Jay Shankar

Pseudopodial Actin Dynamics Control Epithelial-Mesenchymal Transition in Metastatic Cancer Cells

A key cellular process associated with the invasive or metastatic program in many cancers is the transformation of epithelial cells toward a mesenchymal state, a process called epithelial to mesenchymal transition or EMT. Actin-dependent protrusion of cell pseudopodia is a critical element of mesenchymal cell migration and therefore of cancer metastasis. However, whether EMT occurs in human cancers and, in particular, whether it is a prerequisite for tumor cell invasion and metastasis, remains a subject of debate. Microarray and proteomic analysis of actin-rich pseudopodia from six metastatic human tumor cell lines identified 384 mRNAs and 64 proteins common to the pseudopodia of six metastatic human tumor cell lines of various cancer origins leading to the characterization of 19 common pseudopod-specific proteins. Four of these (AHNAK, septin-9, eIF4E, and S100A11) are shown to be essential for pseudopod protrusion and tumor cell migration and invasion. Knockdown of each of these proteins in metastatic cells resulted in reduced actin cytoskeleton dynamics and induction of mesenchymal-epithelial transition (MET) that could be prevented by the stabilization of the actin cytoskeleton. Actin-dependent pseudopodial protrusion and tumor cell migration are therefore determinants of EMT. Protein regulators of pseudopodial actin dynamics may represent unique molecular targets to induce MET and thereby inhibit the metastatic potential of tumor cells.

Regulation of mitophagy by the Gp78 E3 ubiquitin ligase

The Gp78 E3 ubiquitin ligase is shown to target the mitofusin mitochondrial fusion proteins for degradation, inducing mitochondrial fission and mitofusin 1–dependent mitophagy of uncoupled mitochondria. Mitophagy induced by endoplasmic reticulum–associated gp78 defines a distinct cellular pathway to eliminate damaged mitochondria.

The galectin lattice at a glance

ABSTRACT Galectins are a family of widely expressed β-galactoside-binding lectins in metazoans. The 15 mammalian galectins have either one or two conserved carbohydrate recognition domains (CRDs), with galectin-3 being able to pentamerize; they form complexes that crosslink glycosylated ligands to form a dynamic lattice. The galectin lattice regulates the diffusion, compartmentalization and endocytosis of plasma membrane glycoproteins and glycolipids. The galectin lattice also regulates the selection, activation and arrest of T cells, receptor kinase signaling and the functionality of membrane receptors, including the glucagon receptor, glucose and amino acid transporters, cadherins and integrins. The affinity of transmembrane glycoproteins to the galectin lattice is proportional to the number and branching of their N-glycans; with branching being mediated by Golgi N-acetylglucosaminyltransferase-branching enzymes and the supply of UDP-GlcNAc through metabolite flux through the hexosamine biosynthesis pathway. The relative affinities of glycoproteins for the galectin lattice depend on the activities of the Golgi enzymes that generate the epitopes of their ligands and, thus, provide a means to analyze biological function of lectins and of the ‘glycome’ more broadly. Summary: Galectin crosslinking of plasma membrane glycans regulates diffusion, compartmentalization and endocytosis of glycoproteins and glycolipids impacting immunity, cancer and metabolism.

Actin Cytoskeleton Regulation of Epithelial Mesenchymal Transition in Metastatic Cancer Cells

Epithelial-mesenchymal transition (EMT) is associated with loss of the cell-cell adhesion molecule E-cadherin and disruption of cell-cell junctions as well as with acquisition of migratory properties including reorganization of the actin cytoskeleton and activation of the RhoA GTPase. Here we show that depolymerization of the actin cytoskeleton of various metastatic cancer cell lines with Cytochalasin D (Cyt D) reduces cell size and F-actin levels and induces E-cadherin expression at both the protein and mRNA level. Induction of E-cadherin was dose dependent and paralleled loss of the mesenchymal markers N-cadherin and vimentin. E-cadherin levels increased 2 hours after addition of Cyt D in cells showing an E-cadherin mRNA response but only after 10-12 hours in HT-1080 fibrosarcoma and MDA-MB-231 cells in which E-cadherin mRNA level were only minimally affected by Cyt D. Cyt D treatment induced the nuclear-cytoplasmic translocation of EMT-associated SNAI 1 and SMAD1/2/3 transcription factors. In non-metastatic MCF-7 breast cancer cells, that express E-cadherin and represent a cancer cell model for EMT, actin depolymerization with Cyt D induced elevated E-cadherin while actin stabilization with Jasplakinolide reduced E-cadherin levels. Elevated E-cadherin levels due to Cyt D were associated with reduced activation of Rho A. Expression of dominant-negative Rho A mutant increased and dominant-active Rho A mutant decreased E-cadherin levels and also prevented Cyt D induction of E-cadherin. Reduced Rho A activation downstream of actin remodelling therefore induces E-cadherin and reverses EMT in cancer cells. Cyt D treatment inhibited migration and, at higher concentrations, induced cytotoxicity of both HT-1080 fibrosarcoma cells and normal Hs27 fibroblasts, but only induced mesenchymal-epithelial transition in HT-1080 cancer cells. Our studies suggest that actin remodelling is an upstream regulator of EMT in metastatic cancer cells.

