Control of Proliferation and Cancer Growth by the Hippo Signaling Pathway

The control of cell division is essential for normal development and the maintenance of cellular homeostasis. Abnormal cell proliferation is associated with multiple pathological states, including cancer. Although the Hippo/YAP signaling pathway was initially thought to control organ size and growth, increasing evidence indicates that this pathway also plays a major role in the control of proliferation independent of organ size control. In particular, accumulating evidence indicates that the Hippo/YAP signaling pathway functionally interacts with multiple other cellular pathways and serves as a central node in the regulation of cell division, especially in cancer cells. Here, recent observations are highlighted that connect Hippo/YAP signaling to transcription, the basic cell-cycle machinery, and the control of cell division. Furthermore, the oncogenic and tumor-suppressive attributes of YAP/TAZ are reviewed, which emphasizes the relevance of the Hippo pathway in cancer.  127–40.

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title: Elucidation of a Universal Size-Control Mechanism in Drosophila and Mammals
abstract: Coordination 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.

title: Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.
abstract: The Hippo pathway plays a key role in organ size control by regulating cell proliferation and apoptosis in Drosophila. Although recent genetic studies have shown that the Hippo pathway is regulated by the NF2 and Fat tumor suppressors, the physiological regulations of this pathway are unknown. Here we show that in mammalian cells, the transcription coactivator YAP (Yes-associated protein), is inhibited by cell density via the Hippo pathway. Phosphorylation by the Lats tumor suppressor kinase leads to cytoplasmic translocation and inactivation of the YAP oncoprotein. Furthermore, attenuation of this phosphorylation of YAP or Yorkie (Yki), the Drosophila homolog of YAP, potentiates their growth-promoting function in vivo. Moreover, YAP overexpression regulates gene expression in a manner opposite to cell density, and is able to overcome cell contact inhibition. Inhibition of YAP function restores contact inhibition in a human cancer cell line bearing deletion of Salvador (Sav), a Hippo pathway component. Interestingly, we observed that YAP protein is elevated and nuclear localized in some human liver and prostate cancers. Our observations demonstrate that YAP plays a key role in the Hippo pathway to control cell proliferation in response to cell contact.

title: The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation.
abstract: Yes-associated protein (YAP) is a potent transcription coactivator acting via binding to the TEAD transcription factor, and plays a critical role in organ size regulation. YAP is phosphorylated and inhibited by the Lats kinase, a key component of the Hippo tumor suppressor pathway. Elevated YAP protein levels and gene amplification have been implicated in human cancer. In this study, we report that YAP is inactivated during embryonic stem (ES) cell differentiation, as indicated by decreased protein levels and increased phosphorylation. Consistently, YAP is elevated during induced pluripotent stem (iPS) cell reprogramming. YAP knockdown leads to a loss of ES cell pluripotency, while ectopic expression of YAP prevents ES cell differentiation in vitro and maintains stem cell phenotypes even under differentiation conditions. Moreover, YAP binds directly to promoters of a large number of genes known to be important for stem cells and stimulates their expression. Our observations establish a critical role of YAP in maintaining stem cell pluripotency.

title: Metabolic control of YAP and TAZ by the mevalonate pathway
abstract: The YAP and TAZ mediators of the Hippo pathway (hereafter called YAP/TAZ) promote tissue proliferation and organ growth. However, how their biological properties intersect with cellular metabolism remains unexplained. Here, we show that YAP/TAZ activity is controlled by the SREBP/mevalonate pathway. Inhibition of the rate-limiting enzyme of this pathway (HMG-CoA reductase) by statins opposes YAP/TAZ nuclear localization and transcriptional responses. Mechanistically, the geranylgeranyl pyrophosphate produced by the mevalonate cascade is required for activation of Rho GTPases that, in turn, activate YAP/TAZ by inhibiting their phosphorylation and promoting their nuclear accumulation. The mevalonate–YAP/TAZ axis is required for proliferation and self-renewal of breast cancer cells. In Drosophila melanogaster, inhibition of mevalonate biosynthesis and geranylgeranylation blunts the eye overgrowth induced by Yorkie, the YAP/TAZ orthologue. In tumour cells, YAP/TAZ activation is promoted by increased levels of mevalonic acid produced by SREBP transcriptional activity, which is induced by its oncogenic cofactor mutant p53. These findings reveal an additional layer of YAP/TAZ regulation by metabolic cues.

title: The Hippo Signaling Pathway Coordinately Regulates Cell Proliferation and Apoptosis by Inactivating Yorkie, the Drosophila Homolog of YAP
abstract: Coordination between cell proliferation and cell death is essential to maintain homeostasis in multicellular organisms. In Drosophila, these two processes are regulated by a pathway involving the Ste20-like kinase Hippo (Hpo) and the NDR family kinase Warts (Wts; also called Lats). Hpo phosphorylates and activates Wts, which in turn, through unknown mechanisms, negatively regulates the transcription of cell-cycle and cell-death regulators such as cycE and diap1. Here we identify Yorkie (Yki), the Drosophila ortholog of the mammalian transcriptional coactivator yes-associated protein (YAP), as a missing link between Wts and transcriptional regulation. Yki is required for normal tissue growth and diap1 transcription and is phosphorylated and inactivated by Wts. Overexpression of yki phenocopies loss-of-function mutations of hpo or wts, including elevated transcription of cycE and diap1, increased proliferation, defective apoptosis, and tissue overgrowth. Thus, Yki is a critical target of the Wts/Lats protein kinase and a potential oncogene.

title: Herding Hippos: regulating growth in flies and man.
abstract: Control of cell number requires the coordinate regulation of cell proliferation and cell death. Studies in both the fly and mouse have identified the Hippo kinase pathway as a key signaling pathway that controls cell proliferation and apoptosis. Several studies have implicated the Hippo pathway in a variety of cancers. Recent studies have also revealed a role for the Hippo pathway in the control of cell fate decisions during development. In this review, we will cover the current model of Hippo signaling in development. We will explore the differences between the Hippo pathway in invertebrates and mammals, and focus on recent advances in understanding how this conserved pathway is regulated.

title: TAZ: a novel transcriptional co‐activator regulated by interactions with 14‐3‐3 and PDZ domain proteins
abstract: The highly conserved and ubiquitously expressed 14‐3‐3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14‐3‐3, we identified a novel transcriptional co‐activator, TAZ (transcriptional co‐activator with PDZ‐binding motif) as a 14‐3‐3‐binding molecule. TAZ shares homology with Yes‐associated protein (YAP), contains a WW domain and functions as a transcriptional co‐activator by binding to the PPXY motif present on transcription factors. 14‐3‐3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co‐activation through 14‐3‐3‐mediated nuclear export. The C‐terminus of TAZ contains a highly conserved PDZ‐binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ‐stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain‐containing protein NHERF‐2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14‐3‐3.

title: YAP/TAZ Incorporation in the β-Catenin Destruction Complex Orchestrates the Wnt Response
abstract: The Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. Here, we provide biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the β-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for β-TrCP recruitment to the complex and β-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/β-catenin signaling. In line, the β-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression.

title: The Crumbs complex couples cell density sensing to Hippo-dependent control of the TGF-β-SMAD pathway.
abstract: The Hippo pathway senses cell density information to control tissue growth by regulating the localization of the transcriptional regulators TAZ and YAP (TAZ/YAP). TAZ/YAP also regulate TGF-β-SMAD signaling, but whether this role is linked to cell density sensing is unknown. Here we demonstrate that TAZ/YAP dictate the localization of active SMAD complexes in response to cell density-mediated formation of polarity complexes. In high-density cell cultures, the Hippo pathway drives cytoplasmic localization of TAZ/YAP, which sequesters SMAD complexes, thereby suppressing TGF-β signaling. We show that during mouse embryogenesis, this is reflected by differences in TAZ/YAP localization, which define regions of active SMAD2/3 complexes. Interfering with TAZ/YAP phosphorylation drives nuclear accumulation of TAZ/YAP and SMAD2/3. Furthermore, we demonstrate that the Crumbs polarity complex interacts with TAZ/YAP, which relays cell density information by promoting TAZ/YAP phosphorylation, cytoplasmic retention, and suppressed TGF-β signaling. Accordingly, disruption of the Crumbs complex enhances TGF-β signaling and predisposes cells to TGF-β-mediated epithelial-to-mesenchymal transitions.

title: TEAD mediates YAP-dependent gene induction and growth control.
abstract: The YAP transcription coactivator has been implicated as an oncogene and is amplified in human cancers. Recent studies have established that YAP is phosphorylated and inhibited by the Hippo tumor suppressor pathway. Here we demonstrate that the TEAD family transcription factors are essential in mediating YAP-dependent gene expression. TEAD is also required for YAP-induced cell growth, oncogenic transformation, and epithelial-mesenchymal transition. CTGF is identified as a direct YAP target gene important for cell growth. Moreover, the functional relationship between YAP and TEAD is conserved in Drosophila Yki (the YAP homolog) and Scalloped (the TEAD homolog). Our study reveals TEAD as a new component in the Hippo pathway playing essential roles in mediating biological functions of YAP.

title: The Hippo pathway: regulators and regulations.
abstract: Control of cell number is crucial in animal development and tissue homeostasis, and its dysregulation may result in tumor formation or organ degeneration. The Hippo pathway in both Drosophila and mammals regulates cell number by modulating cell proliferation, cell death, and cell differentiation. Recently, numerous upstream components involved in the Hippo pathway have been identified, such as cell polarity, mechanotransduction, and G-protein-coupled receptor (GPCR) signaling. Actin cytoskeleton or cellular tension appears to be the master mediator that integrates and transmits upstream signals to the core Hippo signaling cascade. Here, we review regulatory mechanisms of the Hippo pathway and discuss potential implications involved in different physiological and pathological conditions.

title: Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene.
abstract: Hippo-Lats-Yorkie signaling regulates tissue overgrowth and tumorigenesis in Drosophila. We show that the Mst1 and Mst2 protein kinases, the mammalian Hippo orthologs, are cleaved and constitutively activated in the mouse liver. Combined Mst1/2 deficiency in the liver results in loss of inhibitory Ser127 phosphorylation of the Yorkie ortholog, Yap1, massive overgrowth, and hepatocellular carcinoma (HCC). Reexpression of Mst1 in HCC-derived cell lines promotes Yap1 Ser127 phosphorylation and inactivation and abrogates their tumorigenicity. Notably, Mst1/2 inactivates Yap1 in liver through an intermediary kinase distinct from Lats1/2. Approximately 30% of human HCCs show low Yap1(Ser127) phosphorylation and a majority exhibit loss of cleaved, activated Mst1. Mst1/2 inhibition of Yap1 is an important pathway for tumor suppression in liver relevant to human HCC.

title: Hippo-independent activation of YAP by the GNAQ uveal melanoma oncogene through a trio-regulated rho GTPase signaling circuitry.
abstract: Mutually exclusive activating mutations in the GNAQ and GNA11 oncogenes, encoding heterotrimeric Gαq family members, have been identified in ∼ 83% and ∼ 6% of uveal and skin melanomas, respectively. However, the molecular events underlying these GNAQ-driven malignancies are not yet defined, thus limiting the ability to develop cancer-targeted therapies. Here, we focused on the transcriptional coactivator YAP, a critical component of the Hippo signaling pathway that controls organ size. We found that Gαq stimulates YAP through a Trio-Rho/Rac signaling circuitry promoting actin polymerization, independently of phospholipase Cβ and the canonical Hippo pathway. Furthermore, we show that Gαq promotes the YAP-dependent growth of uveal melanoma cells, thereby identifying YAP as a suitable therapeutic target in uveal melanoma, a GNAQ/GNA11-initiated human malignancy.

title: The Hippo Transducer TAZ Confers Cancer Stem Cell-Related Traits on Breast Cancer Cells
abstract: Cancer stem cells (CSCs) are proposed to drive tumor initiation and progression. Yet, our understanding of the cellular and molecular mechanisms that underlie CSC properties is limited. Here we show that the activity of TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in breast CSCs. TAZ protein levels and activity are elevated in prospective CSCs and in poorly differentiated human tumors and have prognostic value. Gain of TAZ endows self-renewal capacity to non-CSCs. In epithelial cells, TAZ forms a complex with the cell-polarity determinant Scribble, and loss of Scribble--or induction of the epithelial-mesenchymal transition (EMT)--disrupts the inhibitory association of TAZ with the core Hippo kinases MST and LATS. This study links the CSC concept to the Hippo pathway in breast cancer and reveals a mechanistic basis of the control of Hippo kinases by cell polarity.

title: Pro-Invasive Activity of the Hippo Pathway Effectors YAP and TAZ in Cutaneous Melanoma
abstract: YAP and its paralog protein TAZ are downstream effectors of the Hippo pathway. Both are amplified in many human cancers and promote cell proliferation and epithelial–mesenchymal transition. Little is known about the status of the Hippo pathway in cutaneous melanoma. We profiled Hippo pathway component expression in a panel of human melanoma cell lines and melanocytic lesions, and characterized the capacity of YAP and TAZ to control melanoma cell behavior. YAP and TAZ immuno-staining in human samples revealed mixed cytoplasmic and nuclear staining for both proteins in benign nevi and superficial spreading melanoma. TAZ was expressed at higher levels than YAP1/2 in all cell lines and in those with high invasive potential. Stable YAP or TAZ knockdown dramatically reduced the expression of the classical Hippo target CCN2/connective-tissue growth factor (CTGF), as well as anchorage-independent growth, capacity to invade Matrigel, and ability form lung metastases in mice following tail-vein injection. YAP knockdown also reduced invasion in a model of skin reconstruct. Inversely, YAP overexpression increased melanoma cell invasiveness, associated with increased TEA domain–dependent transcription and CCN2/CTGF expression. Together, these results demonstrate that both YAP and TAZ contribute to the invasive and metastatic capacity of melanoma cells and may represent worthy targets for therapeutic intervention.

title: The transcriptional coactivator TAZ regulates mesenchymal differentiation in malignant glioma.
abstract: Recent molecular classification of glioblastoma (GBM) has shown that patients with a mesenchymal (MES) gene expression signature exhibit poor overall survival and treatment resistance. Using regulatory network analysis of available expression microarray data sets of GBM, including The Cancer Genome Atlas (TCGA), we identified the transcriptional coactivator with PDZ-binding motif (TAZ), to be highly associated with the MES network. TAZ expression was lower in proneural (PN) GBMs and lower-grade gliomas, which correlated with CpG island hypermethylation of the TAZ promoter compared with MES GBMs. Silencing of TAZ in MES glioma stem cells (GSCs) decreased expression of MES markers, invasion, self-renewal, and tumor formation. Conversely, overexpression of TAZ in PN GSCs as well as murine neural stem cells (NSCs) induced MES marker expression and aberrant osteoblastic and chondrocytic differentiation in a TEAD-dependent fashion. Using chromatin immunoprecipitation (ChIP), we show that TAZ is directly recruited to a majority of MES gene promoters in a complex with TEAD2. The coexpression of TAZ, but not a mutated form of TAZ that lacks TEAD binding, with platelet-derived growth factor-B (PDGF-B) resulted in high-grade tumors with MES features in a murine model of glioma. Our studies uncover a direct role for TAZ and TEAD in driving the MES differentiation of malignant glioma.

title: hippo Encodes a Ste-20 Family Protein Kinase that Restricts Cell Proliferation and Promotes Apoptosis in Conjunction with salvador and warts
abstract: The coordination between cell proliferation and cell death is essential to maintain homeostasis within multicellular organisms. The mechanisms underlying this regulation are yet to be completely understood. Here, we report the identification of hippo (hpo) as a gene that regulates both cell proliferation and cell death in Drosophila. hpo encodes a Ste-20 family protein kinase that binds to and phosphorylates the tumor suppressor protein Salvador (Sav), which is known to interact with the Warts (Wts) protein kinase. Loss of hpo results in elevated transcription of the cell cycle regulator cyclin E and the cell-death inhibitor diap1, leading to increased proliferation and reduced apoptosis. Further, we show that hpo, sav, and wts define a pathway that regulates diap1 at the transcriptional level. A human homolog of hpo completely rescues the overgrowth phenotype of Drosophila hpo mutants, suggesting that hpo might play a conserved role for growth control in mammals.

title: The Drosophila Mst Ortholog, hippo, Restricts Growth and Cell Proliferation and Promotes Apoptosis
abstract: Establishing and maintaining homeostasis is critical to the well-being of an organism and is determined by the balance of cell proliferation and death. Two genes that function together to regulate growth, proliferation, and apoptosis in Drosophila are warts (wts), encoding a serine/threonine kinase, and salvador (sav), encoding a WW domain containing Wts-interacting protein. However, the mechanisms by which sav and wts regulate growth and apoptosis are not well understood. Here, we describe mutations in hippo (hpo), which encodes a protein kinase most related to mammalian Mst1 and Mst2. Like wts and sav, hpo mutations result in increased tissue growth and impaired apoptosis characterized by elevated levels of the cell cycle regulator cyclin E and apoptosis inhibitor DIAP1. Hpo, Sav, and Wts interact physically and functionally, and regulate DIAP1 levels, likely by Hpo-mediated phosphorylation and subsequent degradation. Thus, Hpo links Sav and Wts to a key regulator of apoptosis.

title: The Hippo pathway and human cancer
abstract: The Hippo pathway controls organ size in diverse species, whereas pathway deregulation can induce tumours in model organisms and occurs in a broad range of human carcinomas, including lung, colorectal, ovarian and liver cancer. Despite this, somatic or germline mutations in Hippo pathway genes are uncommon, with only the upstream pathway gene neurofibromin 2 (NF2) recognized as a bona fide tumour suppressor gene. In this Review, we appraise the evidence for the Hippo pathway as a cancer signalling network, and discuss cancer-relevant biological functions, potential mechanisms by which Hippo pathway activity is altered in cancer and emerging therapeutic strategies.

