Hippo Activity Research Articles

The Iroquois homeobox proteins IRX3 and IRX5 have distinct roles in Wilms tumour development and human nephrogenesis.

Wilms tumour is a paediatric malignancy with features of halted kidney development. Here, we demonstrate that the Iroquois homeobox genes IRX3 and IRX5 are essential for mammalian nephrogenesis and govern the differentiation of Wilms tumour. Knock‐out Irx3 − /Irx5 − mice showed a strongly reduced embryonic nephron formation. In human foetal kidney and Wilms tumour, IRX5 expression was already activated in early proliferative blastema, whereas IRX3 protein levels peaked at tubular differentiation. Accordingly, an orthotopic xenograft mouse model of Wilms tumour showed that IRX3 −/− cells formed bulky renal tumours dominated by immature mesenchyme and active canonical WNT/β‐catenin‐signalling. In contrast, IRX5 −/− cells displayed activation of Hippo and non‐canonical WNT‐signalling and generated small tumours with abundant tubulogenesis. Our findings suggest that promotion of IRX3 signalling or inhibition of IRX5 signalling could be a route towards differentiation therapy for Wilms tumour, in which WNT5A is a candidate molecule for enforced tubular maturation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth

The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that yap -/- mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis. Transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in yap -/- mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improves Yap deficiency phenotypes, indicating functional importance of glucose-fueled nucleotide biosynthesis. Yap-regulated glut1 expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signaling in glucose metabolic homeostasis.

Abstract 214: A Microrna-hippo Pathway Functions in Cardiac Conduction System Homeostasis and Regeneration

The cardiac conduction system (CCS) is required for initiating and maintaining regular rhythmic heartbeats. CCS defects can give rise to cardiac arrhythmia, a leading cause for morbidity worldwide. Hippo signaling, a known organ size and growth regulator, has recently been shown to inhibit cardiomyocyte proliferation and renewal. However, the role for Hippo signaling in the CCS is unknown. Here, we found that adult mice display cardiac arrhythmias when Hippo signaling is specifically disrupted in the CCS, indicating an important role for Hippo in maintaining CCS homeostasis. We discovered that miR-17-92 represses Hippo signaling by directly repressing Lats2 , a core Hippo pathway component and that Hippo activity is increased in miR-17-92 deleted hearts. Importantly, disruption of miR-17-92 in the CCS gives rise to cardiac arrhythmias in adult mice. Notably, specific injury of the CCS causes severe lethal cardiac arrhythmias, whereas inactivation of Lats efficiently rescues the phenotype. In addition, based on data from the chromatin immunoprecipitation deep sequencing with an antibody against the Hippo signaling effector Yap, we found that Hippo signaling regulates genes involved in the CCS homeostasis and regeneration. Together, our findings indicate that maintenance of CCS homeostasis requires proper levels of Hippo signaling, and that repression of Hippo signaling protects the hearts from CCS damage and subsequent arrhythmogenesis.

Abstract 456: YAP-TEAD1-OSM Amplification Loop Plays a Critical Role in Mediating Cardiomyocyte De-differentiation

