RASSF1A/Hippo (SARAH) domains that can homo- and heterodimerize. Here, we report the crystal structures of human Mst2 alone and bound to RASSF5. Mst2 undergoes activation through transautophosphorylation at its activation loop, which requires SARAH-mediated homodimerization. RASSF5 disrupts Mst2 homodimer and blocks Mst2 autoactivation. Binding of RASSF5 to already activated Mst2, however, does not inhibit its kinase activity. Thus, RASSF5 can act as an inhibitor or a potential positive regulator of Mst2, depending on whether it binds to Mst2 before or after activation-loop phosphorylation. We propose that these temporally sensitive functions of RASSFs enable the Hippo pathway to respond to and integrate diverse cellular signals.

Human chronic cholestatic liver diseases are characterized by cholangiocyte proliferation, hepatocyte injury, and fibrosis. Yes-associated protein (YAP), the effector of the Hippo tumor-suppressor pathway, has been shown to play a critical role in promoting cholangiocyte and hepatocyte proliferation and survival during embryonic liver development and hepatocellular carcinogenesis. Therefore, the aim of this study was to examine whether YAP participates in the regenerative response after cholestatic injury. First, we examined human liver tissue from patients with chronic cholestasis. We found more-active nuclear YAP in the bile ductular reactions of primary sclerosing cholangitis and primary biliary cirrhosis patient liver samples. Next, we used the murine bile duct ligation (BDL) model to induce cholestatic liver injury. We found significant changes in YAP activity after BDL in wild-type mice. The function of YAP in the hepatic response after BDL was further evaluated with liver-specific Yap conditional deletion in mice. Ablating Yap in the mouse liver not only compromised bile duct proliferation, but also enhanced hepatocyte necrosis and suppressed hepatocyte proliferation after BDL. Furthermore, primary hepatocytes and cholangiocytes isolated from Yap-deficient livers showed reduced proliferation in response to epidermal growth factor in vitro. Finally, we demonstrated that YAP likely mediates its biological effects through the modulation of Survivin expression. Conclusion: Our data suggest that YAP promotes cholangiocyte and hepatocyte proliferation and prevents parenchymal damage after cholestatic injury in mice and thus may mediate the response to cholestasis-induced human liver disease. (Hepatology 2012;56:1097–1107)

The control of cell and organ growth is fundamental to the development of multicellular organisms. Here, we show that dPTEN, a Drosophila homolog of the mammalian PTEN tumor suppressor gene, plays an essential role in the control of cell size, cell number, and organ size. In mosaic animals, dPTEN− cells proliferate faster than their heterozygous siblings, show an autonomous increase in cell size, and form organs of increased size, whereas overexpression of dPTEN results in opposite phenotypes. The loss-of-function phenotypes of dPTEN are suppressed by mutations in the PI3K target Dakt1 and the translational initiation factor eif4A, suggesting that dPTEN acts through the PI3K signaling pathway to regulate translation. Although activation of PI3K and Akt has been reported to increase rates of cellular growth but not proliferation, loss of dPTEN stimulates both of these processes, suggesting that PTEN regulates overall growth through PI3K/Akt-dependent and -independent pathways. Furthermore, we show that dPTEN does not play a major role in cell survival during Drosophila development. Our results provide a potential explanation for the high frequency of PTEN mutation in human cancer.