title: Yap1 Activation Enables Bypass of Oncogenic Kras Addiction in Pancreatic Cancer
abstract: Activating mutations in KRAS are among the most frequent events in diverse human carcinomas and are particularly prominent in human pancreatic ductal adenocarcinoma (PDAC). An inducible Kras(G12D)-driven mouse model of PDAC has established a critical role for sustained Kras(G12D) expression in tumor maintenance, providing a model to determine the potential for and the underlying mechanisms of Kras(G12D)-independent PDAC recurrence. Here, we show that some tumors undergo spontaneous relapse and are devoid of Kras(G12D) expression and downstream canonical MAPK signaling and instead acquire amplification and overexpression of the transcriptional coactivator Yap1. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving Kras(G12D)-independent tumor maintenance. The Yap1/Tead2 complex acts cooperatively with E2F transcription factors to activate a cell cycle and DNA replication program. Our studies, along with corroborating evidence from human PDAC models, portend a novel mechanism of escape from oncogenic Kras addiction in PDAC.

title: Comprehensive genomic characterization of head and neck squamous cell carcinomas
abstract: The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus-associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss-of-function TP53 mutations and CDKN2A inactivation with frequent copy number alterations including amplification of 3q26/28 and 11q13/22. A subgroup of oral cavity tumours with favourable clinical outcomes displayed infrequent copy number alterations in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and TP53. Other distinct subgroups contained loss-of-function alterations of the chromatin modifier NSD1, WNT pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumours. Therapeutic candidate alterations were identified in most HNSCCs.

title: Frequent hypermethylation of MST1 and MST2 in soft tissue sarcoma
abstract: The RASSF1A tumor suppressor is involved in regulation of apoptosis and cell cycle progression. RASSF1A is localized to microtubules and binds the apoptotic kinases MST1 and MST2. It has been shown that this interaction is mediated by the Sav‐RASSF‐Hpo domain, which is an interaction domain characterized for the Drosophila proteins Sav (human WW45), Hpo (human MST1 and MST2) and Warts/LATS (large tumor suppressor). Previously, we have reported that RASSF1A hypermethylation occurs frequently in soft tissue sarcoma and is associated with an unfavorable prognosis for cancer patients. In our study, we performed methylation analysis of the CpG island promoter of MST1, MST2, WW45, LATS1 and LATS2 in soft tissue sarcomas by methylation‐specific PCR. No or a very low methylation frequency was detected for WW45, LATS1 and LATS2 (<7%). In 19 out of 52 (37%) sarcomas, a methylated promoter of MST1 was detected and 12 out of 60 (20%) samples showed methylation of the MST2 promoter. Methylation status of MST1 was confirmed by bisulfite sequencing. In tumors harboring a methylated promoter of MST1, a reduction of MST1 expression was observed by RT‐PCR. In leiomyosarcomas, MST1 and MST2 or RASSF1A methylation were mutually exclusive (P = 0.007 and P = 0.025, respectively). Surprisingly, a significantly increased risk for tumor‐related death was found for patients with an unmethylated MST1 promoter (P = 0.036). In summary, our results suggest that alteration of the Sav‐RASSF1‐Hpo tumor suppressor pathway may occur through hypermethylation of the CpG island promoter of MST1, MST2 and/or RASSF1A in human sarcomas.

title: Hippo Pathway Activity Influences Liver Cell Fate
abstract: The Hippo-signaling pathway is an important regulator of cellular proliferation and organ size. However, little is known about the role of this cascade in the control of cell fate. Employing a combination of lineage tracing, clonal analysis, and organoid culture approaches, we demonstrate that Hippo pathway activity is essential for the maintenance of the differentiated hepatocyte state. Remarkably, acute inactivation of Hippo pathway signaling in vivo is sufficient to dedifferentiate, at very high efficiencies, adult hepatocytes into cells bearing progenitor characteristics. These hepatocyte-derived progenitor cells demonstrate self-renewal and engraftment capacity at the single-cell level. We also identify the NOTCH-signaling pathway as a functional important effector downstream of the Hippo transducer YAP. Our findings uncover a potent role for Hippo/YAP signaling in controlling liver cell fate and reveal an unprecedented level of phenotypic plasticity in mature hepatocytes, which has implications for the understanding and manipulation of liver regeneration.

title: The Hippo tumor suppressor pathway regulates intestinal stem cell regeneration
abstract: Identification of the signaling pathways that control the proliferation of stem cells (SCs), and whether they act in a cell or non-cell autonomous manner, is key to our understanding of tissue homeostasis and cancer. In the adult Drosophila midgut, the Jun N-Terminal Kinase (JNK) pathway is activated in damaged enterocyte cells (ECs) following injury. This leads to the production of Upd cytokines from ECs, which in turn activate the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway in Intestinal SCs (ISCs), stimulating their proliferation. In addition, the Hippo pathway has been recently implicated in the regulation of Upd production from the ECs. Here, we show that the Hippo pathway target, Yorkie (Yki), also plays a crucial and cell-autonomous role in ISCs. Activation of Yki in ISCs is sufficient to increase ISC proliferation, a process involving Yki target genes that promote division, survival and the Upd cytokines. We further show that prior to injury, Yki activity is constitutively repressed by the upstream Hippo pathway members Fat and Dachsous (Ds). These findings demonstrate a cell-autonomous role for the Hippo pathway in SCs, and have implications for understanding the role of this pathway in tumorigenesis and cancer stem cells.

title: The Hippo signaling pathway restricts the oncogenic potential of an intestinal regeneration program.
abstract: Although a developmental role for Hippo signaling in organ size control is well appreciated, how this pathway functions in tissue regeneration is largely unknown. Here we address this issue using a dextran sodium sulfate (DSS)-induced colonic regeneration model. We find that regenerating crypts express elevated Yes-associated protein (YAP) levels. Inactivation of YAP causes no obvious intestinal defects under normal homeostasis, but severely impairs DSS-induced intestinal regeneration. Conversely, hyperactivation of YAP results in widespread early-onset polyp formation following DSS treatment. Thus, the YAP oncoprotein must be exquisitely controlled in tissue regeneration to allow compensatory proliferation and prevent the intrinsic oncogenic potential of a tissue regeneration program.

title: Hippo signaling regulates differentiation and maintenance in the exocrine pancreas.
abstract: The Hippo signaling pathway is a context-dependent regulator of cell proliferation, differentiation, and apoptosis in species ranging from Drosophila to humans. In this study, we investigated the role of the core Hippo kinases-Mst1 and Mst2-in pancreatic development and homeostasis.



We used a Cre/LoxP system to create mice with pancreas-specific disruptions in Mst1 and Mst2 (Pdx1-Cre;Mst1(-/-);Mst2(fl/fl) mice), the mammalian orthologs of Drosophila Hippo. We used a transgenic approach to overexpress Yap, the downstream mediator of Hippo signaling, in the developing pancreas of mice.



Contrary to expectations, the pancreatic mass of Pdx1-Cre;Mst1(-/-);Mst2(fl/fl) mice was reduced compared with wild-type mice, largely because of postnatal de-differentiation of acinar cells into duct-like cells. Development of this phenotype coincided with postnatal reactivation of YAP expression. Ectopic expression of YAP during the secondary transition (a stage at which YAP is normally absent) blocked differentiation of the endocrine and exocrine compartments, whereas loss of a single Yap allele reduced acinar de-differentiation. The phenotype of Pdx1-Cre;Mst1(-/-);Mst2(fl/fl) mice recapitulated cellular and molecular changes observed during chemical-induced pancreatitis in mice.



The mammalian Hippo kinases, and YAP, maintain postnatal pancreatic acinar differentiation in mice.

title: The hippo pathway effector Yap controls patterning and differentiation of airway epithelial progenitors.
abstract: How epithelial progenitor cells integrate local signals to balance expansion with differentiation during organogenesis is still little understood. Here, we provide evidence that the Hippo pathway effector Yap is a key regulator of this process in the developing lung. We show that when epithelial tubules are forming and branching, a nucleocytoplasmic shift in Yap localization marks the boundary between the airway and the distal lung compartments. At this transition zone, Yap specifies a transcriptional program that controls Sox2 expression and ultimately generates the airway epithelium. Without Yap, epithelial progenitors are unable to properly respond to local TGF-β-induced cues and control levels and distribution of Sox2 to form airways. Yap levels and subcellular localization also markedly influence Sox2 expression and differentiation of adult airway progenitors. Our data reveal a role for the Hippo-Yap pathway in integrating growth-factor-induced cues in the developing and adult lung potentially key for homeostasis and regeneration repair.

title: YAP and TAZ regulate skin wound healing.
abstract: The Hippo signaling pathway regulates organ size, tissue regeneration, and stem cell self-renewal. The two key downstream transcription coactivators in this pathway, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), mediate the major gene regulation and biological functions of the Hippo pathway. The biological functions of YAP and TAZ in many tissues are known; however, their roles in skin wound healing remain unclear. To analyze whether YAP and/or TAZ are required for cutaneous wound healing, we performed small interfering RNA (siRNA)-mediated knockdown of YAP/TAZ in full-thickness skin wounds. YAP is strongly expressed in the nucleus and cytoplasm in the epidermis and hair follicle. Interestingly, YAP is expressed in the nucleus in the dermis at 2 and 7 days after wounding. TAZ normally localizes to the cytoplasm in the dermis but is distributed in both the nucleus and cytoplasm at 1 day after wounding. The knockdown of YAP and TAZ markedly delayed the rate of wound closure and reduced the transforming growth factor-β1 (TGF-β1) expression in the wound. YAP and TAZ also modulate the expression of TGF-β1 signaling pathway components such as Smad-2, p21, and Smad-7. These results suggest that YAP and TAZ localization to the nucleus is required for skin wound healing.

title: Defects in Yolk Sac Vasculogenesis, Chorioallantoic Fusion, and Embryonic Axis Elongation in Mice with Targeted Disruption of Yap65
abstract: YAP is a multifunctional adapter protein and transcriptional coactivator with several binding partners well described in vitro and in cell culture. To explore in vivo requirements for YAP, we generated mice carrying a targeted disruption of the Yap gene. Homozygosity for the Yap tm1Smil allele (Yap −/− ) caused developmental arrest around E8.5. Phenotypic characterization revealed a requirement for YAP in yolk sac vasculogenesis. Yolk sac endothelial and erythrocyte precursors were specified as shown by histology, PECAM1 immunostaining, and alpha globin expression. Nonetheless, development of an organized yolk sac vascular plexus failed in Yap −/− embryos. In striking contrast, vasculogenesis proceeded in both the allantois and the embryo proper. Mutant embryos showed patterned gene expression domains along the anteroposterior neuraxis, midline, and streak/tailbud. Despite this evidence of proper patterning and tissue specification, Yap −/− embryos showed developmental perturbations that included a notably shortened body axis, convoluted anterior neuroepithelium, caudal dysgenesis, and failure of chorioallantoic fusion. These results reveal a vital requirement for YAP in the developmental processes of yolk sac vasculogenesis, chorioallantoic attachment, and embryonic axis elongation.

title: Hippo signaling is a potent in vivo growth and tumor suppressor pathway in the mammalian liver
abstract: How organ size is controlled in mammals is not currently understood. In Drosophila the Hippo signaling pathway functions to suppress growth in imaginal discs and has been suggested to control organ size. To investigate the role of hippo signaling in regulation of mammalian organ size we have generated conditional alleles of Sav1, mst1, and mst2, orthologs of Drosophila Salvador and hippo, respectively. Specific deletion of both mst1 and mst2 in hepatocytes results in significantly enlarged livers due to excessive proliferation. By the age of 5–6 months, mst1/2 conditional mutant livers have multiple foci of liver tumors, indicating that the combined activities of mst1 and mst2 act as redundant tumor suppressors in hepatocytes. Similar findings were obtained with liver-specific deletion of Sav1, a second core Hippo signaling component that facilitates activation of mst1 and mst2. Tumors from sav1 mutants exhibited varied morphology, suggesting a mixed-lineage origin of tumor-initiating cells. Transcriptional profiling of liver tissues from both mst1/2 and sav1 conditional mutants revealed a network of Hippo signaling regulated genes with specific enrichment for genes involved in immune and inflammatory responses. Histological and immunological characterization of mst1/2 double mutant liver tissues revealed abundant accumulation of adult facultative stem cells termed oval cells in periductal regions. Because oval cells induction is commonly associated with liver injury and tumor formation, it is likely that these cells contribute to the enlarged livers and hepatomas that we observe in sav1 and mst1/2 mutants. Taken together, our results demonstrate that the Hippo signaling pathway is a critical regulator of mammalian liver growth and a potent suppressor of liver tumor formation.

title: YAP1 Increases Organ Size and Expands Undifferentiated Progenitor Cells
abstract: The mechanisms that regulate mammalian organ size are poorly understood. It is unclear whether the pathways that control organ size also impinge on stem/progenitor cells. A highly expressed gene in stem cells is YAP1, the ortholog of Drosophila Yorkie, a downstream component of the Hippo pathway. Mutations in components of this pathway produce tissue overgrowth phenotypes in the fly whereas mammalian orthologs, like salvador, merlin, LATS, and YAP1, have been implicated in tumorigenesis. We report here that YAP1 increases organ size and causes aberrant tissue expansion in mice. YAP1 activation reversibly increases liver size more than 4-fold. In the intestine, expression of endogenous YAP1 is restricted to the progenitor/stem cell compartment, and activation of YAP1 expands multipotent undifferentiated progenitor cells, which differentiate upon cessation of YAP1 expression. YAP1 stimulates Notch signaling, and administration of gamma-secretase inhibitors suppressed the intestinal dysplasia caused by YAP1. Human colorectal cancers expressing higher levels of YAP1 share molecular aspects with YAP1-induced dysplastic growth in the mouse. Our data show that the Hippo signaling pathway regulates organ size in mammals and can act on stem cell compartments, indicating a potential link between stem/progenitor cells, organ size, and cancer.

title: Dual role of YAP and TAZ in renewal of the intestinal epithelium
abstract: The rapidly self-renewing intestinal epithelium represents an exquisite model for stem cell biology. So far, genetic studies in mice have uncovered crucial roles for several signalling pathways in the tissue. Here we show, by using intestine-specific gene transfer (iGT), that Hippo signalling effectors, YAP and TAZ, promote both the proliferation of intestinal stem/progenitor cells and their differentiation into goblet cells. These functions of YAP/TAZ are regulated by the upstream Hippo pathway kinases MST1/2 and LATS1/2. Moreover, we identify TEADs and Klf4 as partner transcription factors of YAP/TAZ in the proliferation and differentiation processes, respectively. These results indicate that Hippo signalling plays a dual role in renewal of the intestinal epithelium through the regulation of two different processes, stem/progenitor cell proliferation and differentiation into goblet cells, using two different types of transcription factor. Moreover, iGT should provide a robust platform to elucidate molecular mechanisms of intestinal epithelium self-renewal.

title: A crucial role of WW45 in developing epithelial tissues in the mouse
abstract: The role and molecular mechanisms of a new Hippo signalling pathway are not fully understood in mammals. Here, we generated mice that lack WW45 and revealed a crucial role for WW45 in cell‐cycle exit and epithelial terminal differentiation. Many organs in the mutant mouse embryos displayed hyperplasia accompanied by defects in terminal differentiation of epithelial progenitor cells owing to impaired proliferation arrest rather than intrinsic acceleration of proliferation during differentiation. Importantly, the MST1 signalling pathway is specifically activated in differentiating epithelial cells. Moreover, WW45 is required for MST1 activation and translocation to the nucleus for subsequent LATS1/2 activation upon differentiation signal. LATS1/2 phosphorylates YAP, which, in turn, translocates from the nucleus into the cytoplasm, resulting in cell‐cycle exit and terminal differentiation of epithelial progenitor cells. Collectively, these data provide compelling evidence that WW45 is a key mediator of MST1 signalling in the coordinate coupling of proliferation arrest with terminal differentiation for proper epithelial tissue development in mammals.

title: SOX2 regulates YAP1 to maintain stemness and determine cell fate in the osteo-adipo lineage.
abstract: The osteoblastic and adipocytic lineages arise from mesenchymal stem cells (MSCs), but few regulators of self-renewal and early cell-fate decisions are known. Here, we show that the Hippo pathway effector YAP1 is a direct target of SOX2 and can compensate for the self-renewal defect caused by SOX2 inactivation in osteoprogenitors and MSCs. Osteogenesis is blocked by high SOX2 or YAP1, accelerated by depletion of either one, and the inhibition of osteogenesis by SOX2 requires YAP1. SOX2 favors adipogenesis and induces PPARγ, but adipogenesis can only occur with moderate levels of YAP1. YAP1 induction by SOX2 is restrained in adipogenesis, and both YAP1 overexpression and depletion inhibit the process. YAP1 binds β-catenin and directly induces the Wnt antagonist Dkk1 to dampen pro-osteogenic Wnt signals. We demonstrate a Hippo-independent regulation of YAP1 by SOX2 that cooperatively antagonizes Wnt/β-catenin signals and regulates PPARγ to determine osteogenic or adipocytic fates.