The Hippo pathway regulates organ size by inhibiting cell proliferation and promoting apoptosis through negative regulation of YAP, a transcription factor co-factor. Suppression of Hippo and activation of YAP promotes myocardial regeneration after myocardial infarction. However, our recent results suggest that Hippo downregulation and activation of YAP induce de-differentiation of cardiomyocytes (CMs) through activation of genes regulated by TEAD1, which in turn induces contractile dysfunction in the presence of pressure overload (PO). Since oncostatin M (OSM), a member of the interleukin-6 family of cytokines, is a mediator of CM de-differentiation, we evaluated whether OSM plays an important role in mediating CM de-differentiation in WW45 cKO mice, a mouse model of Hippo downregulation, during PO. RNA sequencing analyses showed that OSM receptors (OSMRs) are upregulated in WW45 cKO mice in response to PO. Immunoblot analyses showed that both OSM and OSMR are upregulated by PO and that their levels are further increased in WW45 cKO mice. Upregulation of OSM and OSMR was also observed in adult cardiomyocytes (ACM) isolated from WW45 cKO mice after PO. Downregulation of YAP and TEAD1 significantly decreased OSM reporter gene activity in vitro , suggesting that OSM and OSMR are downstream targets of the YAP-TEAD1 pathway in CMs. Treatment of mice with OSM increased the levels of MYH7 and ACTA2, as well as YAP and TEAD1, in ACM at baseline, indicating the existence of an amplification loop composed of YAP, TEAD1 and OSM to promote CM de-differentiation. Suppression of any component of the amplification loop inhibited CM de-differentiation and cardiac dysfunction in WW45 cKO mice in response to PO. These results indicate that the YAP-TEAD1-OSM loop plays a critical role in mediating CM de-differentiation.

Hippo Signaling Pathway Dysregulation in Human Huntington\u2019s Disease Brain and Neuronal Stem Cells

The Hippo signaling pathway is involved in organ size regulation and tumor suppression. Although inhibition of Hippo leads to tumorigenesis, activation of Hippo may play a role in neurodegeneration. Specifically, activation of the upstream regulator, mammalian sterile 20 (STE20)-like kinase 1 (MST1), reduces activity of the transcriptional co-activator Yes-Associated Protein (YAP), thereby mediating oxidative stress-induced neuronal death. Here, we investigated the possible role of this pathway in Huntington’s disease (HD) pathogenesis. Our results demonstrate a significant increase in phosphorylated MST1, the active form, in post-mortem HD cortex and in the brains of CAG knock-in HdhQ111/Q111 mice. YAP nuclear localization was also decreased in HD post-mortem cortex and in neuronal stem cells derived from HD patients. Moreover, there was a significant increase in phosphorylated YAP, the inactive form, in HD post-mortem cortex and in HdhQ111/Q111 brain. In addition, YAP was found to interact with huntingtin (Htt) and the chaperone 14-3-3, however this interaction was not altered in the presence of mutant Htt. Lastly, YAP/TEAD interactions and expression of Hippo pathway genes were altered in HD. Together, these results demonstrate that activation of MST1 together with a decrease in nuclear YAP could significantly contribute to transcriptional dysregulation in HD.

Abstract 4485: VGLL3 is a novel outlier in African American prostate tumors and its expression is associated with a tumor suppressor phenotype in prostate cancer

Abstract Higher incidence, progression and mortality of prostate cancer (PCa) have been noted in African American (AA) men compared with Caucasian (CA) men. In fact AA race is an independent predictor of biochemical recurrence for all stage of PCa (Dr. Schaeffer's data analysis, n=1634 of AA patients). The etiology of PCa racial disparities is likely multi-factorial with increasing support of biologic contributors to these differences. By analyzing large cohort of PCa, we have demonstrated that AA PCa exhibits a unique spectrum of molecular subtypes. For example, AA men presented with PCa have a significant decrease in fusion proteins such as ERG, ETS and SPINK, with an enrichment of fusion unknown (FU; triple negative) cases. Given the increased mortality of PCa in AA populations and the enrichment of FU cases among AA tumors, there is an urgent need to define and explore individual outlier as race-based modifiers of disease aggressiveness. By utilizing a large multi-institutional cohort of prostate cancer cases of mixed racial composition, we have identified novel outliers enriched in AA among FU group by COPA outlier analysis.VGLL3 is one of the top outliers enriched in AA. VGLL3 positive cases are associated with low stage (less LNI, p=0.001; SVI, p<0.001; and EPE, p=0.009) and low grade disease (less Gleason >7, p=0.012) of higher protective phenotype of adverse cancer grade and stage. This preliminary work on VGLL3 expression suggests it may be associated with a tumor suppressor-like phenotype with reductions in expression with increasingly aggressive PCa. Studies of VGLL3 in cancer are limited and the role of VGLL3 in prostate has not been explored at all. The goal of this study is to investigate the role of VGLL3 as a novel outlier in AA prostate cancer that modulates disease aggressiveness. We found that mRNA expression levels of VGLL3 were lower in prostate cancer epithelial cells than in normal epithelial cells or stromal cells. By establishing and characterizing PCa models overexpressing VGLL3 in vitro and in vivo, our data demonstrate that VGLL3 significantly reduced migration and invasion of PCa cells and inhibited primary tumor cell growth. Our preliminary data also indicate VGLL3 inhibits PCa metastasis. Furthermore, VGLL3 seems to negatively affect both hippo and AR activity in PCa cells and these might be the mechanisms that VGLL3 inhibits tumor cell growth. These data is consistent with our clinical data suggesting tumor suppressive role of VGLL3 in advanced PCa. Citation Format: Hongjun Kang. VGLL3 is a novel outlier in African American prostate tumors and its expression is associated with a tumor suppressor phenotype in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4485.

