Hippo-YAP Signaling Research Articles

Pi3kcb links Hippo-YAP and PI3K-AKT signaling pathways to promote cardiomyocyte proliferation and survival.

Yes-associated protein (YAP), the nuclear effector of Hippo signaling, regulates cellular growth and survival in multiple organs, including the heart, by interacting with TEA (transcriptional enhancer activator)-domain sequence-specific DNA-binding proteins. Recent studies showed that YAP stimulates cardiomyocyte proliferation and survival. However, the direct transcriptional targets through which YAP exerts its effects are poorly defined. To identify direct YAP targets that mediate its mitogenic and antiapoptotic effects in the heart. We identified direct YAP targets by combining differential gene expression analysis in YAP gain- and loss-of-function with genome-wide identification of YAP-bound loci using chromatin immunoprecipitation and high throughput sequencing. This screen identified Pik3cb, encoding p110β, a catalytic subunit of phosphoinositol-3-kinase, as a candidate YAP effector that promotes cardiomyocyte proliferation and survival. YAP and TEA-domain occupied a conserved enhancer within the first intron of Pik3cb, and this enhancer drove YAP-dependent reporter gene expression. Yap gain- and loss-of-function studies indicated that YAP is necessary and sufficient to activate the phosphoinositol-3-kinase-Akt pathway. Like Yap, Pik3cb gain-of-function stimulated cardiomyocyte proliferation, and Pik3cb knockdown dampened YAP mitogenic activity. Reciprocally, impaired heart function in Yap loss-of-function was significantly rescued by adeno-associated virus-mediated Pik3cb expression. Pik3cb is a crucial direct target of YAP, through which the YAP activates phosphoinositol-3-kinase-AKT pathway and regulates cardiomyocyte proliferation and survival.

The Hippo-YAP signaling pathway and contact inhibition of growth

The Hippo-YAP pathway mediates the control of cell proliferation by contact inhibition as well as other attributes of the physical state of cells in tissues. Several mechanisms sense the spatial and physical organization of cells, and function through distinct upstream modules to stimulate Hippo-YAP signaling: adherens junction or cadherin–catenin complexes, epithelial polarity and tight junction complexes, the FAT-Dachsous morphogen pathway, as well as cell shape, actomyosin or mechanotransduction. Soluble extracellular factors also regulate Hippo pathway signaling, often inhibiting its activity. Indeed, the Hippo pathway mediates a reciprocal relationship between contact inhibition and mitogenic signaling. As a result, cells at the edges of a colony, a wound in a tissue or a tumor are more sensitive to ambient levels of growth factors and more likely to proliferate, migrate or differentiate through a YAP and/or TAZ-dependent process. Thus, the Hippo-YAP pathway senses and responds to the physical organization of cells in tissues and coordinates these physical cues with classic growth-factor-mediated signaling pathways. This Commentary is focused on the biological significance of Hippo-YAP signaling and how upstream regulatory modules of the pathway interact to produce biological outcomes.

The Hippo-YAP signaling pathway and contact inhibition of growth

The Hippo-YAP pathway mediates the control of cell proliferation by contact inhibition as well as other attributes of the physical state of cells in tissues. Several mechanisms sense the spatial and physical organization of cells, and function through distinct upstream modules to stimulate Hippo-YAP signaling: adherens junction or cadherin-catenin complexes, epithelial polarity and tight junction complexes, the FAT-Dachsous morphogen pathway, as well as cell shape, actomyosin or mechanotransduction. Soluble extracellular factors also regulate Hippo pathway signaling, often inhibiting its activity. Indeed, the Hippo pathway mediates a reciprocal relationship between contact inhibition and mitogenic signaling. As a result, cells at the edges of a colony, a wound in a tissue or a tumor are more sensitive to ambient levels of growth factors and more likely to proliferate, migrate or differentiate through a YAP and/or TAZ-dependent process. Thus, the Hippo-YAP pathway senses and responds to the physical organization of cells in tissues and coordinates these physical cues with classic growth-factor-mediated signaling pathways. This Commentary is focused on the biological significance of Hippo-YAP signaling and how upstream regulatory modules of the pathway interact to produce biological outcomes.

A functional interaction between Hippo-YAP signalling and FoxO1 mediates the oxidative stress response.