title: Yap tunes airway epithelial size and architecture by regulating the identity, maintenance, and self-renewal of stem cells.
abstract: Our understanding of how stem cells are regulated to maintain appropriate tissue size and architecture is incomplete. We show that Yap (Yes-associated protein 1) is required for the actual maintenance of an adult mammalian stem cell. Without Yap, adult airway basal stem cells are lost through their unrestrained differentiation, resulting in the simplification of a pseudostratified epithelium into a columnar one. Conversely, Yap overexpression increases stem cell self-renewal and blocks terminal differentiation, resulting in epithelial hyperplasia and stratification. Yap overexpression in differentiated secretory cells causes them to partially reprogram and adopt a stem cell-like identity. In contrast, Yap knockdown prevents the dedifferentiation of secretory cells into stem cells. We then show that Yap functionally interacts with p63, the cardinal transcription factor associated with myriad epithelial basal stem cells. In aggregate, we show that Yap regulates all of the cardinal behaviors of airway epithelial stem cells and determines epithelial architecture.

title: Restriction of intestinal stem cell expansion and the regenerative response by YAP
abstract: A remarkable feature of regenerative processes is their ability to halt proliferation once an organ’s structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)—a protein known for its powerful growth-inducing and oncogenic properties—has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN–APC–GSK-3β complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.

title: Hippo Pathway Inhibits Wnt Signaling to Restrain Cardiomyocyte Proliferation and Heart Size
abstract: Heart size is controlled through an antagonistic interaction between Hippo and Wnt signaling pathways. Genetic regulation of mammalian heart size is poorly understood. Hippo signaling represents an organ-size control pathway in Drosophila, where it also inhibits cell proliferation and promotes apoptosis in imaginal discs. To determine whether Hippo signaling controls mammalian heart size, we inactivated Hippo pathway components in the developing mouse heart. Hippo-deficient embryos had overgrown hearts with elevated cardiomyocyte proliferation. Gene expression profiling and chromatin immunoprecipitation revealed that Hippo signaling negatively regulates a subset of Wnt target genes. Genetic interaction studies indicated that β-catenin heterozygosity suppressed the Hippo cardiomyocyte overgrowth phenotype. Furthermore, the Hippo effector Yap interacts with β-catenin on Sox2 and Snai2 genes. These data uncover a nuclear interaction between Hippo and Wnt signaling that restricts cardiomyocyte proliferation and controls heart size.

title: The Hippo pathway regulates Wnt/beta-catenin signaling.
abstract: Several developmental pathways contribute to processes that regulate tissue growth and organ size. The Hippo pathway has emerged as one such critical regulator. However, how Hippo signaling is integrated with other pathways to coordinate these processes remains unclear. Here, we show that the Hippo pathway restricts Wnt/beta-Catenin signaling by promoting an interaction between TAZ and DVL in the cytoplasm. TAZ inhibits the CK1delta/epsilon-mediated phosphorylation of DVL, thereby inhibiting Wnt/beta-Catenin signaling. Abrogation of TAZ levels or Hippo signaling enhances Wnt3A-stimulated DVL phosphorylation, nuclear beta-Catenin, and Wnt target gene expression. Mice lacking Taz develop polycystic kidneys with enhanced cytoplasmic and nuclear beta-Catenin. Moreover, in Drosophila, Hippo signaling modulates Wg target gene expression. These results uncover a cytoplasmic function of TAZ in regulating Wnt signaling and highlight the role of the Hippo pathway in coordinating morphogenetic signaling with growth control.

title: TAZ-mediated crosstalk between Wnt and Hippo signaling.
abstract: Previously, research addressing Hippo signaling focused on the inactivation of the proto-oncoproteins TAZ and YAP caused by the sequestration of supposedly inactive phospho-TAZ/YAP in the cytoplasm. In this issue of Developmental Cell, the Attisano laboratory now shows that cytoplasmic TAZ inhibits canonical Wnt signaling, thereby highlighting that the Hippo pathway can control other signaling cascades.

title: Insulin/IGF signaling drives cell proliferation in part via Yorkie/YAP.
abstract: The insulin/IGF signaling (IIS) pathway is a potent inducer of cell proliferation in normal development and in cancer. The mechanism by which this occurs, however, is not completely understood. The Hippo signaling pathway regulates cell proliferation via the transcriptional co-activator Yorkie/YAP, however the signaling inputs regulating Hippo activity are not fully elucidated. Here we present evidence linking these two conserved, oncogenic pathways in Drosophila and in mammalian cells. We find that activation of IIS and of Yorkie signaling correlate positively in hepatocellular carcinoma. We show that IIS activates Yorkie in vivo, and that Yorkie plays an important role in the ability of IIS to drive cell proliferation. Interestingly, we also find the converse--that Yorkie signaling activates components of the insulin/TOR pathway. In sum, this crosstalk between IIS and Yorkie leads to coordinated regulation of these two oncogenic pathways.

title: YAP1 is amplified and up-regulated in hedgehog-associated medulloblastomas and mediates Sonic hedgehog-driven neural precursor proliferation.
abstract: Medulloblastoma is the most common solid malignancy of childhood, with treatment side effects reducing survivors' quality of life and lethality being associated with tumor recurrence. Activation of the Sonic hedgehog (Shh) signaling pathway is implicated in human medulloblastomas. Cerebellar granule neuron precursors (CGNPs) depend on signaling by the morphogen Shh for expansion during development, and have been suggested as a cell of origin for certain medulloblastomas. Mechanisms contributing to Shh pathway-mediated proliferation and transformation remain poorly understood. We investigated interactions between Shh signaling and the recently described tumor-suppressive Hippo pathway in the developing brain and medulloblastomas. We report up-regulation of the oncogenic transcriptional coactivator yes-associated protein 1 (YAP1), which is negatively regulated by the Hippo pathway, in human medulloblastomas with aberrant Shh signaling. Consistent with conserved mechanisms between brain tumorigenesis and development, Shh induces YAP1 expression in CGNPs. Shh also promotes YAP1 nuclear localization in CGNPs, and YAP1 can drive CGNP proliferation. Furthermore, YAP1 is found in cells of the perivascular niche, where proposed tumor-repopulating cells reside. Post-irradiation, YAP1 was found in newly growing tumor cells. These findings implicate YAP1 as a new Shh effector that may be targeted by medulloblastoma therapies aimed at eliminating medulloblastoma recurrence.

title: Yes-associated protein up-regulates Jagged-1 and activates the Notch pathway in human hepatocellular carcinoma.
abstract: Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP activity are unclear.



By using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as a downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAP(S127A)), and measured protein levels in HCC, colorectal and pancreatic tumor samples from patients.



Human HCC cell lines and mouse hepatocytes that overexpress YAP(S127A) up-regulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEA domain family member 4 (TEAD4); TEAD4 binding required the Mst1/2 but not β-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and correlated with shorter survival times of patients with HCC or colorectal cancer.



The transcriptional regulator YAP up-regulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer. Transcript profiling: microarray information was deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jxepvsumwosqkve&acc=GSE35004).

title: Regulation of the Hippo-YAP Pathway by G-Protein-Coupled Receptor Signaling
abstract: The Hippo pathway is crucial in organ size control, and its dysregulation contributes to tumorigenesis. However, upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here, we report that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2, thereby activating YAP and TAZ transcription coactivators, which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression, cell migration, and proliferation. In contrast, stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity, thereby inhibiting YAP function. Thus, GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR.

title: Identification of serum-derived sphingosine-1-phosphate as a small molecule regulator of YAP.
abstract: 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.

title: Mutant Gq/11 promote uveal melanoma tumorigenesis by activating YAP.
abstract: Uveal melanoma (UM) is the most common cancer in adult eyes. Approximately 80% of UMs harbor somatic activating mutations in GNAQ or GNA11 (encoding Gq or G11, respectively). Herein, we show in both cell culture and human tumors that cancer-associated Gq/11 mutants activate YAP, a major effector of the Hippo tumor suppressor pathway that is also regulated by G protein-coupled receptor signaling. YAP mediates the oncogenic activity of mutant Gq/11 in UM development, and the YAP inhibitor verteporfin blocks tumor growth of UM cells containing Gq/11 mutations. This study reveals an essential role of the Hippo-YAP pathway in Gq/11-induced tumorigenesis and suggests YAP as a potential drug target for UM patients carrying mutations in GNAQ or GNA11.

title: Regulation of the Hippo-YAP pathway by protease-activated receptors (PARs).
abstract: The Hippo signaling pathway plays a crucial role in tissue growth and tumorigenesis. Core components of the Hippo pathway include the MST1/2 and Lats1/2 kinases. Acting downstream from the Hippo pathway are the YAP/TAZ transcription coactivators, which are inhibited through phosphorylation by Lats. However, upstream signals that regulate the Hippo pathway have not been well delineated. Here we report that stimulation of protease-activated receptors (PARs) activates YAP/TAZ by decreasing phosphorylation and increasing nuclear localization. PAR1 acts through G(12/13) and Rho GTPase to inhibit the Lats1/2 kinase. Our observations establish thrombin as a physiological signal for the Hippo pathway and implicate Hippo-YAP as a key downstream signaling branch of PAR activation.

title: Regulation of Hippo pathway by mitogenic growth factors via phosphoinositide 3-kinase and phosphoinositide-dependent kinase-1
abstract: The Hippo signaling pathway inhibits cell growth and regulates organ size through a kinase cascade that leads to the phosphorylation and nuclear exclusion of the growth-promoting transcriptional coactivator Yes-associated protein (YAP)/Yorkie. It mediates contact inhibition of cell growth downstream of cadherin adhesion molecules and other cell surface proteins. Contact inhibition is often antagonized by mitogenic growth factor signaling. We report an important mechanism for this antagonism, inhibition of Hippo pathway signaling by mitogenic growth factors. EGF treatment of immortalized mammary cells triggers the rapid translocation of YAP into the nucleus along with YAP dephosphorylation, both of which depend on Lats, the terminal kinase in the Hippo pathway. A small-molecule inhibitor screen of downstream effector pathways shows that EGF receptor inhibits the Hippo pathway through activation of PI3-kinase (PI3K) and phosphoinositide-dependent kinase (PDK1), but independent of AKT activity. The PI3K-PDK1 pathway also mediates YAP nuclear translocation downstream of lysophosphatidic acid and serum as a result of constitutive oncogenic activation of PI3K. PDK1 associates with the core Hippo pathway-kinase complex through the scaffold protein Salvador. The entire Hippo core complex dissociates in response to EGF signaling in a PI3K-PDK1–dependent manner, leading to inactivation of Lats, dephosphorylation of YAP, and YAP nuclear accumulation and transcriptional activation of its target gene, CTGF. These findings show that an important activity of mitogenic signaling pathways is to inactivate the growth-inhibitory Hippo pathway and provide a mechanism for antagonism between contact inhibition and growth factor action.

title: Regulation of Hippo signaling by EGFR-MAPK signaling through Ajuba family proteins.
abstract: EGFR and Hippo signaling pathways both control growth and, when dysregulated, contribute to tumorigenesis. We find that EGFR activates the Hippo pathway transcription factor Yorkie and demonstrate that Yorkie is required for the influence of EGFR on cell proliferation in Drosophila. EGFR regulates Yorkie through the influence of its Ras-MAPK branch on the Ajuba LIM protein Jub. Jub is epistatic to EGFR and Ras for Yorkie regulation, Jub is subject to MAPK-dependent phosphorylation, and EGFR-Ras-MAPK signaling enhances Jub binding to the Yorkie kinase Warts and the adaptor protein Salvador. An EGFR-Hippo pathway link is conserved in mammals, as activation of EGFR or RAS activates the Yorkie homolog YAP, and EGFR-RAS-MAPK signaling promotes phosphorylation of the Ajuba family protein WTIP and also enhances WTIP binding to the Warts and Salvador homologs LATS and WW45. Our observations implicate the Hippo pathway in EGFR-mediated tumorigenesis and identify a molecular link between these pathways.

title: YAP-dependent induction of amphiregulin identifies a non-cell-autonomous component of the Hippo pathway
abstract: The Hippo signalling pathway regulates cellular proliferation and survival, thus has profound effects on normal cell fate and tumorigenesis. The pivotal effector of this pathway is YAP (yes-associated protein), a transcriptional co-activator amplified in mouse and human cancers, where it promotes epithelial to mesenchymal transition (EMT) and malignant transformation. So far, studies of YAP target genes have focused on cell-autonomous mediators; here we show that YAP-expressing MCF10A breast epithelial cells enhance the proliferation of neighbouring untransfected cells, implicating a non-cell-autonomous mechanism. We identify the gene for the epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) as a transcriptional target of YAP, whose induction contributes to YAP-mediated cell proliferation and migration, but not EMT. Knockdown of AREG or addition of an EGFR kinase inhibitor abrogates the proliferative effects of YAP expression. Suppression of the negative YAP regulators LATS1 and 2 (large tumour suppressor 1 and 2) is sufficient to induce AREG expression, consistent with physiological regulation of AREG by the Hippo pathway. Genetic interaction between the Drosophila YAP orthologue Yorkie and Egfr signalling components supports the link between these two highly conserved signalling pathways. Thus, YAP-dependent secretion of AREG indicates that activation of EGFR signalling is an important non-cell-autonomous effector of the Hippo pathway, which has implications for the regulation of both physiological and malignant cell proliferation.

title: Neuregulin 1–activated ERBB4 interacts with YAP to induce Hippo pathway target genes and promote cell migration
abstract: Two pathways regulating tissue growth and oncogenic signaling are functionally linked. ERBB4 Signals to YAP Whereas the Hippo pathway limits cell growth in response to mechanical signals or cell-cell contact, growth factors are soluble signals that stimulate cell proliferation. Kinases in the Hippo pathway phosphorylate and inhibit YAP, sequestering YAP in the cytosol and limiting organ size and tissue growth. Neuregulin stimulates the cleavage of the epidermal growth factor receptor family member ERBB4. Haskins et al. found that the cleaved intracellular domain (ICD) of ERBB4 activated YAP-mediated transcription by interacting with YAP. In breast cancer cells, knocking down YAP prevented cell migration induced by neuregulin. These results suggest that in addition to receptor tyrosine kinase signaling, ERBB4 can promote tumor aggressiveness by stimulating YAP. The receptor tyrosine kinase ERBB4, a member of the epidermal growth factor receptor (EGFR) family, is unusual in that ERBB4 can undergo intramembrane proteolysis, releasing a soluble intracellular domain (ICD) that modulates transcription in the nucleus. We found that ERBB4 activated the transcriptional coactivator YAP, which promotes organ and tissue growth and is inhibited by the Hippo tumor-suppressor pathway.  ERBB4 in cultured mammary epithelial cells or adding the ERBB4 ligand neuregulin 1 (NRG1) to breast cancer cell cultures promoted the expression of genes regulated by YAP, such as CTGF. Knocking down YAP or ERBB4 prevented the induction of CTGF expression by NRG1, as did treating cells with the ERBB inhibitors lapatinib or erlotinib, which reduced ERBB4 cleavage. NRG1 stimulated YAP activity to an extent comparable to that of EGF (epidermal growth factor) or LPA (lysophosphatidic acid), known activators of YAP. NRG1 stimulated YAP-dependent cell migration in breast cancer cell lines. These observations connect the unusual nuclear function of a growth factor receptor with a mechanosensory pathway and suggest that NRG1-ERBB4-YAP signaling contributes to the aggressive behavior of tumor cells.

title: Integration of mechanical and chemical signals by YAP and TAZ transcription coactivators
abstract: YAP and TAZ are transcription coactivators and effectors of the Hippo pathway, which play a key role in organ size control. Through interaction with transcription factors such as TEADs, they activate gene transcription and thus promote cell proliferation, inhibit apoptosis, and regulate cell differentiation. Dysregulation of YAP/TAZ was found to correlate with human cancers. The oncogenic roles of these proteins were also demonstrated in animal models. The growth promoting activity of YAP/TAZ is limited by the Hippo tumor suppressor pathway through phosphorylation-induced cytoplasmic retention and destabilization. Recently, it was found that YAP and TAZ mediate responses to several extracellular signals including mechanical stress, GPCR signaling, and the Wnt signaling pathway. All these growth-regulating signals play important roles in normal development and cancer. In this review, we would like to discuss the function of YAP and TAZ as effectors of these physiological signals.

title: YAP/TAZ as mechanosensors and mechanotransducers in regulating organ size and tumor growth
abstract: Organ size is controlled by the concerted action of biochemical and physical processes. Although mechanical forces are known to regulate cell and tissue behavior, as well as organogenesis, the precise molecular events that integrate mechanical and biochemical signals to control these processes are not fully known. The recently delineated Hippo‐tumor suppressor network and its two nuclear effectors, YAP and TAZ, shed light on these mechanisms. YAP and TAZ are proto‐oncogene proteins that respond to complex physical milieu represented by the rigidity of the extracellular matrix, cell geometry, cell density, cell polarity and the status of the actin cytoskeleton. Here, we review the current knowledge of how YAP and TAZ function as mechanosensors and mechanotransducers. We also suggest that by deciphering the mechanical and biochemical signals controlling YAP/TAZ function, we will gain insights into new strategies for cancer treatment and organ regeneration.

title: Role of YAP/TAZ in mechanotransduction
abstract: Cells perceive their microenvironment not only through soluble signals but also through physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behaviour, including growth, differentiation and cancer malignant progression, but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) as nuclear relays of mechanical signals exerted by ECM rigidity and cell shape. This regulation requires Rho GTPase activity and tension of the actomyosin cytoskeleton, but is independent of the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of activated YAP overrules physical constraints in dictating cell behaviour. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment.

title: A Mechanical Checkpoint Controls Multicellular Growth through YAP/TAZ Regulation by Actin-Processing Factors
abstract: Key cellular decisions, such as proliferation or growth arrest, typically occur at spatially defined locations within tissues. Loss of this spatial control is a hallmark of many diseases, including cancer. Yet, how these patterns are established is incompletely understood. Here, we report that physical and architectural features of a multicellular sheet inform cells about their proliferative capacity through mechanical regulation of YAP and TAZ, known mediators of Hippo signaling and organ growth. YAP/TAZ activity is confined to cells exposed to mechanical stresses, such as stretching, location at edges/curvatures contouring an epithelial sheet, or stiffness of the surrounding extracellular matrix. We identify the F-actin-capping/severing proteins Cofilin, CapZ, and Gelsolin as essential gatekeepers that limit YAP/TAZ activity in cells experiencing low mechanical stresses, including contact inhibition of proliferation. We propose that mechanical forces are overarching regulators of YAP/TAZ in multicellular contexts, setting responsiveness to Hippo, WNT, and GPCR signaling.

