Regulation of Hippo signaling by EGFR-MAPK signaling through Ajuba family proteins.

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.

Regulation of Drosophila glial cell proliferation by Merlin-Hippo signaling.

Glia perform diverse and essential roles in the nervous system, but the mechanisms that regulate glial cell numbers are not well understood. Here, we identify and characterize a requirement for the Hippo pathway and its transcriptional co-activator Yorkie in controlling Drosophila glial proliferation. We find that Yorkie is both necessary for normal glial cell numbers and, when activated, sufficient to drive glial over-proliferation. Yorkie activity in glial cells is controlled by a Merlin-Hippo signaling pathway, whereas the upstream Hippo pathway regulators Fat, Expanded, Crumbs and Lethal giant larvae have no detectable role. We extend functional characterization of Merlin-Hippo signaling by showing that Merlin and Hippo can be physically linked by the Salvador tumor suppressor. Yorkie promotes expression of the microRNA gene bantam in glia, and bantam promotes expression of Myc, which is required for Yorkie and bantam-induced glial proliferation. Our results provide new insights into the control of glial growth, and establish glia as a model for Merlin-specific Hippo signaling. Moreover, as several of the genes we studied have been linked to human gliomas, our results suggest that this linkage could reflect their organization into a conserved pathway for the control of glial cell proliferation.

Zyxin links fat signaling to the hippo pathway.

The Hippo signaling pathway has a conserved role in growth control and is of fundamental importance during both normal development and oncogenesis. Despite rapid progress in recent years, key steps in the pathway remain poorly understood, in part due to the incomplete identification of components. Through a genetic screen, we identified the Drosophila Zyxin family gene, Zyx102 (Zyx), as a component of the Hippo pathway. Zyx positively regulates the Hippo pathway transcriptional co-activator Yorkie, as its loss reduces Yorkie activity and organ growth. Through epistasis tests, we position the requirement for Zyx within the Fat branch of Hippo signaling, downstream of Fat and Dco, and upstream of the Yorkie kinase Warts, and we find that Zyx is required for the influence of Fat on Warts protein levels. Zyx localizes to the sub-apical membrane, with distinctive peaks of accumulation at intercellular vertices. This partially overlaps the membrane localization of the myosin Dachs, which has similar effects on Fat-Hippo signaling. Co-immunoprecipitation experiments show that Zyx can bind to Dachs and that Dachs stimulates binding of Zyx to Warts. We also extend characterization of the Ajuba LIM protein Jub and determine that although Jub and Zyx share C-terminal LIM domains, they regulate Hippo signaling in distinct ways. Our results identify a role for Zyx in the Hippo pathway and suggest a mechanism for the role of Dachs: because Fat regulates the localization of Dachs to the membrane, where it can overlap with Zyx, we propose that the regulated localization of Dachs influences downstream signaling by modulating Zyx-Warts binding. Mammalian Zyxin proteins have been implicated in linking effects of mechanical strain to cell behavior. Our identification of Zyx as a regulator of Hippo signaling thus also raises the possibility that mechanical strain could be linked to the regulation of gene expression and growth through Hippo signaling.

Influence of fat-hippo and notch signaling on the proliferation and differentiation of Drosophila optic neuroepithelia.

The Drosophila optic lobe develops from neuroepithelial cells, which function as symmetrically dividing neural progenitors. We describe here a role for the Fat-Hippo pathway in controlling the growth and differentiation of Drosophila optic neuroepithelia. Mutation of tumor suppressor genes within the pathway, or expression of activated Yorkie, promotes overgrowth of neuroepithelial cells and delays or blocks their differentiation; mutation of yorkie inhibits growth and accelerates differentiation. Neuroblasts and other neural cells, by contrast, appear unaffected by Yorkie activation. Neuroepithelial cells undergo a cell cycle arrest before converting to neuroblasts; this cell cycle arrest is regulated by Fat-Hippo signaling. Combinations of cell cycle regulators, including E2f1 and CyclinD, delay neuroepithelial differentiation, and Fat-Hippo signaling delays differentiation in part through E2f1. We also characterize roles for Jak-Stat and Notch signaling. Our studies establish that the progression of neuroepithelial cells to neuroblasts is regulated by Notch signaling, and suggest a model in which Fat-Hippo and Jak-Stat signaling influence differentiation by their acceleration of cell cycle progression and consequent impairment of Delta accumulation, thereby modulating Notch signaling. This characterization of Fat-Hippo signaling in neuroepithelial growth and differentiation also provides insights into the potential roles of Yes-associated protein in vertebrate neural development and medullablastoma.

