Hippo Pathway Kinase Research Articles

Cell detachment activates the Hippo pathway via cytoskeleton reorganization to induce anoikis

Cell attachment to the extracellular matrix (ECM) is crucial to cell physiology such as polarity, motility, and proliferation. In normal cells, loss of attachment to the ECM induces a specific type of apoptosis, termed anoikis. Resistance to anoikis in cancer cells promotes their survival in circulation and dispersion to distant anatomic sites, leading to tumor metastasis. The Yes-associated protein (YAP) transcription coactivator is a human oncogene and a key regulator of organ size. The Hippo tumor suppressor pathway phosphorylates and inhibits YAP. However, little is known about the signals that regulate the Hippo pathway. Here we report that through cytoskeleton reorganization, cell detachment activates the Hippo pathway kinases Lats1/2 and leads to YAP phosphorylation and inhibition. The detachment-induced YAP inactivation is required for anoikis in nontransformed cells, whereas in cancer cells with deregulation of the Hippo pathway, knockdown of YAP and TAZ restores anoikis. Furthermore, we provided evidence that Lats1/2 expression level is indeed significantly down-regulated in metastatic prostate cancer. Our findings provide a novel connection between cell attachment and anoikis through the Hippo pathway and have important implications in cancer therapeutics.

The transcriptional regulator YAP induces Notch-pathway activity in hepatocytes and hepatocellular carcinoma cells

Aims: The Hippo-pathway regulates organ size and homeostasis. Studies revealed that deletion of Hippo-pathway kinases (e.g. Mst1/2) or overexpression of the transcriptional co-activator YAP lead to liver cancer; however, the underlying molecular mechanism have not identified so far. We therefore aimed to define possible effector mechanisms mediating YAP-dependent oncogenic activity in liver cancer. Methods: SiRNA-knockdown, expression profiling, and functional assays were performed using HCC cell lines. Gene expression and protein interaction were analyzed by immunoblotting or RT-PCR and co-immunoprecipitation, respectively. YAP transgenic (YAPLAP) and Mst1/2 knock-out mice as well as human HCCs were examined by IHC, immunoblotting, and RT-PCR. Results: Expression profiling after YAP inhibition in HCC cells and of YAPLAP liver tissues identified the Notch-pathway ligand Jag1 as potential target gene for the Hippo pathway. Expression of cleaved Notch receptor (NICD) and Notch target gene Hes1 were reduced in HCC cells after YAP inhibition. SiRNA-knockdown of Jag1 but not of Jag2 as well as inhibition of Notch signalling by γ-secretase phenocopied the biological effects detected after YAP inhibition (e.g., decreased viability). Overexpression of YAP induced tumor cell proliferation, whereas additional Jag1 inhibition partly compensated the effects. Expression of YAP/Jag1 and YAP/Hes1 significantly correlated in human HCCs. YAPLAP-mice developed dysplastic foci with increased Jag1 expression and elevated proliferation. Furthermore, time-resolved analysis showed a simultaneous increase of Jag1 and Hes1 levels after doxycylin-dependent YAP expression in isolated primary hepatocytes. Accordingly, Mst1/2-/- mice-derived HCCs showed high YAP, Jag1, and Hes1 levels. Conclusion: These data demonstrate that activation of YAP induces Notch-pathway activity by Jag1 expression and that Notch-signalling partly facilitates the oncogenic effects of YAP in vitro and in vivo.

Deregulation of Hippo kinase signalling in Human hepatic malignancies

Hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and hepatoblastoma (HB) are the main hepatic malignancies with limited treatment options and high mortality. Recent studies have implicated Hippo kinase pathway in cancer development, but detailed analysis of Hippo kinase signalling in human hepatic malignancies, especially CC and HB, is lacking. We investigated Hippo kinase signalling in HCC, CC and HB using cells and patient samples. Increased expression of yes-associated protein (Yap), the downstream effector of the Hippo kinase pathway, was observed in HCC cells, and siRNA-mediated knockdown of Yap resulted in decreased survival of HCC cells. The density-dependent activation of Hippo kinase pathway characteristic of normal cells was not observed in HCC cells and CCLP cells, a cholangiocarcinoma cell line. Immunohistochemistry of Yap in HCC, CC and HB tissues indicated extensive nuclear localization of Yap in majority of tissues. Western blot analysis performed using total cell extracts from patient samples and normal livers showed extensive activation of Yap. Marked induction of Glypican-3, CTGF and Survivin, the three Yap target genes was observed in the tumour samples. Further analysis revealed significant decrease in expression and activity of Lats kinase, the main upstream regulator of Yap. However, no change in activation of Mst-2 kinase, the upstream regulator of Lats kinase was observed. These data show that Yap induction mediated by inactivation of Lats is observed in hepatic malignancies. These studies highlight Hippo kinase pathway as a novel therapeutic target for hepatic malignancies.