title: Angiomotins link F-actin architecture to Hippo pathway signaling
abstract: Angiomotin proteins, together with LATS kinase, regulate the Hippo pathway transcriptional coactivator YAP in response to changes in the F-actin cytoskeleton. Competition between F-actin and YAP for binding to angiomotins makes YAP regulation responsive to F-actin levels. Phosphorylation by LATS can switch angiomotins from F-actin to YAP binding.

title: A genetic screen identifies an LKB1–MARK signalling axis controlling the Hippo–YAP pathway
abstract: 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.

title: Cytokinesis Failure Triggers Hippo Tumor Suppressor Pathway Activation
abstract: Genetically unstable tetraploid cells can promote tumorigenesis. Recent estimates suggest that ∼37% of human tumors have undergone a genome-doubling event during their development. This potentially oncogenic effect of tetraploidy is countered by a p53-dependent barrier to proliferation. However, the cellular defects and corresponding signaling pathways that trigger growth suppression in tetraploid cells are not known. Here, we combine RNAi screening and in vitro evolution approaches to demonstrate that cytokinesis failure activates the Hippo tumor suppressor pathway in cultured cells, as well as in naturally occurring tetraploid cells in vivo. Induction of the Hippo pathway is triggered in part by extra centrosomes, which alter small G protein signaling and activate LATS2 kinase. LATS2 in turn stabilizes p53 and inhibits the transcriptional regulators YAP and TAZ. These findings define an important tumor suppression mechanism and uncover adaptive mechanisms potentially available to nascent tumor cells that bypass this inhibitory regulation.

title: Spectrin regulates Hippo signaling by modulating cortical actomyosin activity
abstract: 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.

title: Remodelling the extracellular matrix in development and disease
abstract: The extracellular matrix (ECM) is a highly dynamic structure that is present in all tissues and continuously undergoes controlled remodelling. This process involves quantitative and qualitative changes in the ECM, mediated by specific enzymes that are responsible for ECM degradation, such as metalloproteinases. The ECM interacts with cells to regulate diverse functions, including proliferation, migration and differentiation. ECM remodelling is crucial for regulating the morphogenesis of the intestine and lungs, as well as of the mammary and submandibular glands. Dysregulation of ECM composition, structure, stiffness and abundance contributes to several pathological conditions, such as fibrosis and invasive cancer. A better understanding of how the ECM regulates organ structure and function and of how ECM remodelling affects disease progression will contribute to the development of new therapeutics.

title: Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling
abstract: Tumors are characterized by extracellular matrix (ECM) remodeling and stiffening. The importance of ECM remodeling to cancer is appreciated; the relevance of stiffening is less clear. We found that breast tumorigenesis is accompanied by collagen crosslinking, ECM stiffening, and increased focal adhesions. Induction of collagen crosslinking stiffened the ECM, promoted focal adhesions, enhanced PI3 kinase (PI3K) activity, and induced the invasion of an oncogene-initiated epithelium. Inhibition of integrin signaling repressed the invasion of a premalignant epithelium into a stiffened, crosslinked ECM and forced integrin clustering promoted focal adhesions, enhanced PI3K signaling, and induced the invasion of a premalignant epithelium. Consistently, reduction of lysyl oxidase-mediated collagen crosslinking prevented MMTV-Neu-induced fibrosis, decreased focal adhesions and PI3K activity, impeded malignancy, and lowered tumor incidence. These data show how collagen crosslinking can modulate tissue fibrosis and stiffness to force focal adhesions, growth factor signaling and breast malignancy.

title: Mechano-transduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer associated fibroblasts
abstract: To learn more about cancer-associated fibroblasts (CAFs), we have isolated fibroblasts from different stages of breast cancer progression and analysed their function and gene expression. These analyses reveal that activation of the YAP transcription factor is a signature feature of CAFs. YAP function is required for CAFs to promote matrix stiffening, cancer cell invasion and angiogenesis. Remodelling of the ECM and promotion of cancer cell invasion requires the actomyosin cytoskeleton. YAP regulates the expression of several cytoskeletal regulators, including ANLN and DIAPH3, and controls the protein levels of MYL9 (also known as MLC2). Matrix stiffening further enhances YAP activation, thus establishing a feed-forward self-reinforcing loop that helps to maintain the CAF phenotype. Actomyosin contractility and Src function are required for YAP activation by stiff matrices. Further, transient ROCK inhibition is able to disrupt the feed-forward loop, leading to a long-lasting reversion of the CAF phenotype.

title: The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis
abstract: Merlin, the protein product of the Neurofibromatosis type-2 gene, acts as a tumour suppressor in mice and humans. Merlin is an adaptor protein with a FERM domain and it is thought to transduce a growth-regulatory signal. However, the pathway through which Merlin acts as a tumour suppressor is poorly understood. Merlin, and its function as a negative regulator of growth, is conserved in Drosophila, where it functions with Expanded, a related FERM domain protein. Here, we show that Drosophila Merlin and Expanded are components of the Hippo signalling pathway, an emerging tumour-suppressor pathway. We find that Merlin and Expanded, similar to other components of the Hippo pathway, are required for proliferation arrest and apoptosis in developing imaginal discs. Our genetic and biochemical data place Merlin and Expanded upstream of Hippo and identify a pathway through which they act as tumour-suppressor genes.

title: Kibra functions as a tumor suppressor protein that regulates Hippo signaling in conjunction with Merlin and Expanded.
abstract: The 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.

title: Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein.
abstract: The Yes-associated protein (YAP) is a transcription coactivator that plays a crucial role in organ size control by promoting cell proliferation and inhibiting apoptosis. The Hippo tumor suppressor pathway inhibits YAP through phosphorylation-induced cytoplasmic retention and degradation. Here we report a novel mechanism of YAP regulation by angiomotin (AMOT) family proteins via a direct interaction. Knockdown of AMOT family protein AMOTL2 in polarized Madin-Darby canine kidney (MDCK) cells leads to YAP activation, as indicated by decreased YAP tight junction localization, attenuated YAP phosphorylation, accumulation of nuclear YAP, and induction of YAP target gene expression. Transcriptional coactivator with PDZ-binding motif (TAZ), the YAP paralog, is also regulated by AMOT in a similar fashion. Furthermore, AMOTL2 knockdown results in loss of cell contact inhibition in a manner dependent on the functions of YAP and TAZ. Our results indicate a potential tumor-suppressing role of AMOT family proteins as components of the Hippo pathway, and demonstrate a novel mechanism of YAP and TAZ inhibition by AMOT-mediated tight junction localization. These observations provide a potential link between the Hippo pathway and cell contact inhibition.

title: E-cadherin mediates contact inhibition of proliferation through Hippo signaling-pathway components
abstract: Contact inhibition of cell growth is essential for embryonic development and maintenance of tissue architecture in adult organisms, and the growth of tumors is characterized by a loss of contact inhibition of proliferation. The recently identified Hippo signaling pathway has been implicated in contact inhibition of proliferation as well as organ size control. The modulation of the phosphorylation and nuclear localization of Yes-associated protein (YAP) by the highly conserved kinase cascade of the Hippo signaling pathway has been intensively studied. However, cell-surface receptors regulating the Hippo signaling pathway in mammals are not well understood. In this study, we show that Hippo signaling pathway components are required for E-cadherin–dependent contact inhibition of proliferation. Knockdown of the Hippo signaling components or overexpression of YAP inhibits the decrease in cell proliferation caused by E-cadherin homophilic binding at the cell surface, independent of other cell–cell interactions. We also demonstrate that the E-cadherin/catenin complex functions as an upstream regulator of the Hippo signaling pathway in mammalian cells. Expression of E-cadherin in MDA-MB-231 cells restores the density-dependent regulation of YAP nuclear exclusion. Knockdown of β-catenin in densely cultured MCF10A cells, which mainly depletes E-cadherin–bound β-catenin, induces a decrease in the phosphorylation of S127 residue of YAP and its nuclear accumulation. Moreover, E-cadherin homophilic binding independent of other cell interactions is sufficient to control the subcellular localization of YAP. Therefore, Our results indicate that, in addition to its role in cell–cell adhesion, E-cadherin-mediated cell–cell contact directly regulates the Hippo signaling pathway to control cell proliferation.

title: Epithelial cell polarity, stem cells and cancer
abstract: Fernando Martin‑Belmonte 1 and Mirna Perez‑Moreno 2 Abstract | After years of extensive scientific discovery much has been learned about the networks that regulate epithelial homeostasis. Loss of expression or functional activity of cell adhesion and cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related to advanced stages of tumour progression and invasiveness. But the key roles of these proteins in crosstalk with the Hippo and liver kinase B1 (LKB1)- AMPK pathways and in epithelial function and proliferation indicate that they may also be associated with the early stages of tumorigenesis. For example, deregulation of adhesion and polarity proteins can cause misoriented cell divisions and increased self-renewal of adult epithelial stem cells. In this Review, we highlight some advances in the understanding of how loss of epithelial cell polarity contributes to tumorigenesis.

title: Spatial Organization of Hippo Signaling at the Plasma Membrane Mediated by the Tumor Suppressor Merlin/NF2
abstract: Although 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.

title: The two faces of Hippo: targeting the Hippo pathway for regenerative medicine and cancer treatment
abstract: The Hippo signalling pathway is an emerging growth control and tumour suppressor pathway that regulates cell proliferation and stem cell functions. Defects in Hippo signalling and hyperactivation of its downstream effectors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) contribute to the development of cancer, which suggests that pharmacological inhibition of YAP and TAZ activity may be an effective anticancer strategy. Conversely, YAP and TAZ can also have beneficial roles in stimulating tissue repair and regeneration following injury, so their activation may be therapeutically useful in these contexts. A complex network of intracellular and extracellular signalling pathways that modulate YAP and TAZ activities have recently been identified. Here, we review the regulation of the Hippo signalling pathway, its functions in normal homeostasis and disease, and recent progress in the identification of small-molecule pathway modulators.

title: Disease implications of the Hippo/YAP pathway.
abstract: The Hippo signaling pathway is important for controlling organ size and tissue homeostasis. Originally identified in Drosophila melanogaster, the core components of the Hippo pathway are highly conserved in mammals. The Hippo pathway can be modulated by a wide range of stimuli, including G protein-coupled receptor (GPCR) signaling, changes in the actin cytoskeleton, cell-cell contact, and cell polarity. When activated, the Hippo pathway functions as a tumor suppressor to limit cell growth. However, dysregulation by genetic inactivation of core pathway components or amplification or gene fusion of its downstream effectors results in increased cell proliferation and decreased apoptosis and differentiation. Unsurprisingly, this can lead to tissue overgrowth, tumorigenesis, and many other diseases.

title: The emerging roles of YAP and TAZ in cancer
abstract: Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are the major downstream effectors of the Hippo pathway, which regulates tissue homeostasis, organ size, regeneration and tumorigenesis. In this Progress article, we summarize the current understanding of the biological functions of YAP and TAZ, and how the regulation of these two proteins can be disrupted in cancer. We also highlight recent findings on their expanding role in cancer progression and describe the potential of these targets for therapeutic intervention.

title: Merlin/NF2 Suppresses Tumorigenesis by Inhibiting the E3 Ubiquitin Ligase CRL4DCAF1 in the Nucleus
abstract: Current models imply that the FERM domain protein Merlin, encoded by the tumor suppressor NF2, inhibits mitogenic signaling at or near the plasma membrane. Here, we show that the closed, growth-inhibitory form of Merlin accumulates in the nucleus, binds to the E3 ubiquitin ligase CRL4(DCAF1), and suppresses its activity. Depletion of DCAF1 blocks the promitogenic effect of inactivation of Merlin. Conversely, enforced expression of a Merlin-insensitive mutant of DCAF1 counteracts the antimitogenic effect of Merlin. Re-expression of Merlin and silencing of DCAF1 implement a similar, tumor-suppressive program of gene expression. Tumor-derived mutations invariably disrupt Merlin's ability to interact with or inhibit CRL4(DCAF1). Finally, depletion of DCAF1 inhibits the hyperproliferation of Schwannoma cells from NF2 patients and suppresses the oncogenic potential of Merlin-deficient tumor cell lines. We propose that Merlin suppresses tumorigenesis by translocating to the nucleus to inhibit CRL4(DCAF1).

title: Merlin/NF2 loss-driven tumorigenesis linked to CRL4(DCAF1)-mediated inhibition of the hippo pathway kinases Lats1 and 2 in the nucleus.
abstract: It is currently unclear whether Merlin/NF2 suppresses tumorigenesis by activating upstream components of the Hippo pathway at the plasma membrane or by inhibiting the E3 ubiquitin ligase CRL4(DCAF1) in the nucleus. We found that derepressed CRL4(DCAF1) promotes YAP- and TEAD-dependent transcription by ubiquitylating and, thereby, inhibiting Lats1 and 2 in the nucleus. Genetic epistasis experiments and analysis of tumor-derived missense mutations indicate that this signaling connection sustains the oncogenicity of Merlin-deficient tumor cells. Analysis of clinical samples confirms that this pathway operates in NF2-mutant tumors. We conclude that derepressed CRL4(DCAF1) promotes activation of YAP by inhibiting Lats1 and 2 in the nucleus.

title: Nf2/Merlin controls progenitor homeostasis and tumorigenesis in the liver.
abstract: The molecular signals that control the maintenance and activation of liver stem/progenitor cells are poorly understood, and the role of liver progenitor cells in hepatic tumorigenesis is unclear. We report here that liver-specific deletion of the neurofibromatosis type 2 (Nf2) tumor suppressor gene in the developing or adult mouse specifically yields a dramatic, progressive expansion of progenitor cells throughout the liver without affecting differentiated hepatocytes. All surviving mice eventually developed both cholangiocellular and hepatocellular carcinoma, suggesting that Nf2(-/-) progenitors can be a cell of origin for these tumors. Despite the suggested link between Nf2 and the Hpo/Wts/Yki signaling pathway in Drosophila, and recent studies linking the corresponding Mst/Lats/Yap pathway to mammalian liver tumorigenesis, our molecular studies suggest that Merlin is not a major regulator of YAP in liver progenitors, and that the overproliferation of Nf2(-/-) liver progenitors is instead driven by aberrant epidermal growth factor receptor (EGFR) activity. Indeed, pharmacologic inhibition of EGFR blocks the proliferation of Nf2(-/-) liver progenitors in vitro and in vivo, consistent with recent studies indicating that the Nf2-encoded protein Merlin can control the abundance and signaling of membrane receptors such as EGFR. Together, our findings uncover a critical role for Nf2/Merlin in controlling homeostasis of the liver stem cell niche.

title: The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals.
abstract: The conserved Hippo signaling pathway regulates organ size in Drosophila and mammals. While a core kinase cascade leading from the protein kinase Hippo (Hpo) (Mst1 and Mst2 in mammals) to the transcription coactivator Yorkie (Yki) (YAP in mammals) has been established, upstream regulators of the Hippo kinase cascade are less well defined, especially in mammals. Using conditional knockout mice, we demonstrate that the Merlin/NF2 tumor suppressor and the YAP oncoprotein function antagonistically to regulate liver development. While inactivation of Yap led to loss of hepatocytes and biliary epithelial cells, inactivation of Nf2 led to hepatocellular carcinoma and bile duct hamartoma. Strikingly, the Nf2-deficient phenotypes in multiple tissues were largely suppressed by heterozygous deletion of Yap, suggesting that YAP is a major effector of Merlin/NF2 in growth regulation. Our studies link Merlin/NF2 to mammalian Hippo signaling and implicate YAP activation as a mediator of pathologies relevant to Neurofibromatosis 2.

title: The Angiomotins – From discovery to function
abstract: Angiomotins were originally identified as angiostatin binding proteins and implicated in the regulation of endothelial cell migration. Recent studies have shed light on the role of Angiomotins and other members of the Motin protein family in epithelial cells and in pathways directly linked to the pathogenesis of cancer. In particular, Motins have been shown to play a role in signaling pathways regulated by small G‐proteins and the Hippo–YAP pathway. In this review the role of the Motin protein family in these signaling pathways will be described and open questions will be discussed.

title: Actin-binding and Cell Proliferation Activities of Angiomotin Family Members Are Regulated by Hippo Pathway-mediated Phosphorylation*
abstract: LATS kinase, one of the core kinases of Hippo pathway, phosphorylates and inactivates the downstream coactivator YAP/TAZ.  The angiomotin (Amot) family members are phosphorylated by LATS kinase.  Phosphorylation of Amots by LATS kinase inhibits actin-binding, stabilizes Amot, and inhibits cell proliferation.  Besides phosphorylating YAP/TAZ, LATS kinase may phosphorylate other components of the Hippo pathway. Whether the Hippo pathway has downstream targets other than YAP and TAZ is unknown. In this report, we have identified angiomotin (Amot) family members as novel substrates of Hippo core kinases. The N-terminal regions of Amot proteins contain a conserved HXRXXS consensus site for LATS1/2-mediated phosphorylation. Phospho-specific antibodies showed that Hippo core kinases could mediate phosphorylation of endogenous as well as exogenous Amot family members. Knockdown of LATS1 and LATS2 endogenously reduced the phosphorylation of Amots detected by the phospho-specific antibodies. Mutation of the serine to alanine within this HXRXXS site in Amot and AmotL2 established that this site was essential for Hippo core kinase-mediated phosphorylation. Wild-type and non-phosphorylated Amot (Amot-S175A) were targeted to actin filaments, whereas phospho-mimic Amot (Amot-S175D) failed to be localized with actin. Overexpression of LATS2 caused dissociation of Amot from actin but not Amot-S175A. Mapping of the actin-binding site of Amot showed that serine 175 of Amot was important for the actin-binding activity. Amot-S175A promoted, whereas Amot and Amot-S175D inhibited, cell proliferation. These results collectively suggest that the Hippo pathway negatively regulates the actin-binding activity of Amot family members through direct phosphorylation.