Dynamic Fluctuations in Subcellular Localization of the Hippo Pathway Effector Yorkie In Vivo

The Hippo pathway is an evolutionarily conserved signaling network that integrates diverse cues to control organ size and cell fate. The central downstream pathway protein in Drosophila is the transcriptional co-activator Yorkie (YAP and TAZ in humans), which regulates gene expression with the Scalloped/TEA domain family member (TEAD) transcription factors [1-8]. A central regulatory step in the Hippo pathway is phosphorylation of Yorkie by the NDR family kinase Warts, which promotes Yorkie cytoplasmic localization by stimulating association with 14-3-3 proteins [9-12]. Numerous reports have purported a static model of Hippo signaling whereby, upon Hippo activation, Yorkie/YAP/TAZ become cytoplasmic and therefore inactive, and upon Hippo repression, Yorkie/YAP/TAZ transit to the nucleus and are active. However, we have little appreciation for the dynamics of Yorkie/YAP/TAZ subcellular localization because most studies have been performed in fixed cells and tissues. To address this, we used live multiphoton microscopy to investigate the dynamics of an endogenously tagged Yorkie-Venus protein in growing epithelial organs. We found that the majority of Yorkie rapidly traffics between the cytoplasm and nucleus, rather than being statically localized in either compartment. In addition, discrete cell populations within the same organ display different rates of Yorkie nucleo-cytoplasmic shuttling. By assessing Yorkie dynamics in warts mutant tissue, we found that the Hippo pathway regulates Yorkie subcellular distribution by regulating its rate of nuclear import. Furthermore, Yorkie's localization fluctuates dramatically throughout the cell cycle, being predominantly cytoplasmic during interphase and, unexpectedly, chromatin enriched during mitosis. Yorkie's association with mitotic chromatin is Scalloped dependent, suggesting a potential role in mitotic bookmarking.

Glypican 3 (GPC3)‐CD81 axis regulates Ezrin mediated Hippo pathway via cross talking with HGF/c‐Met axis in hepatocytes and hepatocellular carcinoma (HCC)