The Hippo pathway is an evolutionarily conserved regulator of organ size and tumorigenesis that negatively regulates cell growth and survival. Here we report that YAP, the terminal effector of the Hippo pathway, interacts with FoxO1 in the nucleus of cardiomyocytes, thereby promoting survival. YAP and FoxO1 form a functional complex on the promoters of the catalase and MnSOD antioxidant genes and stimulate their transcription. Inactivation of YAP, induced by Hippo activation, suppresses FoxO1 activity and decreases antioxidant gene expression, suggesting that Hippo signaling modulates the FoxO1-mediated antioxidant response. In the setting of ischemia/reperfusion (I/R) in the heart, activation of Hippo antagonizes YAP-FoxO1, leading to enhanced oxidative stress-induced cell death through downregulation of catalase and MnSOD. Conversely, restoration of YAP activity protects against I/R injury. These results suggest that YAP is a nuclear co-factor of FoxO1 and that the Hippo pathway negatively affects cardiomyocyte survival by inhibiting the function of YAP-FoxO1.

An emerging role for Hippo-YAP signaling in cardiovascular development

The Hippo signaling pathway was originally discovered in Drosophila and shown to be critical for organ size control and tumorigenesis. This pathway consists of a cascade of kinases and several adaptors that lead to the phosphorylation and inhibition, through nuclear exclusion, of the transcriptional cofactor Yorkie in Drosophila or YAP (yes associated protein) in mammals. Recent studies demonstrate that cardiac-specific deletion of the Hippo pathway kinase Mst (STE20-like protein kinases) co-activator WW45 (WW domain-containing adaptor 45), Mst1, Mst2, or Lats2 (large tumor suppressor homologue 2) in mice result in over-grown hearts with elevated cardiomyocyte proliferation. Consistent with these observations, over-expression of YAP in the mouse embryonic heart increases heart size and promotes cardiac regeneration and contractility after myocardial infarction by inducing cardiomyocyte proliferation, whereas deletion of YAP in the mouse heart impedes cardiomyocyte proliferation, causing myocardial hypoplasia and embryonic or premature lethality. YAP has also been shown to play an important role in the vascular system. Specific-deletion of YAP from vascular smooth muscle cells in mice results in aberrant development of large arteries with a hypoplastic arterial wall phenotype. Hippo-YAP signaling cross-talks with other signaling pathways such as IGF (insulin-like growth factor) and Wnt signaling to promote heart growth by increasing expression of cell cycle genes. The purpose of this review is to summarize these recent findings and discuss potential diagnostic or therapeutic strategies in cardiovascular system based on manipulating the Hippo-YAP signaling.

Dual function of Yap in the regulation of lens progenitor cells and cellular polarity

Hippo-Yap signaling has been implicated in organ size determination via its regulation of cell proliferation, growth and apoptosis (Pan, 2007). The vertebrate lens comprises only two major cell types, lens progenitors and differentiated fiber cells, thereby providing a relatively simple system for studying size-controlling mechanisms. In order to investigate the role of Hippo-Yap signaling in lens size regulation, we conditionally ablated Yap in the developing mouse lens. Lens progenitor-specific deletion of Yap led to near obliteration of the lens primarily due to hypocellularity in the lens epithelium (LE) and accompanying lens fiber (LF) defects. A significantly reduced LE progenitor pool resulted mainly from failed self-renewal and increased apoptosis. Additionally, Yap-deficient lens progenitor cells precociously exited the cell cycle and expressed the LF marker, β-Crystallin. The mutant progenitor cells also exhibited multiple cellular and subcellular alterations including cell and nuclear shape change, organellar polarity disruption, and disorganized apical polarity complex and junction proteins such as Crumbs, Pals1, Par3 and ZO-1. Yap-deficient LF cells failed to anchor to the overlying LE layer, impairing their normal elongation and packaging. Furthermore, our localization study results suggest that, in the developing LE, Yap participates in the cell context-dependent transition from the proliferative to differentiation-competent state by integrating cell density information. Taken together, our results shed new light on Yap's indispensable and novel organizing role in mammalian organ size control by coordinating multiple events including cell proliferation, differentiation, and polarity.

A genetic screen identifies an LKB1–MARK signalling axis controlling the Hippo–YAP pathway

The Hippo-YAP pathway is an emerging signalling cascade involved in the regulation of stem cell activity and organ size. To identify components of this pathway, we performed an RNAi-based kinome screen in human cells. Our screen identified several kinases not previously associated with Hippo signalling that control multiple cellular processes. One of the hits, LKB1, is a common tumour suppressor whose mechanism of action is only partially understood. We demonstrate that LKB1 acts through its substrates of the microtubule affinity-regulating kinase family to regulate the localization of the polarity determinant Scribble and the activity of the core Hippo kinases. Our data also indicate that YAP is functionally important for the tumour suppressive effects of LKB1. Our results identify a signalling axis that links YAP activation with LKB1 mutations, and have implications for the treatment of LKB1-mutant human malignancies. In addition, our findings provide insight into upstream signals of the Hippo-YAP signalling cascade.