title: Phosphorylation of Angiomotin by Lats1/2 Kinases Inhibits F-actin Binding, Cell Migration, and Angiogenesis*
abstract: Substrates of the Hippo pathway kinases Lats1/2 are largely unknown besides YAP/TAZ.  Phosphorylation of angiomotin by Lats1/2 inhibits interaction with F-actin thus impairs cell migration and angiogenesis.  AMOTp130 is a physiological and functional substrate of Lats1/2 and the Hippo pathway.  Demonstrating how identification of novel substrates would facilitate understanding the physiology of the Hippo pathway. The Hippo tumor suppressor pathway plays important roles in organ size control through Lats1/2 mediated phosphorylation of the YAP/TAZ transcription co-activators. However, YAP/TAZ independent functions of the Hippo pathway are largely unknown. Here we report a novel role of the Hippo pathway in angiogenesis. Angiomotin p130 isoform (AMOTp130) is phosphorylated on a conserved HXRXXS motif by Lats1/2 downstream of GPCR signaling. Phosphorylation disrupts AMOT interaction with F-actin and correlates with reduced F-actin stress fibers and focal adhesions. Furthermore, phosphorylation of AMOT by Lats1/2 inhibits endothelial cell migration in vitro and angiogenesis in zebrafish embryos in vivo. Thus AMOT is a direct substrate of Lats1/2 mediating functions of the Hippo pathway in endothelial cell migration and angiogenesis.

title: Serum deprivation inhibits the transcriptional co-activator YAP and cell growth via phosphorylation of the 130-kDa isoform of Angiomotin by the LATS1/2 protein kinases
abstract: This study defines a unique mechanism controlling the activation of Hippo signaling and consequent inhibition of cell growth. Specifically, serum starvation is found to induce the large tumor suppressor (LATS)1/2 kinases to phosphorylate and thus stabilize the 130 kDa isoform of the membrane-associated polarity protein angiomotin (Amot130). As a consequence, Amot130 recruits the E3 protein-ubiquitin ligase atrophin-1 interacting protein 4. This multiprotein complex then signals the degradation of Yes-associated protein (YAP) and the inhibition of cell growth. These findings significantly modify our current view that YAP phosphorylation by LATS1/2 is sufficient for its inhibition in mammals and thus for growth arrest. Large tumor suppressor (LATS)1/2 protein kinases transmit Hippo signaling in response to intercellular contacts and serum levels to limit cell growth via the inhibition of Yes-associated protein (YAP). Here low serum and high LATS1 activity are found to enhance the levels of the 130-kDa isoform of angiomotin (Amot130) through phosphorylation by LATS1/2 at serine 175, which then forms a binding site for 14-3-3. Such phosphorylation, in turn, enables the ubiquitin ligase atrophin-1 interacting protein (AIP)4 to bind, ubiquitinate, and stabilize Amot130. Consistently, the Amot130 (S175A) mutant, which lacks LATS phosphorylation, bound AIP4 poorly under all conditions and showed reduced stability. Amot130 and AIP4 also promoted the ubiquitination and degradation of YAP in response to serum starvation, unlike Amot130 (S175A). Moreover, silencing Amot130 expression blocked LATS1 from inhibiting the expression of connective tissue growth factor, a YAP-regulated gene. Concordant with phosphorylated Amot130 specifically mediating these effects, wild-type Amot130 selectively induced YAP phosphorylation and reduced transcription of connective tissue growth factor in an AIP4-dependent manner versus Amot130 (S175A). Further, Amot130 but not Amot130 (S175A) strongly inhibited the growth of MDA-MB-468 breast cancer cells. The dominant-negative effects of Amot130 (S175A) on YAP signaling also support that phosphorylated Amot130 transduces Hippo signaling. Likewise, Amot130 expression provoked premature growth arrest during mammary cell acini formation, whereas Amot130 (S175A)-expressing cells formed enlarged and poorly differentiated acini. Taken together, the phosphorylation of Amot130 by LATS is found to be a key feature that enables it to inhibit YAP-dependent signaling and cell growth.

title: Alpha-Catenins Control Cardiomyocyte Proliferation by Regulating Yap Activity
abstract: Shortly after birth, muscle cells of the mammalian heart lose their ability to divide. Thus, they are unable to effectively replace dying cells in the injured heart. The recent discovery that the transcriptional coactivator Yes-associated protein (Yap) is necessary and sufficient for cardiomyocyte proliferation has gained considerable attention. However, the upstream regulators and signaling pathways that control Yap activity in the heart are poorly understood.  To investigate the role of &agr;-catenins in the heart using cardiac-specific &agr;E- and &agr;T-catenin double knockout mice.  We used 2 cardiac-specific Cre transgenes to delete both &agr;E-catenin (Ctnna1) and &agr;T-catenin (Ctnna3) genes either in the perinatal or in the adult heart. Perinatal depletion of & increased cardiomyocyte number in the postnatal heart. Increased nuclear Yap and the cell cycle regulator cyclin D1 accompanied cardiomyocyte proliferation in the & double knockout hearts. Fetal genes were increased in the & double knockout hearts indicating a less mature cardiac gene expression profile. Knockdown of &agr;-catenins in neonatal rat cardiomyocytes also resulted in increased proliferation, which could be blocked by knockdown of Yap. Finally, inactivation of &agr;-catenins in the adult heart using an inducible Cre led to increased nuclear Yap and cardiomyocyte proliferation and improved contractility after myocardial infarction.  These studies demonstrate that &agr;-catenins are critical regulators of Yap, a transcriptional coactivator essential for cardiomyocyte proliferation. Furthermore, we provide proof of concept that inhibiting &agr;-catenins might be a useful strategy to promote myocardial regeneration after injury.

title: β-Catenin-Driven Cancers Require a YAP1 Transcriptional Complex for Survival and Tumorigenesis
abstract: Joseph Rosenbluh, Deepak Nijhawan, Andrew G. Cox, Xingnan Li, James T. Neal, Eric J. Schafer, Travis I. Zack, Xiaoxing Wang, Aviad Tsherniak, Anna C. Schinzel, Diane D. Shao, Steven E. Schumacher, Barbara A. Weir, Francisca Vazquez, Glenn S. Cowley, David E. Root, Jill P. Mesirov, Rameen Beroukhim, Calvin J. Kuo, Wolfram Goessling, and William C. Hahn* *Correspondence: william_hahn@dfci.harvard.edu http://dx.doi.org/10.1016/j.cell.2013.03.007

title: Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated apoptosis.
abstract: We have used an affinity purification method to identify substrates of protein kinase B/Akt. One protein that associates with 14-3-3 in an Akt-dependent manner is shown here to be the Yes-associated protein (YAP), which is phosphorylated by Akt at serine 127, leading to binding to 14-3-3. Akt promotes YAP localization to the cytoplasm, resulting in loss from the nucleus where it functions as a coactivator of transcription factors including p73. p73-mediated induction of Bax expression following DNA damage requires YAP function and is attenuated by Akt phosphorylation of YAP. YAP overexpression increases, while YAP depletion decreases, p73-mediated apoptosis following DNA damage, in an Akt inhibitable manner. Akt phosphorylation of YAP may thus suppress the induction of the proapoptotic gene expression response following cellular damage.

title: Hippo pathway effector Yap promotes cardiac regeneration
abstract: The adult mammalian heart has limited potential for regeneration. Thus, after injury, cardiomyocytes are permanently lost, and contractility is diminished. In contrast, the neonatal heart can regenerate owing to sustained cardiomyocyte proliferation. Identification of critical regulators of cardiomyocyte proliferation and quiescence represents an important step toward potential regenerative therapies. Yes-associated protein (Yap), a transcriptional cofactor in the Hippo signaling pathway, promotes proliferation of embryonic cardiomyocytes by activating the insulin-like growth factor and Wnt signaling pathways. Here we report that mice bearing mutant alleles of Yap and its paralog WW domain containing transcription regulator 1 (Taz) exhibit gene dosage-dependent cardiac phenotypes, suggesting redundant roles of these Hippo pathway effectors in establishing proper myocyte number and maintaining cardiac function. Cardiac-specific deletion of Yap impedes neonatal heart regeneration, resulting in a default fibrotic response. Conversely, forced expression of a constitutively active form of Yap in the adult heart stimulates cardiac regeneration and improves contractility after myocardial infarction. The regenerative activity of Yap is correlated with its activation of embryonic and proliferative gene programs in cardiomyocytes. These findings identify Yap as an important regulator of cardiac regeneration and provide an experimental entry point to enhance this process.

title: Activation of Mst1 causes dilated cardiomyopathy by stimulating apoptosis without compensatory ventricular myocyte hypertrophy.
abstract: Activation of mammalian sterile 20-like kinase 1 (Mst1) by genotoxic compounds is known to stimulate apoptosis in some cell types. The importance of Mst1 in cell death caused by clinically relevant pathologic stimuli is unknown, however. In this study, we show that Mst1 is a prominent myelin basic protein kinase activated by proapoptotic stimuli in cardiac myocytes and that Mst1 causes cardiac myocyte apoptosis in vitro in a kinase activity-dependent manner. In vivo, cardiac-specific overexpression of Mst1 in transgenic mice results in activation of caspases, increased apoptosis, and dilated cardiomyopathy. Surprisingly, however, Mst1 prevents compensatory cardiac myocyte elongation or hypertrophy despite increased wall stress, thereby obscuring the use of the Frank-Starling mechanism, a fundamental mechanism by which the heart maintains cardiac output in response to increased mechanical load at the single myocyte level. Furthermore, Mst1 is activated by ischemia/reperfusion in the mouse heart in vivo. Suppression of endogenous Mst1 by cardiac-specific overexpression of dominant-negative Mst1 in transgenic mice prevents myocyte death by pathologic insults. These results show that Mst1 works as both an essential initiator of apoptosis and an inhibitor of hypertrophy in cardiac myocytes, resulting in a previously unrecognized form of cardiomyopathy.

title: Regulation of YAP by mTOR and autophagy reveals a therapeutic target of Tuberous Sclerosis Complex
abstract: HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Regulation of YAP by mTOR and autophagy reveals a therapeutic target of Tuberous Sclerosis Complex Ning Liang

title: Mst1 and Mst2 protein kinases restrain intestinal stem cell proliferation and colonic tumorigenesis by inhibition of Yes-associated protein (Yap) overabundance
abstract: Ablation of the kinases Mst1 and Mst2, orthologs of the Drosophila antiproliferative kinase Hippo, from mouse intestinal epithelium caused marked expansion of an undifferentiated stem cell compartment and loss of secretory cells throughout the small and large intestine. Although median survival of mice lacking intestinal Mst1/Mst2 is 13 wk, adenomas of the distal colon are common by this age. Diminished phosphorylation, enhanced abundance, and nuclear localization of the transcriptional coactivator Yes-associated protein 1 (Yap1) is evident in Mst1/Mst2-deficient intestinal epithelium, as is strong activation of β-catenin and Notch signaling. Although biallelic deletion of Yap1 from intestinal epithelium has little effect on intestinal development, inactivation of a single Yap1 allele reduces Yap1 polypeptide abundance to nearly wild-type levels and, despite the continued Yap hypophosphorylation and preferential nuclear localization, normalizes epithelial structure. Thus, supraphysiologic Yap polypeptide levels are necessary to drive intestinal stem cell proliferation. Yap is overexpressed in 68 of 71 human colon cancers and in at least 30 of 36 colon cancer-derived cell lines. In colon-derived cell lines where Yap is overabundant, its depletion strongly reduces β-catenin and Notch signaling and inhibits proliferation and survival. These findings demonstrate that Mst1 and Mst2 actively suppress Yap1 abundance and action in normal intestinal epithelium, an antiproliferative function that frequently is overcome in colon cancer through Yap1 polypeptide overabundance. The dispensability of Yap1 in normal intestinal homeostasis and its potent proliferative and prosurvival actions when overexpressed in colon cancer make it an attractive therapeutic target.

title: Notch and hippo converge on Cdx2 to specify the trophectoderm lineage in the mouse blastocyst.
abstract: The first lineage choice in mammalian embryogenesis is that between the trophectoderm, which gives rise to the trophoblast of the placenta, and the inner cell mass, from which is derived the embryo proper and the yolk sac. The establishment of these lineages is preceded by the inside-versus-outside positioning of cells in the early embryo and stochastic expression of key transcription factors, which is then resolved into lineage-restricted expression. The regulatory inputs that drive this restriction and how they relate to cell position are largely unknown. Here, we show an unsuspected role of Notch signaling in regulating trophectoderm-specific expression of Cdx2 in cooperation with TEAD4. Notch activity is restricted to outer cells and is able to influence positional allocation of blastomeres, mediating preferential localization to the trophectoderm. Our results show that multiple signaling inputs at preimplantation stages specify the first embryonic lineages.

title: Notch Inhibits Yorkie Activity in Drosophila Wing Discs
abstract: During development, tissues and organs must coordinate growth and patterning so they reach the right size and shape. During larval stages, a dramatic increase in size and cell number of Drosophila wing imaginal discs is controlled by the action of several signaling pathways. Complex cross-talk between these pathways also pattern these discs to specify different regions with different fates and growth potentials. We show that the Notch signaling pathway is both required and sufficient to inhibit the activity of Yorkie (Yki), the Salvador/Warts/Hippo (SWH) pathway terminal transcription activator, but only in the central regions of the wing disc, where the TEAD factor and Yki partner Scalloped (Sd) is expressed. We show that this cross-talk between the Notch and SWH pathways is mediated, at least in part, by the Notch target and Sd partner Vestigial (Vg). We propose that, by altering the ratios between Yki, Sd and Vg, Notch pathway activation restricts the effects of Yki mediated transcription, therefore contributing to define a zone of low proliferation in the central wing discs.

title: The Yes-associated protein controls the cell density regulation of Hedgehog signaling
abstract: The evolutionarily conserved Hedgehog (Hh) signaling pathway is essential for correct embryogenesis and is misregulated in several malignancies. In cell culture, Hh-sensitive cells display a striking dependence on cell density with active Hh signaling requiring cell-to-cell contact. As the Hippo/YAP system is tightly linked to cell density control and contact inhibition, we investigated the cross-talk between the two pathways. Our data reveal that the suppression of Hh signaling in the absence of cellular contacts is independent of primary cilia and is mediated by the YAP oncogene. Overexpression of YAP blocks Hh signaling whereas RNA interference-mediated knockdown of YAP enhances Hh/GLI activity. Despite this negative regulation, Hh signaling promotes YAP activity through post-transcriptional mechanisms, resulting in a negative feedback loop. In vivo, we found strong nuclear YAP immunoreactivity restricted to compartments with low Hh pathway activity in human and mouse pancreatic cancer. Finally, we identified protease-activated receptors (PARs) as molecules being able to override the inverse Hippo/Hh regulation, potentially giving tumors a mechanism to utilize both oncogenic pathways in parallel.Oncogenesis (2014) 3, e112; doi:10.1038/oncsis.2014.27; published online 11 August 2014

title: JNK phosphorylates Yes-associated protein (YAP) to regulate apoptosis
abstract: Yes-associated protein (YAP) regulates DNA damage and chemosensitivity, as well as functioning as a pro-growth, cell size regulator. For both of its roles, regulation by phosphorylation is crucial. We undertook an in vitro screen to identify novel YAP kinases to discover new signaling pathways to better understand YAP's function. We identified JNK1 and JNK2 as robust YAP kinases, as well as mapped multiple sites of phosphorylation. Using inhibitors and siRNA, we showed that JNK specifically phosphorylates endogenous YAP in a number of cell types. We show that YAP protects keratinocytes from UV irradiation but promotes UV-induced apoptosis in a squamous cell carcinoma. We defined the mechanism for this dual role to be YAP's ability to bind and stabilize the pro-proliferative ΔNp63α isoform in a JNK-dependent manner. Our report indicates that an evaluation of the expression of the different isoforms of p63 and p73 is crucial in determining YAP's function.

title: Control of the hippo pathway by Set7-dependent methylation of Yap.
abstract: Methylation of nonhistone proteins is emerging as a regulatory mechanism to control protein function. Set7 (Setd7) is a SET-domain-containing lysine methyltransferase that methylates and alters function of a variety of proteins in vitro, but the in vivo relevance has not been established. We found that Set7 is a modifier of the Hippo pathway. Mice that lack Set7 have a larger progenitor compartment in the intestine, coinciding with increased expression of Yes-associated protein (Yap) target genes. Mechanistically, monomethylation of lysine 494 of Yap is critical for cytoplasmic retention. These results identify a methylation-dependent checkpoint in the Hippo pathway.

title: Tyrosine phosphorylation controls Runx2‐mediated subnuclear targeting of YAP to repress transcription
abstract: Src/Yes tyrosine kinase signaling contributes to the regulation of bone homeostasis and inhibits osteoblast activity. Here we show that the endogenous Yes‐associated protein (YAP), a mediator of Src/Yes signaling, interacts with the native Runx2 protein, an osteoblast‐related transcription factor, and suppresses Runx2 transcriptional activity in a dose‐dependent manner. Runx2, through its PY motif, recruits YAP to subnuclear domains in situ and to the osteocalcin (OC) gene promoter in vivo. Inhibition of Src/Yes kinase blocks tyrosine phosphorylation of YAP and dissociates endogenous Runx2–YAP complexes. Consequently, recruitment of the YAP co‐repressor to subnuclear domains is abrogated and expression of the endogenous OC gene is induced. Our results suggest that Src/Yes signals are integrated through organization of Runx2–YAP transcriptional complexes at subnuclear sites to attenuate skeletal gene expression.