The experiments that integrate different pathways in normal and abnormal circumstances will help us to develop optimum targeted therapies because of multiple pathways involved in the solid tumor carcinogenesis. We have shown that activation of CD81 is associated with phosphorylation of Ezrin, which is overexpressed in many types of solid tumors and has been identified as metastasis related protein for many years. We have also shown that GPC3 binds to the membrane tetraspanin CD81, which is one of the portals of entry of hepatitis C virus (HCV). Previous studies have shown that GPC3 is associated with inhibition of hepatocyte growth and liver regeneration by inhibiting CD81‐mediated p‐Ezrin/Hippo/Yap pathway. It also has been shown that HGF/Met axis which promotes growth in HCC can phosphorylate Ezrin as well by different kinases. Since we have demonstrated the predominant GPC+++/CD81− pattern in HCC, the purpose of this study is to explore the regulation of expression and phosphorylation of Ezrin in HCC. We have found that CD81 expression could be detected in membrane and organelle fraction in human hepatoma cell lines HepG2, Huh7 and Hep3B, but immunoprecipitation experiment suggests that CD81 from latter two human hepatoma cell lines has lost association with GPC3. Human hepatoma cell lines have dramatically up regulated Ezrin expression and Yap activity. We found that treatment of primary rat hepatocytes with CD81 agonist antibody or HGF led to higher levels of phosphorylated Ezrin and lower Hippo activity. Mice overexpressing GPC3 had decreased p‐Ezrin but increased Hippo activity. In mice with post‐natal Met knock out there was less Yap expression at baseline level. Rat hepatoma cell lines JM1 and JM2 had dramatically down regulated CD81 expression and Hippo activity, and up regulated p‐Ezrin. Treatment with 20 umol of Ezrin inhibitor led to significant cell death selectively in JM2 cells in vitro.ConclusionsBoth HGF/Met and CD81 mediate p‐Ezrin activity and enhance nuclear Yap levels. These pathways in normal hepatocytes are inhibited by GPC3 and low HGF/Met activity. In human hepatocytes, GPC3 inhibits the CD81‐mediated p‐Ezrin effect on Hippo pathway and cause a decrease in nuclear Yap. The different distribution pattern of HGF/Met and GPC3/CD81 between normal hepatocytes and HCC cells provides the chance to develop precise anti‐HCC therapies. And Ezrin is a promising target of anti‐HCC therapies including primary lesion and metastasis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Salvador has an extended SARAH domain that mediates binding to Hippo kinase

The Hippo pathway controls cell proliferation and differentiation through the precisely tuned activity of a core kinase cassette. The activity of Hippo kinase is modulated by interactions between its C-terminal coiled-coil, termed the SARAH domain, and the SARAH domains of either dRassF or Salvador. Here, we wanted to understand the molecular basis of SARAH domain-mediated interactions and their influence on Hippo kinase activity. We focused on Salvador, a positive effector of Hippo activity and the least well-characterized SARAH domain-containing protein. We determined the crystal structure of a complex between Salvador and Hippo SARAH domains from Drosophila This structure provided insight into the organization of the Salvador SARAH domain including a folded N-terminal extension that expands the binding interface with Hippo SARAH domain. We also found that this extension improves the solubility of the Salvador SARAH domain, enhances binding to Hippo, and is unique to Salvador. We therefore suggest expanding the definition of the Salvador SARAH domain to include this extended region. The heterodimeric assembly observed in the crystal was confirmed by cross-linked MS and provided a structural basis for the mutually exclusive interactions of Hippo with either dRassF or Salvador. Of note, Salvador influenced the kinase activity of Mst2, the mammalian Hippo homolog. In co-transfected HEK293T cells, human Salvador increased the levels of Mst2 autophosphorylation and Mst2-mediated phosphorylation of select substrates, whereas Salvador SARAH domain inhibited Mst2 autophosphorylation in vitro These results suggest Salvador enhances the effects of Hippo kinase activity at multiple points in the Hippo pathway.

Hepatitis C Virus Mimics Effects of Glypican-3 on CD81 and Promotes Development of Hepatocellular Carcinomas via Activation of Hippo Pathway in Hepatocytes