Abstract C87: Regulation of stemness by Hippo-YAP pathway under hypoxia in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and the third most common cause of cancer-related death. The Hippo-YAP signaling pathway is gaining recognition as it plays an important role in both organ size control and tumorigenesis. Liver-specific ablation of WW45 in rodent models (one of the tumor suppressive Hippo pathway components in mammals) has been reported to increase liver size and expand hepatic stem/progenitor cells (oval cells) and to develop HCC eventually. The expression of YAP also has been reported as an independent poor prognostic marker for human HCCs. Amphiregulin is a downstream target of YAP and is a member of the EGF family, which is implicated in cancer stem cells in HCC. Cancer stem cells have the ability to self-renew, differentiate, and proliferate, and have greater tumorigenicity, chemoresistance, and resistance to hypoxia. HCCs expressing stem cell makers have actually been reported to be associated with poor prognosis and aggressive biological behavior. The aim of this study is to investigate the Hippo-YAP pathway and its related oncogenic signaling in HCCs expressing stem cell markers. The effect of amphiregulin on the regulation of the Hippo-YAP pathway was also studied to identify potential molecular therapeutic targets for HCCs expressing stem cell markers. Under hypoxic condition, the expression of cancer stem cell markers (EpCAM, CD133, and CK19) was upregulated in a time dependent manner and the Hippo-YAP pathway was oncogenically activated in four HCC cell lines (HepG2, Hep3B, Huh7, and SK-Hep1). In addition, the treatment of recombinant human amphiregulin dephosphorylated and activated YAP pathway. Therefore, up-regulation of cancer stem cell markers with activation of oncogenic YAP pathway in HCC cell lines might suggest the acquisition of survival advantage of tumor cells under hypoxia. The relationship between the regulatory mechanism of cancer stem cells and the Hippo-YAP pathway can be used for molecular target of chemotherapy for HCC. Citation Format: Ji Su Kim, Ei Yong Ahn, Young Nyun Park. Regulation of stemness by Hippo-YAP pathway under hypoxia in hepatocellular carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C87.

The role of LPA and YAP signaling in long-term migration of human ovarian cancer cells.

BackgroundThe Hippo-YAP signaling pathway is altered and implicated as oncogenic in many human cancers. However, extracellular signals that regulate the mammalian Hippo pathway have remained elusive until very recently when it was shown that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) ligands including lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P). LPA inhibits Lats kinase activity in HEK293 cells, but the potential involvement of a protein phosphatase was not investigated. The extracellular regulators of YAP dephosphorylation (dpYAP) and nuclear translocation in epithelial ovarian cancer (EOC) are essentially unknown.ResultsWe showed here that LPA dose- and time-dependently induced dpYAP in human EOC cell lines OVCA433, OVCAR5, CAOV3, and Monty-1, accompanied by increased YAP nuclear translocation. YAP was involved in LPA-induced migration and invasion of EOC cells and LPA3 was a major LPA receptor mediating the migratory effect. We demonstrated that G13, but not or to a lesser extent G12, Gi or Gq, was necessary for LPA-induced dpYAP and its nuclear translocation and that RhoA-ROCK, but not RhoB, RhoC, Rac1, cdc42, PI3K, ERK, or AKT, were required for the LPA-dpYAP effect. In contrast to results in HEK293 cells, LPA did not inhibit Mst and Lats kinase in OVCA433 EOC cells. Instead, protein phosphatase 1A (PP1A) acted down-stream of RhoA in LPA-induction of dpYAP. In addition, we identified that amphiregulin (AREG), a down-stream target of YAP which activated EGF receptors (EGFR), mediated an LPA-stimulated and EGFR-dependent long-term (16 hr) cell migration. This process was transcription- and translation-dependent and was distinct from a transcription- and YAP-independent short-term (4 hr) cell migration. EOC tissues had reduced pYAP levels compared to normal and benign ovarian tissues, implying the involvement of dpYAP in EOC pathogenesis, as well as its potential marker and/or target values.ConclusionsA novel LPA-LPA3-G13-RhoA-ROCK-PP1A-dpYAP-AREG-EGFR signaling pathway was linked to LPA-induced migration of EOC cells. Reduced pYAP levels were demonstrated in human EOC tumors as compared to both normal ovarian tissues and benign gynecologic masses. Our findings support that YAP is a potential marker and target for developing novel therapeutic strategies against EOC.