title: Yap1 phosphorylation by c-Abl is a critical step in selective activation of proapoptotic genes in response to DNA damage.
abstract: Cells undergo apoptosis upon exposure to severe DNA damage stress. Under this condition, p73 is phosphorylated and activated by c-Abl. The transcription coactivator Yap1 binds p73 to generate a complex that escapes p73 proteasomal degradation and recruits p300 to support transcription of proapoptotic genes. However, the mechanism of selective activation of proapoptotic genes by Yap1 remained unclear. In this study, we show that c-Abl directly phosphorylates Yap1 at position Y357 in response to DNA damage. Tyrosine-phosphorylated Yap1 is a more stable protein that displays higher affinity to p73 and selectively coactivates p73 proapoptotic target genes. Furthermore, we show that Yap1 switches between p73-mediated proapoptotic and growth arrest target genes based on its phosphorylation state. Thus, our data demonstrate that modification of a transcription coactivator, namely the DNA damage-induced phosphorylation of Yap1 by c-Abl, influences the specificity of target gene activation.

title: c-Abl antagonizes the YAP oncogenic function
abstract: YES-associated protein (YAP) is a central transcription coactivator that functions as an oncogene in a number of experimental systems. However, under DNA damage, YAP activates pro-apoptotic genes in conjunction with p73. This program switching is mediated by c-Abl (Abelson murine leukemia viral oncogene) via phosphorylation of YAP at the Y357 residue (pY357). YAP as an oncogene coactivates the TEAD (transcriptional enhancer activator domain) family transcription factors. Here we asked whether c-Abl regulates the YAP–TEAD functional module. We found that DNA damage, through c-Abl activation, specifically depressed YAP–TEAD-induced transcription. Remarkably, c-Abl counteracts YAP-induced transformation by interfering with the YAP–TEAD transcriptional program. c-Abl induced TEAD1 phosphorylation, but the YAP–TEAD complex remained unaffected. In contrast, TEAD coactivation was compromised by phosphomimetic YAP Y357E mutation but not Y357F, as demonstrated at the level of reporter genes and endogenous TEAD target genes. Furthermore, YAP Y357E also severely compromised the role of YAP in cell transformation, migration, anchorage-independent growth, and epithelial-to-mesenchymal transition (EMT) in human mammary MCF10A cells. These results suggest that YAP pY357 lost TEAD transcription activation function. Our results demonstrate that YAP pY357 inactivates YAP oncogenic function and establish a role for YAP Y357 phosphorylation in cell-fate decision.

title: PP1 Cooperates with ASPP2 to Dephosphorylate and Activate TAZ*
abstract: The Hippo pathway regulates organ size by controlling both cell proliferation and apoptosis. TAZ functions as a transcriptional co-activator downstream of the Hippo pathway and has been implicated in human cancer development. A key step in the Hippo-TAZ pathway is phosphorylation of TAZ by LATS kinase, which leads to TAZ inhibition by both cytoplasmic retention and degradation. However, the mechanism of TAZ dephosphorylation and the responsible phosphatase are unknown. Here, we identified PP1 as a bona fide TAZ phosphatase. PP1A dephosphorylates TAZ at Ser-89 and Ser-311, promotes TAZ nuclear translocation, and stabilizes TAZ by disrupting the binding to the SCF E3 ubiquitin ligase. Furthermore, ASPP2 facilitates the interaction between TAZ and PP1 to promote TAZ dephosphorylation. As a result, PP1 and ASPP2 increase TAZ-dependent gene expression. This study demonstrates that PP1A and ASPP2 play a critical role in promoting TAZ function by antagonizing the LATS kinase through TAZ dephosphorylation.

title: PP1A-Mediated Dephosphorylation Positively Regulates YAP2 Activity
abstract: The Hippo/MST1 signaling pathway plays an important role in the regulation of cell proliferation and apoptosis. As a major downstream target of the Hippo/MST1 pathway, YAP2 (Yes-associated protein 2) functions as a transcriptional cofactor that has been implicated in many biological processes, including organ size control and cancer development. MST1/Lats kinase inhibits YAP2's nuclear accumulation and transcriptional activity through inducing the phosphorylation at serine 127 and the sequential association with 14-3-3 proteins. However, the dephosphorylation of YAP2 is not fully appreciated.  In the present study, we demonstrate that PP1A (catalytic subunit of protein phosphatase-1) interacts with and dephosphorylates YAP2 in vitro and in vivo, and PP1A-mediated dephosphorylation induces the nuclear accumulation and transcriptional activation of YAP2. Inhibition of PP1 by okadiac acid (OA) increases the phosphorylation at serine 127 and cytoplasmic translocation of YAP2 proteins, thereby mitigating its transcription activity. PP1A expression enhances YAP2's pro-survival capability and YAP2 knockdown sensitizes ovarian cancer cells to cisplatin treatment.  Our findings define a novel molecular mechanism that YAP2 is positively regulated by PP1-mediated dephosphorylation in the cell survival.

title: Structural insights into the YAP and TEAD complex.
abstract: The Yes-associated protein (YAP) transcriptional coactivator is a key regulator of organ size and a candidate human oncogene inhibited by the Hippo tumor suppressor pathway. The TEAD family of transcription factors binds directly to and mediates YAP-induced gene expression. Here we report the three-dimensional structure of the YAP (residues 50-171)-TEAD1 (residues 194-411) complex, in which YAP wraps around the globular structure of TEAD1 and forms extensive interactions via three highly conserved interfaces. Interface 3, including YAP residues 86-100, is most critical for complex formation. Our study reveals the biochemical nature of the YAP-TEAD interaction, and provides a basis for pharmacological intervention of YAP-TEAD hyperactivation in human diseases.

title: TEADs Mediate Nuclear Retention of TAZ to Promote Oncogenic Transformation*
abstract: The transcriptional coactivators YAP and TAZ are downstream targets inhibited by the Hippo tumor suppressor pathway. The expression level of TAZ is recently shown to be elevated in invasive breast cancer cells and some primary breast cancers. TAZ is important for breast cancer cell migration, invasion, and tumorigenesis, but the underlying mechanism is not defined. In this study, we show that TAZ interacts with TEAD transcriptional factors. Knockdown of TEADs suppresses TAZ-mediated oncogenic transformation of MCF10A cells. Uncoupling TAZ from Hippo regulation by S89A mutation enhances its transforming ability. Several residues located in the N-terminal region of TAZ are identified to be important for interaction with TEADs, and these same residues are equally important for TAZ to transform MCF10A cells. Mechanistically, TAZ mutants defective in interaction with TEADs fail to accumulate in the nucleus. Live cell imaging of enhanced green fluorescent protein-TAZ and its mutant defective in TEAD interaction suggests that TEAD interaction mediates nuclear retention. These results reveal a novel mechanism for TEADs to regulate nuclear retention and thus the transforming ability of TAZ.

title: Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP.
abstract: The Drosophila TEAD ortholog Scalloped is required for Yki-mediated overgrowth but is largely dispensable for normal tissue growth, suggesting that its mammalian counterpart may be exploited for selective inhibition of oncogenic growth driven by YAP hyperactivation. Here we test this hypothesis genetically and pharmacologically. We show that a dominant-negative TEAD molecule does not perturb normal liver growth but potently suppresses hepatomegaly/tumorigenesis resulting from YAP overexpression or Neurofibromin 2 (NF2)/Merlin inactivation. We further identify verteporfin as a small molecule that inhibits TEAD-YAP association and YAP-induced liver overgrowth. These findings provide proof of principle that inhibiting TEAD-YAP interactions is a pharmacologically viable strategy against the YAP oncoprotein.

title: TEAD/TEF transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein localized in the cytoplasm.
abstract: Mammals express four highly conserved TEAD/TEF transcription factors that bind the same DNA sequence, but serve different functions during development. TEAD-2/TEF-4 protein purified from mouse cells was associated predominantly with a novel TEAD-binding domain at the amino terminus of YAP65, a powerful transcriptional coactivator. YAP65 interacted specifically with the carboxyl terminus of all four TEAD proteins. Both this interaction and sequence-specific DNA binding by TEAD were required for transcriptional activation in mouse cells. Expression of YAP in lymphocytic cells that normally do not support TEAD-dependent transcription (e.g., MPC11) resulted in up to 300-fold induction of TEAD activity. Conversely, TEAD overexpression squelched YAP activity. Therefore, the carboxy-terminal acidic activation domain in YAP is the transcriptional activation domain for TEAD transcription factors. However, whereas TEAD was concentrated in the nucleus, excess YAP65 accumulated in the cytoplasm as a complex with the cytoplasmic localization protein, 14-3-3. Because TEAD-dependent transcription was limited by YAP65, and YAP65 also binds Src/Yes protein tyrosine kinases, we propose that YAP65 regulates TEAD-dependent transcription in response to mitogenic signals.

title: Connective tissue growth factor autocriny in human hepatocellular carcinoma: Oncogenic role and regulation by epidermal growth factor receptor/yes‐associated protein–mediated activation
abstract: The identification of molecular mechanisms involved in the maintenance of the transformed phenotype of hepatocellular carcinoma (HCC) cells is essential for the elucidation of therapeutic strategies. Here, we show that human HCC cells display an autocrine loop mediated by connective tissue growth factor (CTGF) that promotes DNA synthesis and cell survival. Expression of CTGF was stimulated by epidermal growth factor receptor (EGFR) ligands and was dependent on the expression of the transcriptional coactivator, Yes‐associated protein (YAP). We identified elements in the CTGF gene proximal promoter that bound YAP‐enclosing complexes and were responsible for basal and EGFR‐stimulated CTGF expression. We also demonstrate that YAP expression can be up‐regulated through EGFR activation not only in HCC cells, but also in primary human hepatocytes. CTGF contributed to HCC cell dedifferentiation, expression of inflammation‐related genes involved in carcinogenesis, resistance toward doxorubicin, and in vivo HCC cell growth. Importantly, CTGF down‐regulated tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) receptor 2 expression and was involved in the reduced sensitivity of these cells toward TRAIL‐mediated apoptosis.  We have identified autocrine CTGF as a novel determinant of HCC cells' neoplastic behavior. Expression of CTGF can be stimulated through the EGFR‐signaling system in HCC cells in a novel cross‐talk with the oncoprotein YAP. Moreover, to our knowledge, this is the first study that identifies a signaling mechanism triggering YAP gene expression in healthy and transformed liver parenchymal cells. (HEPATOLOGY 2011)

title: The biology of YAP/TAZ: hippo signaling and beyond.
abstract: The transcriptional regulators YAP and TAZ are the focus of intense interest given their remarkable biological properties in development, tissue homeostasis and cancer. YAP and TAZ activity is key for the growth of whole organs, for amplification of tissue-specific progenitor cells during tissue renewal and regeneration, and for cell proliferation. In tumors, YAP/TAZ can reprogram cancer cells into cancer stem cells and incite tumor initiation, progression and metastasis. As such, YAP/TAZ are appealing therapeutic targets in cancer and regenerative medicine. Just like the function of YAP/TAZ offers a molecular entry point into the mysteries of tissue biology, their regulation by upstream cues is equally captivating. YAP/TAZ are well known for being the effectors of the Hippo signaling cascade, and mouse mutants in Hippo pathway components display remarkable phenotypes of organ overgrowth, enhanced stem cell content and reduced cellular differentiation. YAP/TAZ are primary sensors of the cell's physical nature, as defined by cell structure, shape and polarity. YAP/TAZ activation also reflects the cell "social" behavior, including cell adhesion and the mechanical signals that the cell receives from tissue architecture and surrounding extracellular matrix (ECM). At the same time, YAP/TAZ entertain relationships with morphogenetic signals, such as Wnt growth factors, and are also regulated by Rho, GPCRs and mevalonate metabolism. YAP/TAZ thus appear at the centerpiece of a signaling nexus by which cells take control of their behavior according to their own shape, spatial location and growth factor context.

title: Mammalian Tead proteins regulate cell proliferation and contact inhibition as transcriptional mediators of Hippo signaling
abstract: Regulation of organ size is important for development and tissue homeostasis. In Drosophila, Hippo signaling controls organ size by regulating the activity of a TEAD transcription factor, Scalloped, through modulation of its co-activator protein Yki. Here, we show that mouse Tead proteins regulate cell proliferation by mediating Hippo signaling. In NIH3T3 cells, cell density and Hippo signaling regulated the activity of endogenous Tead proteins by modulating nuclear localization of a Yki homolog, Yap1, and the resulting change in Tead activity altered cell proliferation. Tead2-VP16 mimicked Yap1 overexpression, including increased cell proliferation, reduced cell death, promotion of EMT, lack of cell contact inhibition and promotion of tumor formation. Growth-promoting activities of various Yap1 mutants correlated with their Tead-co-activator activities. Tead2-VP16 and Yap1 regulated largely overlapping sets of genes. However, only a few of the Tead/Yap1-regulated genes in NIH3T3 cells were affected in Tead1-/-;Tead2-/- or Yap1-/- embryos. Most of the previously identified Yap1-regulated genes were not affected in NIH3T3 cells or mutant mice. In embryos, levels of nuclear Yap1 and Tead1 varied depending on cell type. Strong nuclear accumulation of Yap1 and Tead1 were seen in myocardium, correlating with requirements of Tead1 for proliferation. However, their distribution did not always correlate with proliferation. Taken together, mammalian Tead proteins regulate cell proliferation and contact inhibition as a transcriptional mediator of Hippo signaling, but the mechanisms by which Tead/Yap1 regulate cell proliferation differ depending on the cell type, and Tead, Yap1 and Hippo signaling may play multiple roles in mouse embryos.

title: Rb inactivation accelerates neoplastic growth and substitutes for recurrent amplification of cIAP1, cIAP2 and Yap1 in sporadic mammary carcinoma associated with p53 deficiency
abstract: Genetically defined mouse models offer an important tool to identify critical secondary genetic alterations with relevance to human cancer pathogenesis. We used newly generated MMTV-Cre105Ayn mice to inactivate p53 and/or Rb strictly in the mammary epithelium, and to determine recurrent genomic changes associated with deficiencies of these genes. p53 inactivation led to formation of estrogen receptor-positive raloxifene-responsive mammary carcinomas with features of luminal subtype B. Rb deficiency was insufficient to initiate carcinogenesis but promoted genomic instability and growth rate of neoplasms associated with p53 inactivation. Genome-wide analysis of mammary carcinomas identified a recurrent amplification at chromosome band 9A1, a locus orthologous to human 11q22, which contains protooncogenes cIAP1 (Birc2), cIAP2 (Birc3) and Yap1. It is interesting that this amplicon was preferentially detected in carcinomas carrying wild-type Rb. However, all three genes were overexpressed in carcinomas with p53 and Rb inactivation, likely due to E2F-mediated transactivation, and cooperated in carcinogenesis according to gene knockdown experiments. These findings establish a model of luminal subtype B mammary carcinoma, identify critical role of cIAP1, cIAP2 and Yap1 co-expression in mammary carcinogenesis and provide an explanation for the lack of recurrent amplifications of cIAP1, cIAP2 and Yap1 in some tumors with frequent Rb deficiency, such as mammary carcinoma.

title: Molecular mechanisms underlying RB protein function
abstract: Inactivation of the RB protein is one of the most fundamental events in cancer. Coming to a molecular understanding of its function in normal cells and how it impedes cancer development has been challenging. Historically, the ability of RB to regulate the cell cycle placed it in a central role in proliferative control, and research focused on RB regulation of the E2F family of transcription factors. Remarkably, several recent studies have found additional tumour-suppressor functions of RB, including alternative roles in the cell cycle, maintenance of genome stability and apoptosis. These advances and new structural studies are combining to define the multifunctionality of RB.

title: The retinoblastoma protein and cell cycle control
abstract: pRB, the product of the retinoblastoma tumor suppressor gene, operates in the midst of the cell cycle clock apparatus. Its main role is to act as a signal transducer connecting the cell cycle clock with the transcriptional machinery. In this role, pRB allows the clock to control the expression of banks of genes that mediate advance of the cell through a critical phase of its growth cycle. Loss of pRB function deprives the clock and thus the cell of an important mechanism for braking cell proliferation through modulation of gene expression.

title: Cellular mechanisms of tumour suppression by the retinoblastoma gene
abstract: The retinoblastoma (RB) tumour suppressor gene is functionally inactivated in a broad range of paediatric and adult cancers, and a plethora of cellular functions and partners have been identified for the RB protein. Data from human tumours and studies from mouse models indicate that loss of RB function contributes to both cancer initiation and progression. However, we still do not know the identity of the cell types in which RB normally prevents cancer initiation in vivo, and the specific functions of RB that suppress distinct aspects of the tumorigenic process are poorly understood.

title: Newly identified aspects of tumor suppression by RB
abstract: The retinoblastoma (RB) tumor suppressor belongs to a cellular pathway that plays a crucial role in restricting the G1-S transition of the cell cycle in response to a large number of extracellular and intracellular cues. Research in the last decade has highlighted the complexity of regulatory networks that ensure proper cell cycle progression, and has also identified multiple cellular functions beyond cell cycle regulation for RB and its two family members, p107 and p130. Here we review some of the recent evidence pointing to a role of RB as a molecular adaptor at the crossroads of multiple pathways, ensuring cellular homeostasis in different contexts. In particular, we discuss the pro- and anti-tumorigenic roles of RB during the early stages of cancer, as well as the importance of the RB pathway in stem cells and cell fate decisions.