Glypican (GPC)-3 is overexpressed in hepatocellular carcinomas (HCCs). GPC3 binds to CD81. Forced expression of CD81 in a GPC3-expressing HCC cell line caused activation of Hippo, a decrease in ezrin phosphorylation, and a decrease in yes-associated protein (YAP). CD81 is also associated with hepatitis C virus (HCV) entry into hepatocytes. Activation of CD81 by agonistic antibody causes activation of tyrosine-protein kinase SYK (SYK) and phosphorylation of ezrin, a regulator of the Hippo pathway. In cultures of normal hepatocytes, CD81 agonistic antibody led to enhanced phosphorylation of ezrin and an increase in nuclear YAP. HCV E2 protein mimicked GPC3 and led to enhanced Hippo activity and decreased YAP in cultured normal human hepatocytes. HCC tissue microarray revealed a lack of expression of CD81 in most HCCs, rendering them insusceptible to HCV infection. Activation of CD81 by agonistic antibody suppressed the Hippo pathway and increased nuclear YAP. HCV mimicked GPC3, causing Hippo activation and a decrease in YAP. HCV is thus likely to enhance hepatic neoplasia by acting as a promoter of growth of early CD81-negative neoplastic hepatocytes, which are resistant to HCV infection, and thus have a proliferative advantage to clonally expand as they participate in compensatory regeneration for the required maintenance of 100% of liver weight (hepatostat).

POSH regulates Hippo signaling through ubiquitin-mediated expanded degradation

The Hippo signaling pathway is a master regulator of organ growth, tissue homeostasis, and tumorigenesis. The activity of the Hippo pathway is controlled by various upstream components, including Expanded (Ex), but the precise molecular mechanism of how Ex is regulated remains poorly understood. Here we identify Plenty of SH3s (POSH), an E3 ubiquitin ligase, as a key component of Hippo signaling in DrosophilaPOSH overexpression synergizes with loss of Kibra to induce overgrowth and up-regulation of Hippo pathway target genes. Furthermore, knockdown of POSH impedes dextran sulfate sodium-induced Yorkie-dependent intestinal stem cell renewal, suggesting a physiological role of POSH in modulating Hippo signaling. Mechanistically, POSH binds to the C-terminal of Ex and is essential for the Crumbs-induced ubiquitination and degradation of Ex. Our findings establish POSH as a crucial regulator that integrates the signal from the cell surface to negatively regulate Ex-mediated Hippo activation in Drosophila.

SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK.

The Hippo pathway controls tissue growth and homeostasis through a central MST-LATS kinase cascade. The scaffold protein SAV1 promotes the activation of this kinase cascade, but the molecular mechanisms remain unknown. Here, we discover SAV1-mediated inhibition of the PP2A complex STRIPAKSLMAP as a key mechanism of MST1/2 activation. SLMAP binding to autophosphorylated MST2 linker recruits STRIPAK and promotes PP2A-mediated dephosphorylation of MST2 at the activation loop. Our structural and biochemical studies reveal that SAV1 and MST2 heterodimerize through their SARAH domains. Two SAV1-MST2 heterodimers further dimerize through SAV1 WW domains to form a heterotetramer, in which MST2 undergoes trans-autophosphorylation. SAV1 directly binds to STRIPAK and inhibits its phosphatase activity, protecting MST2 activation-loop phosphorylation. Genetic ablation of SLMAP in human cells leads to spontaneous activation of the Hippo pathway and alleviates the need for SAV1 in Hippo signaling. Thus, SAV1 promotes Hippo activation through counteracting the STRIPAKSLMAP PP2A phosphatase complex.

Hippo Reprograms the Transcriptional Response to Ras Signaling

Hyperactivating mutations in Ras signaling are hallmarks of carcinomas. Ras signaling mediates cell fate decisions as well as proliferation during development. It is not known what dictates whether Ras signaling drives differentiation versus proliferation. Here we show that the Hippo pathway is critical for this decision. Loss of Hippo switches Ras activation from promoting cellular differentiation toaggressive cellular proliferation. Transcriptome analysis combined with genetic tests show that this excessive proliferation depends on the synergistic induction of Ras target genes. Using ChIP-nexus, we find that Hippo signaling keeps Ras targets in check by directly regulating the expression of two key downstream transcription factors of Ras signaling: the ETS-domain transcription factor Pointed and the repressor Capicua. Our results highlight how independent signaling pathways can impinge on each other at the level of transcription factors, thereby providing a safety mechanism to keep proliferation in check under normal developmental conditions.