Identification of Serum-Derived Sphingosine-1-Phosphate as a Small Molecule Regulator of YAP

Hippo signaling represents a tumor suppressor pathway that regulates organ size and tumorigenesis through phosphorylation and inhibition of the transcription coactivator YAP. Here, we show that serum deprivation dramatically induces YAP Ser127 phosphorylation and cytoplasmic retention, independent of cell-cell contact. Through chemical isolation and activity profiling, we identified serum-derived sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) as small molecule activators of YAP. S1P induces YAP nuclear localization through S1P(2) receptor, Rho GTPase activation, and F-actin polymerization, independent of the core Hippo pathway kinases. Bioinformatics studies also showed that S1P stimulation induces YAP target gene expression in mouse liver and human embryonic stem cells. These results revealed potent small molecule regulators of YAP and suggest that S1P and LPA might modulate cell proliferation and tumorigenesis through YAP activation.

Measuring transcripts of components of the BRCA1 DNA repair pathway, components of the hippo-YAP pathway, β-TrCP, HER2, AEG-1, EZH2 and other genes in advanced gastric cancer.

4068 Background: The primary goal of this study was to classify gastric cancer in two different sub-groups. We speculated that HER2-positive gastric cancers could be regulated by β-TrCP, while tumors unaffected by β-TrCP activation could be more dependent on the Hippo-YAP signaling pathway, in which TAZ increases the expression of AXL. Gastric cancer can overexpress AEG-1 and EZH2. The relationship between EZH2 and the Hippo-YAP pathway was explored in this study. We also explored BRCA1 complexes, including upstream components, such as 53BP1 and MMSET. Methods: Paraffin-embedded samples were collected from 132 advanced gastric cancer patients (p) treated with first-line FOLFOX, 58 of whom received second-line docetaxel-based treatment. Gene expression levels of HER2, β-TrCP, TAZ, AXL, EZH2, AEG-1, BRCA1, and genes involved in DNA repair pathways (RAP80, PIAS1, PIAS4, MDC1, 53BP1, MMSET, and UBC9) were quantified by real-time PCR. BIM, IDO, and SPINK1 were also examined. Results: A close correlation was found for the majority of the genes, for example, BRCA1-MMSET (rho=0.28; P=0.002) and AXL-TAZ (rho=0.58; P>0.001). Median overall survival (OS) was 12.5 months (m). Among the 58 p receiving second-line docetaxel, in p with high levels of BRCA1, OS was 24.9 m, while it was 19.1 m in p with intermediate levels and 9.5 m in p with low levels (P=0.009). OS was 28.6 in p with high levels of MMSET, 13.8 in p with intermediate levels and 12.3 in p with low levels (P=0.001). In the multivariate analysis, only BRCA1 was a significant marker for OS (Table). Conclusions: The study showed the predictive value of BRCA1, which could be useful for customizing chemotherapy with or without docetaxel. In addition, MMSET requires further study since it is involved in DNA repair upstream of BRCA1. [Table: see text]

Yap1 Protein Regulates Vascular Smooth Muscle Cell Phenotypic Switch by Interaction with Myocardin

The Hippo-Yap (Yes-associated protein) signaling pathway has emerged as one of the critical pathways regulating cell proliferation, differentiation, and apoptosis in response to environmental and developmental cues. However, Yap1 roles in vascular smooth muscle cell (VSMC) biology have not been investigated. VSMCs undergo phenotypic switch, a process characterized by decreased gene expression of VSMC contractile markers and increased proliferation, migration, and matrix synthesis. The goals of the present studies were to investigate the relationship between Yap1 and VSMC phenotypic switch and to determine the molecular mechanisms by which Yap1 affects this essential process in VSMC biology. Results demonstrated that the expression of Yap1 was rapidly up-regulated by stimulation with PDGF-BB (a known inducer of phenotypic switch in VSMCs) and in the injured vessel wall. Knockdown of Yap1 impaired VSMC proliferation in vitro and enhanced the expression of VSMC contractile genes as well by increasing serum response factor binding to CArG-containing regions of VSMC-specific contractile genes within intact chromatin. Conversely, the interaction between serum response factor and its co-activator myocardin was reduced by overexpression of Yap1 in a dose-dependent manner. Taken together, these results indicate that down-regulation of Yap1 promotes VSMC contractile phenotype by both up-regulating myocardin expression and promoting the association of the serum response factor-myocardin complex with VSMC contractile gene promoters and suggest that the Yap1 signaling pathway is a central regulator of phenotypic switch of VSMCs.