Willin/FRMD6 expression activates the Hippo signaling pathway kinases in mammals and antagonizes oncogenic YAP

The Salvador/Warts/Hippo (Hippo) signaling pathway defines a novel signaling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis and cancer development in mammals. The Drosophila melanogaster protein Expanded acts in the Hippo signaling pathway to control organ size. Previously, willin/FRMD6 has been proposed as the human orthologue of Expanded. Willin lacks C-terminal sequences that are present in Expanded and, to date, little is known about the functional role of willin in mammalian cells. When willin is expressed in D. melanogaster epithelial tissues, it has the same subcellular localization as Expanded, but cannot rescue growth defects associated with expanded deficiency. However, we show that ectopic willin expression causes an increase in phosphorylation of the core Hippo signaling pathway components MST1/2, LATS1 and YAP, an effect that can be antagonized by ezrin. In MCF10A cells, loss of willin expression displays epithelial-to-mesenchymal transition features and willin overexpression antagonizes YAP activity via the N-terminal FERM domain of willin. Therefore, in mammalian cells willin influences Hippo signaling activity by activating the core Hippo pathway kinase cassette.

WW domain interactions regulate the Hippo tumor suppressor pathway

The Hippo kinase pathway is emerging as a conserved signaling pathway that is essential for organ growth and tumorigenesis in Drosophila and mammalians. Although the signaling of the core kinases is relatively well understood, less is known about the upstream inputs, downstream outputs and regulation of the whole cascade. Enrichment of the Hippo pathway components with WW domains and their cognate proline-rich interacting motifs provides a versatile platform for further understanding the mechanisms that regulate organ growth and tumorigenesis. Here, we review recently discovered mechanisms of WW domain-mediated interactions that contribute to the regulation of the Hippo signaling pathway in tumorigenesis. We further discuss new insights and future directions on the emerging role of such regulation.

The Hippo–YAP pathway in organ size control and tumorigenesis: an updated version

The Hippo signaling pathway is gaining recognition as an important player in both organ size control and tumorigenesis, which are physiological and pathological processes that share common cellular signaling mechanisms. Upon activation by stimuli such as high cell density in cell culture, the Hippo pathway kinase cascade phosphorylates and inhibits the Yes-associated protein (YAP)/TAZ transcription coactivators representing the major signaling output of the pathway. Altered gene expression resulting from YAP/TAZ inhibition affects cell number by repressing cell proliferation and promoting apoptosis, thereby limiting organ size. Recent studies have provided new insights into the Hippo signaling pathway, elucidating novel phosphorylation-dependent and independent mechanisms of YAP/Yki inhibition by the Hippo pathway, new Hippo pathway components, novel YAP target transcription factors and target genes, and the three-dimensional structure of the YAP-TEAD complex, and providing further evidence for the involvement of YAP and the Hippo pathway in tumorigenesis.

Yes‐associated protein expression is induced in hepatocellular carcinoma and is responsive to cell density.

Hepatocellular Carcinoma (HCC) is the most common malignancy of the liver associated with high mortality. We are interested in understanding the role of pathways involved in organ size regulation in pathogenesis of HCC. Specifically, we are investigating the role of Hippo Kinase pathway and yes‐associated protein (Yap), the downstream effector of the pathway. Three HCC cell lines investigated, Hep3B, HepG2 and Huh7, demonstrated increase in Yap as compared to normal human hepatocytes. The expression levels and cellular localization of Yap was responsive to cell density. High cell confluency resulted in decreased Yap expression and nuclear export. siRNA‐mediated knockdown of Yap resulted in decreased cell proliferation of Hep3B cells. Immunohistochemical analysis of Yap in phenobarbital+diethylnitrosamine‐induced liver tumors in C57BL/6 mice, spontaneous tumors in Farnesoid X Receptor knockout mice and human HCC biopsies revealed extensive nuclear staining on Yap in highly proliferative tumors. These data indicate that Yap helps the hepatoma cells overcome the cell adhesion‐mediated growth inhibition and plays a critical role in pathogenesis of HCC.

The WW Domain Protein Kibra Acts Upstream of Hippo in Drosophila

The conserved Hippo kinase pathway plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. Whereas the function of the core kinase cascade, consisting of the serine/threonine kinases Hippo and Warts, in phosphorylating and thereby inactivating the transcriptional coactivator Yorkie is well established, much less is known about the upstream events that regulate Hippo signaling activity. The FERM domain proteins Expanded and Merlin appear to represent two different signaling branches that feed into the Hippo pathway. Signaling by the atypical cadherin Fat may act via Expanded, but how Merlin is regulated has remained elusive. Here, we show that the WW domain protein Kibra is a Hippo signaling component upstream of Hippo and Merlin. Kibra acts synergistically with Expanded, and it physically interacts with Merlin. Thus, Kibra predominantly acts in the Merlin branch upstream of the core kinase cascade to regulate Hippo signaling.