Glypican 3 (GPC3)‐CD81 axis regulates spleen tyrosine kinase(Syk)‐Ezrin mediated Hippo pathway via cross talking with HGF/c‐Met axis in hepatocytes and hepatocellular carcinoma (HCC)

The experiments that integrate different pathways in normal and abnormal circumstances will help us to develop better targeted therapies because of multiple pathways involve in the solid tumor carcinogenesis. GPC3 is over‐expressed in HCC. Previous studies have shown that GPC3 is associated with inhibition of hepatocyte growth and GPC3 transgenic mice with targeted expression in hepatocytes have decreased liver regeneration. We have also shown that GPC3 binds to the membrane tetraspanin CD81, which is one of the portals of entry of hepatitis C virus (HCV). Studies elsewhere have shown that activation of CD81 is associated with phosphorylation of Ezrin. The latter has been shown to regulate the Hippo pathway by modulation activity of tumor suppressor gene NF2 (Merlin), responsible for regulating nuclear levels of the protein known as Yes‐associated protein (Yap). It also has been shown that HGF/Met axis which promotes growth in HCC can phosphorylate Ezrin as well by unknown mechanisms. Expression of Yap in the nucleus enhances hepatocyte growth. Mice over‐expressing GPC3 have decreased levels of nuclear Yap. Since we have demonstrated that GPC3 binds to CD81, the purpose of this study is to explore the role of GPC3 in regulation of Yap via CD81 and Hippo pathway and to investigate the possible mechanisms. CD81 expression could be detected in membrane and organelle fraction in human hepatoma cell lines HepG2, Huh7 and Hep3B, but immunoprecipitation experiment suggests that CD81 from latter two human hepatoma cell lines has lost association with GPC3. Human hepatoma cell line had dramatically up regulated Ezrin expression and Yap activity. We found that treatment of primary rat hepatocytes with CD81 agonist antibody or HGF led to higher levels of phosphorylated Ezrin and lower Hippo activity in a Syk dependent manner. While mice over‐expressing GPC3 decreased p‐Ezrin but increased Hippo activity. Transgenic Met knock out mice showed less Yap expression at baseline level, and abrogated up regulated Yap expression which was observed in wild type mice after 2d PHx and increased subsequent apoptosis. Rat hepatoma cell lines JM1 and JM2 had dramatically down regulated CD81 expression and Hippo activity, and up regulated Ezrin activity in vitro. Force expression of Syk expression in JM2 rat hepatoma cells downregulated Hippo activity while upregulated Ezrin activity in vitro.ConclusionsBoth Met‐phosphoEzrin and CD81‐phosphoEzrin signaling enhances nuclear Yap levels. These pathways in normal hepatocytes are inhibited by GPC3 and low HGF/Met activity. In human HCC, loss of CD81 expression and highly activity of HGF/Met promote the CD81‐phosphoEzrin effect on Hippo pathway and cause a increase in nuclear Yap. The different distribution pattern of HGF/Met and GPC3/CD81 between normal hepatocytes and HCC cells provides the chance to develop precise anti‐HCC therapies.

A Middle Pleistocene hippo tracksite at Gombore II-2 (Melka Kunture, Upper Awash, Ethiopia)

In this paper we describe exceptionally well-preserved evidence of hippo activity at Gombore II-2, a 700,000year-old site at Melka Kunture, at 2000masl in the Ethiopian highlands, in the Upper Awash Valley. We excavated and made casts of footprints that had been left after a volcanic eruption. The 3D casts provide a detailed record of the outer anatomy and soft tissues of Pleistocene hippos. They reveal for the first time the shape of the legs and feet of the animals. Gombore II-2 also provides information on the behavior of past hippos. This prominent species of the African wildlife affects the vegetation and the landscape, and acts as a geomorphological agent. The animals wallow beside bodies of water and erode deep trails linking them to pastures. At Gombore II-2 the trampling marks converge toward an erosional feature that we interpret as a fossil trail, documenting an early Middle Pleistocene behavior similar to that of today. Signs of trampling and erosional features interpreted as hippo trails have also been described in Pliocene and early Pleistocene levels at Olduvai and Koobi Fora. We further underline that the hippo population of this tract of the valley was resilient and re-established itself not long time after the volcanic eruption.