Phosphorylation-independent repression of Yorkie in Fat-Hippo signaling.

The Fat-Hippo signaling pathway plays an important role in the regulation of normal organ growth during development, and in pathological growth during cancer. Fat-Hippo signaling controls growth through a transcriptional co-activator protein, Yorkie. A Fat-Hippo pathway has been described in which Yorkie is repressed by phosphorylation, mediated directly by the kinase Warts and indirectly by upstream tumor suppressors that promote Warts kinase activity. We present here evidence for an alternate pathway in which Yorkie activity is repressed by direct physical association with three other pathway components: Expanded, Hippo, and Warts. Each of these Yorkie repressors contains one or more PPXY sequence motifs, and associates with Yorkie via binding of these PPXY motifs to WW domains of Yorkie. This direct binding inhibits Yorkie activity independently from effects on Yorkie phosphorylation, and does so both in vivo and in cultured cell assays. These results emphasize the importance of the relative levels of Yorkie and its upstream tumor suppressors to Yorkie regulation, and suggest a dual repression model, in which upstream tumor suppressors can regulate Yorkie activity both by promoting Yorkie phosphorylation and by direct binding.

The Fat and Warts signaling pathways: new insights into their regulation, mechanism and conservation.

A cassette of cytoplasmic Drosophila tumor suppressors, including the kinases Hippo and Warts, has recently been linked to the transmembrane tumor suppressor Fat. These proteins act within interconnected signaling pathways, the principal functions of which are to control the growth and polarity of developing tissues. Recent studies have enhanced our understanding of the basis for signal transduction by Fat and Warts pathways, including the identification of a DNA-binding protein at the end of the pathway, have established the conservation of Fat and Warts signaling from flies to mammals, and have given us new insights into their regulation and biological functions.

Sialilated beta1,6 branched N-oligosaccharides modulate adhesion, chemotaxis and motility of melanoma cells: Effect on invasion and spontaneous metastasis properties.

B16BL6 cells, selected specifically for invasive characteristics from B16F10 mouse melanoma cells, displayed greater ability to metastasize to lungs and produced larger colonies than the parent cells, when injected intravenously. When the two cell lines were compared for surface beta1,6-branched N-oligosaccharides by flow cytometry using Leuco-Phyto-Heam-Agglutinin, B16BL6 were found to express significantly higher levels. Inhibition of the oligosaccharide expression, by treatment of the cells with swainsonine or antisense-N-acetyl glucosaminyl-transferase-V, significantly reduced metastasis and invasion (>50%). Further, inhibition of oligosaccharides on the molecules like beta1 integrin (one of the major carriers) caused 30-45% reduction in their adherence to extra-cellular-matrix components especially collagen IV and laminin, and chemotaxis towards fibronectin and matrigel. The inhibition also decreased haptotaxis by approximately 50% to fibronectin but surprisingly was enhanced towards laminin by approximately 75%. The cells on which the expression of these oligosaccharides was inhibited failed to exhibit the characteristic spontaneous metastasis and adhesion properties of B16BL6 cells. In none of the cases, however, the secretion of matrix-metallo-proteases correlated with oligosaccharide expression. Sialylation of surface oligosaccharides was found to be accompanied by even higher motility and adherence to the substrates. These results strongly support an important role of cell surface beta1,6-linked N-oligosaccharides, especially the sialylated derivatives, in the processes that influence invasion and metastasis.