Mechanisms by which Wnt pathways integrate the organization of receptors, organelles, and cytoskeletal proteins to confer cell polarity and directional cell movement are incompletely understood. We show that acute responses to Wnt5a involve recruitment of actin, myosin IIB, Frizzled 3, and melanoma cell adhesion molecule into an intracellular structure in a melanoma cell line. In the presence of a chemokine gradient, this Wnt-mediated receptor-actin-myosin polarity (W-RAMP) structure accumulates asymmetrically at the cell periphery, where it triggers membrane contractility and nuclear movement in the direction of membrane retraction. The process requires endosome trafficking, is associated with multivesicular bodies, and is regulated by Wnt5a through the small guanosine triphosphatases Rab4 and RhoB. Thus, cell-autonomous mechanisms allow Wnt5a to control cell orientation, polarity, and directional movement in response to positional cues from chemokine gradients.Wnt signaling controls cell polarity and directional cell movement in developmental systems, as well as cell invasion in certain cancers. Features shared between noncanonical Wnt pathways include recruitment of Frizzled (Fz) receptors to the posterior end of cells, and asymmetric distribution of atypical cell adhesion molecules, often associated with Fz (1, 2). Thus, receptors redistribute, in response to Wnt, to define an axis of asymmetry. In developmental systems, these processes can be regulated by interactions with adjacent cells, which confer orientation with respect to surrounding tissues (1, 3). For example, during endoderm specification in Caenorhabditis elegans, the division plane in the four-cell blastomere is determined by a positional Wnt signal from a near-by P 2 cell (4). In contrast, Wnt pathway mutations in C. elegans that disrupt neuronal cell migration and polarity can be rescued by Wnt overexpression without requiring a localized source of ligand (5, 6). This suggests that directional presentation of Wnt to cells is not always needed for cell polarization, rather that Wnt ligands act permissively to allow cells to respond to environmental cues for longitudinal guidance and directional movement. In such cases, WM239A cells treated with Wnt5a were immunostained for melanoma cell adhesion molecule (MCAM, also known as MUC18 and CD146), an immunoglobulin G-family cell adhesion receptor implicated in melanoma tumorigenesis and metastasis (9). In untreated cells, MCAM was distributed uniformly. However, treatment of cells with Wnt5a for 30 min led to redistribution of MCAM into a polarized structure, which we call the Wnt5a-mediated receptor-actin-myosin polarity (W-RAMP) structure, for reasons described below (Fig. 1, A and B). The W-RAMP structure was also observed in some untreated cells, but this was suppressed by depleting Wnt5a with RNA interference (RNAi) (Fig. 1B), which indicated that the basal signal results from autocrine responses to endogeneous ligand. Similar W-RAMP structures were observed in WM1789 melanoma cells, as...

AbtractEpithelial–mesenchymal transition (EMT) is an important contributor to the invasion and metastasis of epithelial-derived cancers. While considerable effort has focused in the regulators involved in the transition process, we have focused on consequences of EMT to prosurvival signaling. Changes in distinct metastable and ‘epigentically-fixed’ EMT states were measured by correlation of protein, phosphoprotein, phosphopeptide and RNA transcript abundance. The assembly of 1167 modulated components into functional systems or machines simplified biological understanding and increased prediction confidence highlighting four functional groups: cell adhesion and migration, metabolism, transcription nodes and proliferation/survival networks. A coordinate metabolic reduction in a cluster of 17 free-radical stress pathway components was observed and correlated with reduced glycolytic and increased oxidative phosphorylation enzyme capacity, consistent with reduced cell cycling and reduced need for macromolecular biosynthesis in the mesenchymal state. An attenuation of EGFR autophosphorylation and a switch from autocrine to paracrine-competent EGFR signaling was implicated in the enablement of tumor cell chemotaxis. A similar attenuation of IGF1R, MET and RON signaling with EMT was observed. In contrast, EMT increased prosurvival autocrine IL11/IL6-JAK2-STAT signaling, autocrine fibronectin-integrin α5β1 activation, autocrine Axl/Tyro3/PDGFR/FGFR RTK signaling and autocrine TGFβR signaling. A relatively uniform loss of polarity and cell–cell junction linkages to actin cytoskeleton and intermediate filaments was measured at a systems level. A more heterogeneous gain of ECM remodeling and associated with invasion and migration was observed. Correlation to stem cell, EMT, invasion and metastasis datasets revealed the greatest similarity with normal and cancerous breast stem cell populations, CD49fhi/EpCAM-/lo and CD44hi/CD24lo, respectively.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-010-9367-3) contains supplementary material, which is available to authorized users.

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Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect ofPDGF on increasing p7OS6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p7OS6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p7OS6K.

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