AJUBA LIM Proteins Limit Hippo Activity in Proliferating Cells by Sequestering the Hippo Core Kinase Complex in the Cytosol.

The Hippo pathway controls organ growth and is implicated in cancer development. Whether and how Hippo pathway activity is limited to sustain or initiate cell growth when needed is not understood. The members of the AJUBA family of LIM proteins are negative regulators of the Hippo pathway. In mammalian epithelial cells, we found that AJUBA LIM proteins limit Hippo regulation of YAP, in proliferating cells only, by sequestering a cytosolic Hippo kinase complex in which LATS kinase is inhibited. At the plasma membranes of growth-arrested cells, AJUBA LIM proteins do not inhibit or associate with the Hippo kinase complex. The ability of AJUBA LIM proteins to inhibit YAP regulation by Hippo and to associate with the kinase complex directly correlate with their capacity to limit Hippo signaling during Drosophila wing development. AJUBA LIM proteins did not influence YAP activity in response to cell-extrinsic or cell-intrinsic mechanical signals. Thus, AJUBA LIM proteins limit Hippo pathway activity in contexts where cell proliferation is needed.

Disease implication of hyper-Hippo signalling

The Hippo signalling pathway regulates cellular proliferation, apoptosis and differentiation, thus exerting profound effects on cellular homeostasis. Inhibition of Hippo signalling has been frequently implicated in human cancers, indicating a well-known tumour suppressor function of the Hippo pathway. However, it is less certain whether and how hyperactivation of the Hippo pathway affects biological outcome in living cells. This review describes current knowledge of the regulatory mechanisms of the Hippo pathway, mainly focusing on hyperactivation of the Hippo signalling nexus. The disease implications of hyperactivated Hippo signalling have also been discussed, including arrhythmogenic cardiomyopathy, Sveinsson's chorioretinal atrophy, Alzheimer's disease, amyotrophic lateral sclerosis and diabetes. By highlighting the significance of disease-relevant Hippo signalling activation, this review can offer exciting prospects to address the onset and potential reversal of Hippo-related disorders.

Cross-Talk Between Mitochondrial Fusion and the Hippo Pathway in Controlling Cell Proliferation During Drosophila Development.

Cell proliferation and tissue growth depend on the coordinated regulation of multiple signaling molecules and pathways during animal development. Previous studies have linked mitochondrial function and the Hippo signaling pathway in growth control. However, the underlying molecular mechanisms are not fully understood. Here we identify a Drosophila mitochondrial inner membrane protein ChChd3 as a novel regulator for tissue growth. Loss of ChChd3 leads to tissue undergrowth and cell proliferation defects. ChChd3 is required for mitochondrial fusion and removal of ChChd3 increases mitochondrial fragmentation. ChChd3 is another mitochondrial target of the Hippo pathway, although it is only partially required for Hippo pathway-mediated overgrowth. Interestingly, lack of ChChd3 leads to inactivation of Hippo activity under normal development, which is also dependent on the transcriptional coactivator Yorkie (Yki). Furthermore, loss of ChChd3 induces oxidative stress and activates the JNK pathway. In addition, depletion of other mitochondrial fusion components, Opa1 or Marf, inactivates the Hippo pathway as well. Taken together, we propose that there is a cross-talk between mitochondrial fusion and the Hippo pathway, which is essential in controlling cell proliferation and tissue homeostasis in Drosophila.