title: The retinoblastoma tumor suppressor and stem cell biology.
abstract: Stem cells play a critical role during embryonic development and in the maintenance of homeostasis in adult individuals. A better understanding of stem cell biology, including embryonic and adult stem cells, will allow the scientific community to better comprehend a number of pathologies and possibly design novel approaches to treat patients with a variety of diseases. The retinoblastoma tumor suppressor RB controls the proliferation, differentiation, and survival of cells, and accumulating evidence points to a central role for RB activity in the biology of stem and progenitor cells. In some contexts, loss of RB function in stem or progenitor cells is a key event in the initiation of cancer and determines the subtype of cancer arising from these pluripotent cells by altering their fate. In other cases, RB inactivation is often not sufficient to initiate cancer but may still lead to some stem cell expansion, raising the possibility that strategies aimed at transiently inactivating RB might provide a novel way to expand functional stem cell populations. Future experiments dedicated to better understanding how RB and the RB pathway control a stem cell's decisions to divide, self-renew, or give rise to differentiated progeny may eventually increase our capacity to control these decisions to enhance regeneration or help prevent cancer development.

title: Aberrant Regulation of Survivin by the RB/E2F Family of Proteins*
abstract: Survivin is a putative oncogene that is aberrantly expressed in cancer cells. It has been hypothesized to play a central role in cancer progression and resistance to therapy in diverse tumor types. Although some of the transcriptional processes regulating its expression have been established, the diversity of genes that may be controlling the levels of its expression in both normal cells as well as in cancer cells has not been fully explored. The most common genetically mutated pathways in human malignancies are the p53 tumor suppressor pathway and the RB/E2F pathway. Both of these pathways, when intact, provide essential checkpoints in the maintenance of normal cell growth and protect the cell from DNA damage. Using non-transformed embryonic fibroblasts, we provide evidence of a molecular link between the regulation of survivin transcription and the RB/E2F family of proteins. We demonstrate that both pRB and p130 can interact with the survivin promoter and can repress survivin transcription. We also show that the E2F activators (E2F1, E2F2, and E2F3) can bind to the survivin promoter and induce survivin transcription. Genetically modified cells that harbor deletions in various members of the RB/E2F family confirm our data from the wild-type cells. Our findings implicate several members of the RB/E2F pathway in an intricate mechanism of survivin gene regulation that, when genetically altered during the process of tumorigenesis, may function within cancer cells to aberrantly alter survivin levels and enhance tumor progression.

title: Organ size control is dominant over Rb family inactivation to restrict proliferation in vivo.
abstract: In mammals, a cell's decision to divide is thought to be under the control of the Rb/E2F pathway. We previously found that inactivation of the Rb family of cell cycle inhibitors (Rb, p107, and p130) in quiescent liver progenitors leads to uncontrolled division and cancer initiation. Here, we show that, in contrast, deletion of the entire Rb gene family in mature hepatocytes is not sufficient for their long-term proliferation. The cell cycle block in Rb family mutant hepatocytes is independent of the Arf/p53/p21 checkpoint but can be abrogated upon decreasing liver size. At the molecular level, we identify YAP, a transcriptional regulator involved in organ size control, as a factor required for the sustained expression of cell cycle genes in hepatocytes. These experiments identify a higher level of regulation of the cell cycle in vivo in which signals regulating organ size are dominant regulators of the core cell cycle machinery.

title: A YAP/TAZ-Regulated Molecular Signature Is Associated with Oral Squamous Cell Carcinoma
abstract: Oral squamous cell carcinoma (OSCC) is a prevalent form of cancer that develops from the epithelium of the oral cavity. OSCC is on the rise worldwide, and death rates associated with the disease are particularly high. Despite progress in understanding the mutational and expression landscape associated with OSCC, advances in deciphering these alterations for the development of therapeutic strategies have been limited. Further insight into the molecular cues that contribute to OSCC is therefore required. Here, we show that the transcriptional regulators YAP (YAP1) and TAZ (WWTR1), which are key effectors of the Hippo pathway, drive protumorigenic signals in OSCC. Regions of premalignant oral tissues exhibit aberrant nuclear YAP accumulation, suggesting that dysregulated YAP activity contributes to the onset of OSCC. Supporting this premise, we determined that nuclear YAP and TAZ activity drives OSCC cell proliferation, survival, and migration in vitro, and is required for OSCC tumor growth and metastasis in vivo. Global gene expression profiles associated with YAP and TAZ knockdown revealed changes in the control of gene expression implicated in protumorigenic signaling, including those required for cell cycle progression and survival. Notably, the transcriptional signature regulated by YAP and TAZ significantly correlates with gene expression changes occurring in human OSCCs identified by The Cancer Genome Atlas (TCGA), emphasizing a central role for YAP and TAZ in OSCC biology.  This study defines a YAP/TAZ-regulated transcriptional program in OSCC and reveals novel roles for nuclear YAP/TAZ activity in the onset and progression of this cancer.

title: Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth
abstract: YAP/TAZ are nuclear effectors of the Hippo pathway regulating organ growth and tumorigenesis. Yet, their function as transcriptional regulators remains underinvestigated. By ChIP-seq analyses in breast cancer cells, we discovered that the YAP/TAZ transcriptional response is pervasively mediated by a dual element: TEAD factors, through which YAP/TAZ bind to DNA, co-occupying chromatin with activator protein-1 (AP-1, dimer of JUN and FOS proteins) at composite cis-regulatory elements harbouring both TEAD and AP-1 motifs. YAP/TAZ/TEAD and AP-1 form a complex that synergistically activates target genes directly involved in the control of S-phase entry and mitosis. This control occurs almost exclusively from distal enhancers that contact target promoters through chromatin looping. YAP/TAZ-induced oncogenic growth is strongly enhanced by gain of AP-1 and severely blunted by its loss. Conversely, AP-1-promoted skin tumorigenesis is prevented in YAP/TAZ conditional knockout mice. This work highlights a new layer of signalling integration, feeding on YAP/TAZ function at the chromatin level.

title: YAP Regulates S-Phase Entry in Endothelial Cells
abstract: The Hippo pathway regulates cell proliferation and apoptosis through the Yes-associated protein (YAP) transcriptional activator. YAP has a well-described role in promoting cell proliferation and survival, but the precise mechanisms and transcriptional targets that underlie these properties are still unclear and likely context-dependent. We found, using siRNA-mediated knockdown, that YAP is required for proliferation in endothelial cells but not HeLa cells. Specifically, YAP is required for S-phase entry and its absence causes cells to accumulate in G1. Microarray analysis suggests that YAP mediates this effect by regulating the transcription of genes involved in the assembly and/or firing of replication origins and homologous recombination of DNA. These findings thus provide insight into the molecular mechanisms by which YAP regulates cell cycle progression.

title: Cell cycle regulation of the E2F transcription factor involves an interaction with cyclin A
abstract: We have examined E2F binding activity in extracts of synchronized NIH 3T3 cells. During the G0 to G1 transition, there is a marked increase in the level of active E2F. Subsequently, there are changes in the nature of E2F-containing complexes. A G1-specific complex increases in abundance, disappears, and is then replaced by another complex as S phase begins. Analysis of extracts of thymidine-blocked cells confirms that the complexes are cell cycle regulated. We also show that the cyclin A protein is a component of the S phase complex. Each complex can be dissociated by the adenovirus E1A 12S product, releasing free E2F. The release of E2F from the cyclin A complex coincides with the stimulation of an E2F-dependent promoter. We suggest that these interactions control the activity of E2F and that disruption of the complexes by E1A contributes to a loss of cellular proliferation control.

title: Hippo signalling in the G2/M cell cycle phase: lessons learned from the yeast MEN and SIN pathways.
abstract: Over the past decade Hippo kinase signalling has been established as an essential tumour suppressor pathway controlling tissue growth in flies and mammals. All members of the Hippo core signalling cassette are conserved from yeast to humans, whereby the yeast analogues of Hippo, Mats and Lats are central components of the mitotic exit network and septation initiation network in budding and fission yeast, respectively. Here, we discuss how far core Hippo signalling components in Drosophila melanogaster and mammals have reported similar mitotic functions as already established for their highly conserved yeast counterparts.

title: Lats2 Is an Essential Mitotic Regulator Required for the Coordination of Cell Division*
abstract: Tumor suppressor Lats2 is a member of the conserved Dbf2 kinase family. It localizes to the centrosome and has been implicated in regulation of the cell cycle and apoptosis. However, the in vivo function of this kinase remains unclear. Here, we show that complete disruption of the gene encoding Lats2 in mice causes developmental defects in the nervous system and embryonic lethality. Furthermore, mutant cells derived from total LATS2-knock-out embryos exhibit mitotic defects including centrosome fragmentation and cytokinesis defects, followed by nuclear enlargement and multinucleation. We show that the Mob1 family, a regulator of mitotic exit, associates with Lats2 to induce its activation. We also show that the complete LATS2-knock-out cells exhibit an acceleration of exit from mitosis and marked down-regulation of critical mitotic regulators. These results suggest that Lats2 plays an essential mitotic role in coordinating accurate cytokinesis completion, governing the stabilization of other mitotic regulators.

title: A kinase shRNA screen links LATS2 and the pRB tumor suppressor.
abstract: pRB-mediated inhibition of cell proliferation is a complex process that depends on the action of many proteins. However, little is known about the specific pathways that cooperate with the Retinoblastoma protein (pRB) and the variables that influence pRB's ability to arrest tumor cells. Here we describe two shRNA screens that identify kinases that are important for pRB to suppress cell proliferation and pRB-mediated induction of senescence markers. The results reveal an unexpected effect of LATS2, a component of the Hippo pathway, on pRB-induced phenotypes. Partial knockdown of LATS2 strongly suppresses some pRB-induced senescence markers. Further analysis shows that LATS2 cooperates with pRB to promote the silencing of E2F target genes, and that reduced levels of LATS2 lead to defects in the assembly of DREAM (DP, RB [retinoblastoma], E2F, and MuvB) repressor complexes at E2F-regulated promoters. Kinase assays show that LATS2 can phosphorylate DYRK1A, and that it enhances the ability of DYRK1A to phosphorylate the DREAM subunit LIN52. Intriguingly, the LATS2 locus is physically linked with RB1 on 13q, and this region frequently displays loss of heterozygosity in human cancers. Our results reveal a functional connection between the pRB and Hippo tumor suppressor pathways, and suggest that low levels of LATS2 may undermine the ability of pRB to induce a permanent cell cycle arrest in tumor cells.

title: TAZ induces growth factor-independent proliferation through activation of EGFR ligand amphiregulin
abstract: The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. We previously showed that the pivotal effector of this pathway, YAP, is amplified in tumors and promotes epithelial-to-mesenchymal transition (EMT) and malignant transformation. Here, we report that overexpression of TAZ, a paralog of YAP, in human mammary epithelial cells promotes EMT and, in particular, some invasive structures in 3D cultures. TAZ also leads to cell migration and anchorage-independent growth in soft agar. Furthermore, we identified amphiregulin (AREG), an epidermal growth factor receptor (EGFR) ligand, as a target of TAZ. We show that AREG functions in a non-cell-autonomous manner to mediate EGF-independent growth and malignant behavior of mammary epithelial cells. In addition, ablation of TEAD binding completely abolishes the TAZ-induced phenotype. Last, analysis of breast cancer patient samples reveals a positive correlation between TAZ and AREG in vivo. In summary, TAZ-dependent secretion of AREG indicates that activation of the EGFR signaling is an important non-cell-autonomous effector of the Hippo pathway, and TAZ as well as its targets may play significant roles in breast tumorigenesis and metastasis.

title: Nuclear CDKs Drive Smad Transcriptional Activation and Turnover in BMP and TGF-β Pathways
abstract: TGF-beta 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-beta pathways.

title: KRAS and YAP1 Converge to Regulate EMT and Tumor Survival
abstract: Cancer cells that express oncogenic alleles of RAS typically require sustained expression of the mutant allele for survival, but the molecular basis of this oncogene dependency remains incompletely understood. To identify genes that can functionally substitute for oncogenic RAS, we systematically expressed 15,294 open reading frames in a human KRAS-dependent colon cancer cell line engineered to express an inducible KRAS-specific shRNA. We found 147 genes that promoted survival upon KRAS suppression. In particular, the transcriptional coactivator YAP1 rescued cell viability in KRAS-dependent cells upon suppression of KRAS and was required for KRAS-induced cell transformation. Acquired resistance to Kras suppression in a Kras-driven murine lung cancer model also involved increased YAP1 signaling. KRAS and YAP1 converge on the transcription factor FOS and activate a transcriptional program involved in regulating the epithelial-mesenchymal transition (EMT). Together, these findings implicate transcriptional regulation of EMT by YAP1 as a significant component of oncogenic RAS signaling.

title: The transcriptional coactivator Yes-associated protein drives p73 gene-target specificity in response to DNA Damage.
abstract: The transcriptional coactivator Yes-associated protein (YAP) has been shown to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Here, we show that YAP requires the promyelocytic leukemia gene (PML) and nuclear body localization to coactivate p73. YAP imparts selectivity to p73 by promoting the activation of a subset of p53 and/or p73 target promoters. Endogenous p73, YAP, and p300 proteins are concomitantly recruited onto the regulatory regions of the apoptotic target gene p53AIP1 only when cells are exposed to apoptotic conditions. Silencing of YAP by specific siRNA impairs p300 recruitment and reduces histone acetylation on the p53AIP1 target gene, resulting in delayed or reduced apoptosis mediated by p73. We also found that YAP contributes to the DNA damage-induced accumulation of p73 and potentiates the p300-mediated acetylation of p73. Altogether, our findings identify YAP as a key determinant of p73 gene targeting in response to DNA damage.

title: RESCUE OF HIPPO CO-ACTIVATOR YAP1 TRIGGERS DNA DAMAGE-INDUCED APOPTOSIS IN HEMATOLOGICAL CANCERS
abstract: Oncogene-induced DNA damage elicits genomic instability in epithelial cancer cells, but apoptosis is blocked through inactivation of the tumor suppressor p53. In hematological cancers, the relevance of ongoing DNA damage and the mechanisms by which apoptosis is suppressed are largely unknown. We found pervasive DNA damage in hematologic malignancies, including multiple myeloma, lymphoma and leukemia, which leads to activation of a p53-independent, proapoptotic network centered on nuclear relocalization of ABL1 kinase. Although nuclear ABL1 triggers cell death through its interaction with the Hippo pathway coactivator YAP1 in normal cells, we show that low YAP1 levels prevent nuclear ABL1-induced apoptosis in these hematologic malignancies. YAP1 is under the control of a serine-threonine kinase, STK4. Notably, genetic inactivation of STK4 restores YAP1 levels, triggering cell death in vitro and in vivo. Our data therefore identify a new synthetic-lethal strategy to selectively target cancer cells presenting with endogenous DNA damage and low YAP1 levels.

title: The Hippo signaling pathway in stem cell biology and cancer
abstract: The Hippo signaling pathway, consisting of a highly conserved kinase cascade (MST and Lats) and downstream transcription coactivators (YAP and TAZ), plays a key role in tissue homeostasis and organ size control by regulating tissue‐specific stem cells. Moreover, this pathway plays a prominent role in tissue repair and regeneration. Dysregulation of the Hippo pathway is associated with cancer development. Recent studies have revealed a complex network of upstream inputs, including cell density, mechanical sensation, and G‐protein‐coupled receptor (GPCR) signaling, that modulate Hippo pathway activity. This review focuses on the role of the Hippo pathway in stem cell biology and its potential implications in tissue homeostasis and cancer.

title: TAZ, a Transcriptional Modulator of Mesenchymal Stem Cell Differentiation
abstract: Mesenchymal stem cells (MSCs) are a pluripotent cell type that can differentiate into several distinct lineages. Two key transcription factors, Runx2 and peroxisome proliferator–activated receptor γ (PPARγ), drive MSCs to differentiate into either osteoblasts or adipocytes, respectively. How these two transcription factors are regulated in order to specify these alternate cell fates remains a pivotal question. Here we report that a 14-3-3–binding protein, TAZ (transcriptional coactivator with PDZ-binding motif), coactivates Runx2-dependent gene transcription while repressing PPARγ-dependent gene transcription. By modulating TAZ expression in model cell lines, mouse embryonic fibroblasts, and primary MSCs in culture and in zebrafish in vivo, we observed alterations in osteogenic versus adipogenic potential. These results indicate that TAZ functions as a molecular rheostat that modulates MSC differentiation.

title: Rb regulates fate choice and lineage commitment in vivo
abstract: Mutation of the retinoblastoma gene (RB1) tumour suppressor occurs in one-third of all human tumours and is particularly associated with retinoblastoma and osteosarcoma. Numerous functions have been ascribed to the product of the human RB1 gene, the retinoblastoma protein (pRb). The best known is pRb’s ability to promote cell-cycle exit through inhibition of the E2F transcription factors and the transcriptional repression of genes encoding cell-cycle regulators. In addition, pRb has been shown in vitro to regulate several transcription factors that are master differentiation inducers. Depending on the differentiation factor and cellular context, pRb can either suppress or promote their transcriptional activity. For example, pRb binds to Runx2 and potentiates its ability to promote osteogenic differentiation in vitro. In contrast, pRb acts with E2F to suppress peroxisome proliferator-activated receptor γ subunit (PPAR-γ), the master activator of adipogenesis. Because osteoblasts and adipocytes can both arise from mesenchymal stem cells, these observations suggest that pRb might play a role in the choice between these two fates. However, so far, there is no evidence for this in vivo. Here we use mouse models to address this hypothesis in mesenchymal tissue development and tumorigenesis. Our data show that Rb status plays a key role in establishing fate choice between bone and brown adipose tissue in vivo.