A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCFβ-TRCP

The Yes-associated protein (YAP) transcription coactivator is a key regulator of organ size and a candidate human oncogene. YAP is inhibited by the Hippo pathway kinase cascade, at least in part via phosphorylation of Ser 127, which results in YAP 14-3-3 binding and cytoplasmic retention. Here we report that YAP is phosphorylated by Lats on all of the five consensus HXRXXS motifs. Phosphorylation of Ser 381 in one of them primes YAP for subsequent phosphorylation by CK1delta/epsilon in a phosphodegron. The phosphorylated phosphodegron then recruits the SCF(beta-TRCP) E3 ubiquitin ligase, which catalyzes YAP ubiquitination, ultimately leading to YAP degradation. The phosphodegron-mediated degradation and the Ser 127 phosphorylation-dependent translocation coordinately suppress YAP oncogenic activity. Our study identified CK1delta/epsilon as new regulators of YAP and uncovered an intricate mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation (nuclear-cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation).

Phosphorylation of the Tumor Suppressor Fat Is Regulated by Its Ligand Dachsous and the Kinase Discs Overgrown

The Drosophila tumor suppressor gene fat encodes a large cadherin that regulates growth and a form of tissue organization known as planar cell polarity (PCP). Fat regulates growth via the Hippo kinase pathway, which controls expression of genes promoting cell proliferation and inhibiting apoptosis (reviewed in). The Hippo pathway is highly conserved and is implicated in the regulation of mammalian growth and cancer development. Genetic studies suggest that Fat activity is regulated by binding to another large cadherin, Dachsous (Ds). The tumor suppressor discs overgrown (dco)/Casein Kinase I delta/epsilon also regulates Hippo activity and PCP. The biochemical nature of how Fat, Ds, and Dco interact to regulate these pathways is poorly understood. Here we demonstrate that Fat is cleaved to generate 450 kDa and 110 kDa fragments (Fat(450) and Fat(110)). Fat(110) contains the cytoplasmic and transmembrane domain. The cytoplasmic domain of Fat binds Dco and is phosphorylated by Dco at multiple sites. Importantly, we show Fat forms cis-dimers and that Fat phosphorylation is regulated by Dachsous and Dco in vivo. We propose that Ds regulates Dco-dependent phosphorylation of Fat and Fat-associated proteins to control Fat signaling in growth and PCP.

Role of Hippo Kinase Pathway In Pathogenesis of Hepatocellular Carcinoma.

The Hippo Kinase pathway regulates organ size in mammals and is also implicated in pathogenesis of various cancers. We have investigated the role of Hippo Kinase pathway in pathogenesis of hepatocellular carcinoma (HCC). Three HCC cell lines investigated, Hep3B, HepG2 and Huh7, demonstrated increase in Yes‐associated protein (YAP), the downstream effector of the Hippo Kinase pathway as compared to normal human hepatocytes. Increase in Ser127‐phorphorylated of YAP, which is shown to be an anti‐apoptotic and pro‐survival signal in cancer cells, was observed in all HCC cell lines. Large tumor suppressor‐1 (Lats1), an upstream kinase known to phosphorylate YAP was induced in HCC cell lines along with MOB1, a protein known to cooperate with Lats1 in phosphorylation of YAP. Mst2, a member of STE20‐kinase family, which activates Lats1, was also increased in the HCC cells. Immunofluorescence analysis indicated YAP localization both in nucleus and cytoplasm while Lats1 was mainly cytoplasmic. These data indicate that Hippo Kinase pathway is activated in the HCC cell lines, may help the HCC cells to escape apoptosis and could be an important target in treatment of primary as well as metastatic HCC.

The FERM-Domain Protein Expanded Regulates Hippo Pathway Activity via Direct Interactions with the Transcriptional Activator Yorkie

The Hippo kinase pathway plays a central role in growth regulation and tumor suppression from flies to man. The Hippo/Mst kinase phosphorylates and activates the NDR family kinase Warts/Lats, which phosphorylates and inhibits the transcriptional activator Yorkie/YAP. Current models place the FERM-domain protein Expanded upstream of Hippo kinase in growth control. To understand how Expanded regulates Hippo pathway activity, we used affinity chromatography and mass spectrometry to identify Expanded-binding proteins. Surprisingly we find that Yorkie is the major Expanded-binding protein in Drosophila S2 cells. Expanded binds Yorkie at endogenous levels via WW-domain-PPxY interactions, independently of Yorkie phosphorylation at S168, which is critical for 14-3-3 binding. Expanded relocalizes Yorkie from the nucleus, abrogating its nuclear activity, and it can regulate growth downstream of warts in vivo. These data lead to a new model whereby Expanded functions downstream of Warts, in concert with 14-3-3 proteins to sequester Yorkie in the cytoplasm, inhibiting growth activity of the Hippo pathway.