Glypican 3 (GPC3), CD81 and HCV E2 protein control Yap and Hippo Pathway

Liver failure is a common outcome of end‐stage liver diseases. Modern stem cell and tissue engineering techniques can't achieve functional unique liver with multi cell types. Transplantation can only save limited amount of patients, the realistic approach for the majority of patients is to use residual functional liver tissue to restore impaired liver function. Maintaining dynamic balance, mitogenic and mitoinhibitory signals coexist in the human organs. This balance determines the fate of single cell, and determines the size and morphologic characterization of tissue or organ finally. Therefore, theoretically, restoration of functional liver mass in liver failure setting depends on either negative modulation of mitoinhibitory effect or positive modulation of mitogenic effect. One group of Japanese liver failure patients showed aberrantly up regulation of mitogenic signals such as HGF, indicates this blocked regeneration might be dominantly mediated by mitoinhibitory signals. Thus, the investigation of the mechanism of involving mitoinhibitory signaling may provide novel therapeutic targets for liver failure treatment.Previous studies from our laboratory have shown that GPC3 is associated with inhibition of hepatocyte growth and GPC3 transgenic mice with targeted expression in hepatocytes have decreased liver regeneration. Our previous investigation also showed that GPC3 overexpress intensively both in fulminant hepatic failure human liver tissues. We have also shown that GPC3 binds to the membrane tetraspanin known as CD81. Studies from other sources have shown that activation of CD81 is associated with highly targeted phosphorylation of the protein Ezrin. The latter has been shown to regulate the Hippo pathway, responsible for regulating nuclear levels of the protein known as Yes‐associated protein (Yap). Expression of Yap in the nucleus relates to enhanced liver size and activation of hepatocyte growth. Mice over‐expressing GPC3 with associated suppressed liver regeneration have decreased levels of nuclear Yap. We also found that treatment of primary rat hepatocytes with CD81 agonist antibody led to higher levels of phosphorylated Ezrin and lower Hippo activity, and this lower Hippo activity could be reversed by Spleen tyrosine kinase (Syk) inhibitor in highly differentiated hepatocytes. Partial hepatectomy (PHx) exhibited that loss of GPC3 antagonistic modulation during early liver regeneration, CD81 activated Ezrin and down regulated Hippo activity in vivo. Treatment with Hepatitis C virus (HCV) envelope protein E2 led to more Hippo activity and decrease in nuclear Yap in Hepa RG cells and primary human hepatocytes.ConclusionsDependent on Syk mediated CD81, GPC3 and HCV may function as growth inhibitory factor by modulating YAP expression pattern. The CD81‐Ezrin signaling enhances nuclear Yap levels. This pathway in normal hepatocytes is inhibited by GPC3. In human hepatocytes, GPC3 and the E2 protein of HCV also inhibit the CD81‐Ezrin effect on Hippo pathway and cause a decrease in nuclear Yap. Persistent HCV infection blocks the potential of liver regeneration by E2 protein interacting with CD81.Support or Funding InformationNIH/NCI R01‐CA103958

Cell Competition Drives the Growth of Intestinal Adenomas in Drosophila

SummaryTumor-host interactions play an increasingly recognized role in modulating tumor growth. Thus, understanding the nature and impact of this complex bidirectional communication is key to identifying successful anti-cancer strategies. It has been proposed that tumor cells compete with and kill neighboring host tissue to clear space that they can expand into; however, this has not been demonstrated experimentally. Here we use the adult fly intestine to investigate the existence and characterize the role of competitive tumor-host interactions. We show that APC−/−-driven intestinal adenomas compete with and kill surrounding cells, causing host tissue attrition. Importantly, we demonstrate that preventing cell competition, by expressing apoptosis inhibitors, restores host tissue growth and contains adenoma expansion, indicating that cell competition is essential for tumor growth. We further show that JNK signaling is activated inside the tumor and in nearby tissue and is required for both tumor growth and cell competition. Lastly, we find that APC−/− cells display higher Yorkie (YAP) activity than host cells and that this promotes tumor growth, in part via cell competition. Crucially, we find that relative, rather than absolute, Hippo activity determines adenoma growth. Overall, our data indicate that the intrinsic over-proliferative capacity of APC−/− cells is not uncontrolled and can be constrained by host tissues if cell competition is inhibited, suggesting novel possible therapeutic approaches.