title: Pax3 and Hippo Signaling Coordinate Melanocyte Gene Expression in Neural Crest.
abstract: Loss of Pax3, a developmentally regulated transcription factor expressed in pre-migratory neural crest, results in severe developmental defects and embryonic lethality. Although Pax3 mutations produce profound phenotypes, the intrinsic transcriptional activation exhibited by Pax3 is surprisingly modest. We postulated the existence of transcriptional co-activators that function with Pax3 to mediate developmental functions. A high-throughput screen identified the Hippo effector proteins Taz and Yap65 as Pax3 co-activators. Synergistic co-activation of target genes by Pax3Taz/Yap65 requires DNA binding by Pax3, is Tead-independent, and is regulated by Hippo kinases Mst1 and Lats2. In vivo, Pax3 and Yap65 co-localize in the nucleus of neural crest progenitors in the dorsal neural tube. Neural crest deletion of Taz and Yap65 results in embryonic lethal neural crest defects and decreased expression of the Pax3 target gene, Mitf. These results suggest that Pax3 activity is regulated by the Hippo pathway and that Pax factors are Hippo effectors.

title: A WW domain protein TAZ is a critical coactivator for TBX5, a transcription factor implicated in Holt-Oram syndrome.
abstract: The T-box transcription factor TBX5 plays essential roles in cardiac and limb development. Various mutations in the TBX5 gene have been identified in patients with Holt-Oram syndrome, which is characterized by congenital defects in the heart and upper extremities. In this study, we identified a WW-domain-containing transcriptional regulator TAZ as a potent TBX5 coactivator. TAZ directly associates with TBX5 and markedly stimulates TBX5-dependent promoters by interacting with the histone acetyltransferases p300 and PCAF. YAP, a TAZ-related protein with conserved functional domains, also stimulates TBX5-dependent transcription, possibly by forming a heterodimer with TAZ. TBX5 lacks a PY motif, which mediates the association of other proteins with TAZ, and interacts with TAZ through multiple domains including its carboxyl-terminal structure. Truncation mutants of TBX5 identified in patients with Holt-Oram syndrome were markedly impaired in their ability to associate with and be stimulated by TAZ. These findings reveal key roles for TAZ and YAP in the control of TBX5-dependent transcription and suggest the involvement of these coactivators in cardiac and limb development.

title: Hippo Signaling Influences HNF4A and FOXA2 Enhancer Switching during Hepatocyte Differentiation
abstract: Cell fate acquisition is heavily influenced by direct interactions between master regulators and tissue-specific enhancers. However, it remains unclear how lineage-specifying transcription factors, which are often expressed in both progenitor and mature cell populations, influence cell differentiation. Using in vivo mouse liver development as a model, we identified thousands of enhancers that are bound by the master regulators HNF4A and FOXA2 in a differentiation-dependent manner, subject to chromatin remodeling, and associated with differentially expressed target genes. Enhancers exclusively occupied in the embryo were found to be responsive to developmentally regulated TEAD2 and coactivator YAP1. Our data suggest that Hippo signaling may affect hepatocyte differentiation by influencing HNF4A and FOXA2 interactions with temporal enhancers. In summary, transcription factor-enhancer interactions are not only tissue specific but also differentiation dependent, which is an important consideration for researchers studying cancer biology or mammalian development and/or using transformed cell lines.

title: Downstream of Mutant KRAS, the Transcription Regulator YAP Is Essential for Neoplastic Progression to Pancreatic Ductal Adenocarcinoma
abstract: Without the transcription regulator YAP, KRAS-mutant pancreatic neoplasia cannot progress into invasive cancer. Targeting YAP to Promote the Antitumor Response Pancreatic ductal adenocarcinoma is frequently associated with mutations in KRAS, but targeting RAS proteins is clinically challenging. From studies with genetically engineered mouse models and correlations in human samples, Zhang et al. determined that the protein YAP mediated the transition from pancreatic metaplasia to invasive adenocarcinoma by transcriptionally activating genes induced by KRAS signaling. Deleting Yap in Kras or Kras:Trp53 mutant mouse pancreatic epithelia prevented their proliferation, decreased the expression and secretion of KRAS-regulated proteins that mediate inflammation and migration, and promoted immune cell infiltration in the tumor microenvironment in mice. Because Yap deletion did not affect normal pancreatic development or function, the findings indicate that Yap may be a viable target for KRAS-mutant PDAC. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates and frequently carries oncogenic KRAS mutation. However, KRAS has thus far not been a viable therapeutic target. We found that the abundance of YAP mRNA, which encodes Yes-associated protein (YAP), a protein regulated by the Hippo pathway during tissue development and homeostasis, was increased in human PDAC tissue compared with that in normal pancreatic epithelia. In genetically engineered KrasG12D and KrasG12D:Trp53R172H mouse models, pancreas-specific deletion of Yap halted the progression of early neoplastic lesions to PDAC without affecting normal pancreatic development and endocrine function. Although Yap was dispensable for acinar to ductal metaplasia (ADM), an initial step in the progression to PDAC, Yap was critically required for the proliferation of mutant Kras or Kras:Trp53 neoplastic pancreatic ductal cells in culture and for their growth and progression to invasive PDAC in mice. Yap functioned as a critical transcriptional switch downstream of the oncogenic KRAS–mitogen-activated protein kinase (MAPK) pathway, promoting the expression of genes encoding secretory factors that cumulatively sustained neoplastic proliferation, a tumorigenic stromal response in the tumor microenvironment, and PDAC progression in Kras and Kras:Trp53 mutant pancreas tissue. Together, our findings identified Yap as a critical oncogenic KRAS effector and a promising therapeutic target for PDAC and possibly other types of KRAS-mutant cancers.

title: The Hippo effector YAP promotes resistance to RAF- and MEK-targeted cancer therapies
abstract: Resistance to RAF- and MEK-targeted therapy is a major clinical challenge. RAF and MEK inhibitors are initially but only transiently effective in some but not all patients with BRAF gene mutation and are largely ineffective in those with RAS gene mutation because of resistance. Through a genetic screen in BRAF-mutant tumor cells, we show that the Hippo pathway effector YAP (encoded by YAP1) acts as a parallel survival input to promote resistance to RAF and MEK inhibitor therapy. Combined YAP and RAF or MEK inhibition was synthetically lethal not only in several BRAF-mutant tumor types but also in RAS-mutant tumors. Increased YAP in tumors harboring BRAF V600E was a biomarker of worse initial response to RAF and MEK inhibition in patients, establishing the clinical relevance of our findings. Our data identify YAP as a new mechanism of resistance to RAF- and MEK-targeted therapy. The findings unveil the synthetic lethality of combined suppression of YAP and RAF or MEK as a promising strategy to enhance treatment response and patient survival.

title: The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain
abstract: The transcriptional coactivator Yes-associated protein (YAP) is a major regulator of organ size and proliferation in vertebrates. As such, YAP can act as an oncogene in several tissue types if its activity is increased aberrantly. Although no activating mutations in the yap1 gene have been identified in human cancer, yap1 is located on the 11q22 amplicon, which is amplified in several human tumors. In addition, mutations or epigenetic silencing of members of the Hippo pathway, which represses YAP function, have been identified in human cancers. Here we demonstrate that, in addition to increasing tumor growth, increased YAP activity is potently prometastatic in breast cancer and melanoma cells. Using a Luminex-based approach to multiplex in vivo assays, we determined that the domain of YAP that interacts with the TEAD/TEF family of transcription factors but not the WW domains or PDZ-binding motif, is essential for YAP-mediated tumor growth and metastasis. We further demonstrate that, through its TEAD-interaction domain, YAP enhances multiple processes known to be important for tumor progression and metastasis, including cellular proliferation, transformation, migration, and invasion. Finally, we found that the metastatic potential of breast cancer and melanoma cells is strongly correlated with increased TEAD transcriptional activity. Together, our results suggest that increased YAP/TEAD activity plays a causal role in cancer progression and metastasis.

title: Targeting Hippo pathway by specific interruption of YAP‐TEAD interaction using cyclic YAP‐like peptides
abstract: Hippo signaling pathway is emerging as a novel target for anticancer therapy because it plays key roles in organ size control and tumorigenesis. As the downstream effectors, Yes‐associated protein (YAP)‐transcriptional enhancer activation domain family member (TEAD) association is essential for YAP‐driven oncogenic activity, while TEAD is largely dispensable for normal tissue growth. We present the design of YAP‐like peptides (17mer) to occupy the interface 3 on TEAD. Introducing cysteines at YAP sites 87 and 96 can induce disulfide formation, as confirmed by crystallography. The engineered peptide significantly improves the potency in disrupting YAP‐TEAD interaction in vitro. To confirm that blocking YAP‐TEAD complex formation by directly targeting on TEAD is a valid approach, we report a significant reduction in tumor growth rate in a hepatocellular carcinoma xenograft model after introducing the dominant‐negative mutation (Y406H) of TEAD1 to abolish YAP‐TEAD interaction. Our results suggest that targeting TEAD is a promising strategy against YAP‐induced oncogenesis.—Zhou, Z., Hu, T., Xu, Z., Lin, Z., Zhang, Z., Feng, T., Zhu, L., Rong, Y., Shen, H., Luk, J. M., Zhang, X., Qin, N. Targeting Hippo pathway by specific interruption of YAP‐TEAD interaction using cyclic YAP‐like peptides. FASEB J. 29, 724‐732 (2015). www.fasebj.org

title: VGLL4 functions as a new tumor suppressor in lung cancer by negatively regulating the YAP-TEAD transcriptional complex
abstract: Lung cancer is one of the most devastating diseases worldwide with high incidence and mortality. Hippo (Hpo) pathway is a conserved regulator of organ size in both Drosophila and mammals. Emerging evidence has suggested the significance of Hpo pathway in cancer development. In this study, we identify VGLL4 as a novel tumor suppressor in lung carcinogenesis through negatively regulating the formation of YAP-TEAD complex, the core component of Hpo pathway. Our data show that VGLL4 is frequently observed to be lowly expressed in both mouse and human lung cancer specimens. Ectopic expression of VGLL4 significantly suppresses the growth of lung cancer cells in vitro. More importantly, VGLL4 significantly inhibits lung cancer progression in de novo mouse model. We further find that VGLL4 inhibits the activity of the YAP-TEAD transcriptional complex. Our data show that VGLL4 directly competes with YAP in binding to TEADs and executes its growth-inhibitory function through two TDU domains. Collectively, our study demonstrates that VGLL4 is a novel tumor suppressor for lung cancer through negatively regulating the YAP-TEAD complex formation and thus the Hpo pathway.

title: A peptide mimicking VGLL4 function acts as a YAP antagonist therapy against gastric cancer.
abstract: The Hippo pathway has been implicated in suppressing tissue overgrowth and tumor formation by restricting the oncogenic activity of YAP. However, transcriptional regulators that inhibit YAP activity have not been well studied. Here, we uncover clinical importance for VGLL4 in gastric cancer suppression and find that VGLL4 directly competes with YAP for binding TEADs. Importantly, VGLL4's tandem Tondu domains are not only essential but also sufficient for its inhibitory activity toward YAP. A peptide mimicking this function of VGLL4 potently suppressed tumor growth in vitro and in vivo. These findings suggest that disruption of YAP-TEADs interaction by a VGLL4-mimicking peptide may be a promising therapeutic strategy against YAP-driven human cancers.

title: YAP Inhibition Restores Hepatocyte Differentiation in Advanced HCC, Leading to Tumor Regression.
abstract: Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC). Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat in maintaining metabolic specialization, differentiation, and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs that control hepatocyte quiescence and differentiation. Importantly, treatment with small interfering RNA-lipid nanoparticles (siRNA-LNPs) targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of the positional identity of hepatocytes, supports targeting of YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype that is potentially responsive to this approach.

title: Yes-associated protein (YAP) functions as a tumor suppressor in breast
abstract: Yes-associated protein (YAP) has been shown to positively regulate p53 family members and to be negatively regulated by the AKT proto-oncogene product in promoting apoptosis. On the basis of this function and its location at 11q22.2, a site of frequent loss of heterozygosity (LOH) in breast cancer, we investigated whether YAP is a tumor suppressor in breast. Examination of tumors by immunohistochemistry demonstrated significant loss of YAP protein. LOH analysis revealed that protein loss correlates with specific deletion of the YAP gene locus. Functionally, short hairpin RNA knockdown of YAP in breast cell lines suppressed anoikis, increased migration and invasiveness, inhibited the response to taxol and enhanced tumor growth in nude mice. This is the first report indicating YAP as a tumor suppressor, revealing its decreased expression in breast cancer as well as demonstrating the functional implications of YAP loss in several aspects of cancer signaling.

title: A temporal requirement for Hippo signaling in mammary gland differentiation, growth, and tumorigenesis.
abstract: Despite recent progress, the physiological role of Hippo signaling in mammary gland development and tumorigenesis remains poorly understood. Here we show that the Hippo pathway is functionally dispensable in virgin mammary glands but specifically required during pregnancy. In contrast to many other tissues, hyperactivation of YAP in mammary epithelia does not induce hyperplasia but leads to defects in terminal differentiation. Interestingly, loss of YAP causes no obvious defects in virgin mammary glands but potently suppresses oncogene-induced mammary tumors. The selective requirement for YAP in oncogenic growth highlights the potential of YAP inhibitors as molecular targeted therapies against breast cancers.

title: YAP1, the nuclear target of Hippo signaling, stimulates heart growth through cardiomyocyte proliferation but not hypertrophy
abstract: Heart growth is tightly controlled so that the heart reaches a predetermined size. Fetal heart growth occurs through cardiomyocyte proliferation, whereas postnatal heart growth involves primarily physiological cardiomyocyte hypertrophy. The Hippo kinase cascade is an important regulator of organ growth. A major target of this kinase cascade is YAP1, a transcriptional coactivator that is inactivated by Hippo kinase activity. Here, we used both genetic gain and loss of Yap1 function to investigate its role in regulating proliferative and physiologic hypertrophic heart growth. Fetal Yap1 inactivation caused marked, lethal myocardial hypoplasia and decreased cardiomyocyte proliferation, whereas fetal activation of YAP1 stimulated cardiomyocyte proliferation. Enhanced proliferation was particularly dramatic in trabecular cardiomyocytes that normally exit from the cell cycle. Remarkably, YAP1 activation was sufficient to stimulate proliferation of postnatal cardiomyocytes, both in culture and in the intact heart. A dominant negative peptide that blocked YAP1 binding to TEAD transcription factors inhibited YAP1 proliferative activity, indicating that this activity requires YAP1–TEAD interaction. Although Yap1 was a critical regulator of cardiomyocyte proliferation, it did not influence physiological hypertrophic growth of cardiomyocytes, because postnatal Yap1 gain or loss of function did not significantly alter cardiomyocyte size. These studies demonstrate that Yap1 is a crucial regulator of cardiomyocyte proliferation, cardiac morphogenesis, and myocardial trabeculation. Activation of Yap1 in postnatal cardiomyocytes may be a useful strategy to stimulate cardiomyocyte expansion in therapeutic myocardial regeneration.

title: Dynamic alterations in Hippo signaling pathway and YAP activation during liver regeneration.
abstract: The Hippo signaling pathway has been implicated in mammalian organ size regulation and tumor suppression. Specifically, the Hippo pathway plays a critical role regulating the activity of transcriptional coactivator Yes-associated protein (YAP), which modulates a proliferative transcriptional program. Recent investigations have demonstrated that while this pathway is activated in quiescent livers, its inhibition leads to liver overgrowth and tumorigenesis. However, the role of the Hippo pathway during the natural process of liver regeneration remains unknown. Here we investigated alterations in the Hippo signaling pathway and YAP activation during liver regeneration using a 70% partial hepatectomy (PH) rat model. Our results indicate an increase in YAP activation by 1 day following PH as demonstrated by increased YAP nuclear localization and increased YAP target gene expression. Investigation of the Hippo pathway revealed a decrease in the activation of core kinases Mst1/2 by 1 day as well as Lats1/2 and its adapter protein Mob1 by 3 days following PH. Evaluation of liver-to-body weight ratios indicated that the liver reaches its near normal size by 7 days following PH, which correlated with a return to baseline YAP nuclear levels and target gene expression. Additionally, when liver size was restored, Mst1/2 kinase activation returned to levels observed in quiescent livers indicating reactivation of the Hippo signaling pathway. These findings illustrate the dynamic changes in the Hippo signaling pathway and YAP activation during liver regeneration, which stabilize when the liver-to-body weight ratio reaches homeostatic levels.

title: Differences in Yes-associated protein and mRNA levels in regenerating liver and hepatocellular carcinoma.
abstract: Yes-associated protein (YAP) has been identified as an oncoprotein that             regulates organ size by modulating proliferative and apoptotic activities. The             aim of this study was to measure YAP gene expression and protein levels during             rat liver regeneration and in liver cancer, and to explore possible correlations             between YAP levels and cancer cell proliferation following partial hepatectomy.             We examined YAP expression in rat regenerating liver using real-time PCR, Western             blotting and immunohistochemical methods. YAP mRNA and protein levels were measured             in human hepatocellular carcinoma. The results demonstrated that YAP protein levels             were markedly increased in the regenerating liver (2.5-3 times), while YAP mRNA             levels decreased slightly (1.47-2.17 times) (P=0.017). YAP mRNA and protein levels             were both higher in hepatocellular carcinoma than in para-cancerous tissue. In             conclusion, liver regeneration alone elevated YAP protein levels without elevating             YAP mRNA levels. YAP mRNA and protein levels were both elevated in liver cancer             cells.