p38 MAP kinase–dependent phosphorylation of the Gp78 E3 ubiquitin ligase controls ER–mitochondria association and mitochondria motility

Epitope mapping of the 3F3A mAb identified p38 MAPK phosphorylation of Ser-538 of the E3 ubiquitin ligase Gp78. p38 MAPK phosphorylation of Ser-538 prevents Gp78-dependent mitofusin degradation, mitochondrial fission, and ER–mitochondria association, defining a novel regulatory mechanism of Gp78 activity at the ER–mitochondria interface.

Coordinated expression of galectin-3 and caveolin-1 in thyroid cancer†

Galectin‐3 (Gal3) is the single most accurate marker for the diagnosis of differentiated thyroid cancer (DTC). Gal3 overrides the tumour suppressor activity of caveolin‐1 (Cav1) and functions in concert with Cav1 to promote focal adhesion turnover and tumour cell migration and invasion. To study their coordinated role in progression of a human cancer, we investigated the expression of Gal3 and Cav1 in specimens of human benign thyroid lesions, DTC and anaplastic thyroid cancer (ATC). Gal3 and Cav1 expression is significantly associated with DTC and ATC, but not benign nodules. Essentially all Cav1‐positive DTC cancers express Gal3, supporting the synergistic activity of these two proteins in DTC progression. Similarly, coordinated elevated Gal3/Cav1 expression was observed in three DTC‐derived cell lines (papillary TCP1 and KTC1 and follicular FTC133) but only one (ACT1) of five ATC‐derived cell lines. Using siRNA knockdown, Gal3 and Cav1 were shown to be required for RhoA GTPase activation, stabilization of focal adhesion kinase (FAK; a measure of focal adhesion signalling and turnover) and increased migration of the DTC cell lines studied, but not the ATC cell lines, including ACT1, which expresses elevated levels of Gal3 and Cav1. Co‐expression of Gal3 and Cav1 in the T238 anaplastic cell line stabilized FAK‐GFP in focal adhesions. Gal3 and Cav1 therefore function synergistically to promote focal adhesion signalling, migration and progression of DTC. Copyright

Raft endocytosis of AMF regulates mitochondrial dynamics through Rac1 signaling and the Gp78 ubiquitin ligase.

Summary Gp78 is a cell surface receptor that also functions as an E3 ubiquitin ligase in the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. The Gp78 ligand, the glycolytic enzyme phosphoglucose isomerase (PGI; also called autocrine motility factor, AMF), functions as a cytokine upon secretion by tumor cells. AMF is internalized through a PI3K- and dynamin-dependent raft endocytic pathway to the smooth ER; however, the relationship between AMF and Gp78 ubiquitin ligase activity remains unclear. AMF uptake to the smooth ER is inhibited by the dynamin inhibitor, dynasore, is reduced in Gp78 knockdown cells and induces the dynamin-dependent downregulation of its cell surface receptor. AMF uptake is Rac1-dependent and is inhibited by expression of dominant-negative Rac1 and the Rac1 inhibitor NSC23766, and is therefore distinct from Cdc42- and RhoA-dependent raft endocytic pathways. AMF stimulates Rac1 activation, but this is reduced by dynasore treatment and is absent in Gp78-knockdown cells; therefore, AMF activities require Gp78-mediated endocytosis. AMF also prevents Gp78-induced degradation of the mitochondrial fusion proteins, mitofusin 1 and 2 in a dynamin-, Rac1- and phosphoinositide 3-kinase (PI3K)-dependent manner. Gp78 induces mitochondrial clustering and fission in a manner dependent on GP78 ubiquitin ligase activity, and this is also reversed by uptake of AMF. The raft-dependent endocytosis of AMF, therefore, promotes Rac1-PI3K signaling that feeds back to promote AMF endocytosis and also inhibits the ability of Gp78 to target the mitofusins for degradation, thereby preventing Gp78-dependent mitochondrial fission. Through regulation of an ER-localized ubiquitin ligase, the raft-dependent endocytosis of AMF represents an extracellular regulator of mitochondrial fusion and dynamics.

Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

RNA Purification from Tumor Cell Protrusions Using Porous Polycarbonate Filters

Actin-rich cellular protrusions or pseudopodia form via local actin filament polymerization and branching and represent a variety of polarized cellular domains including lamellipodia, filipodia, and neuronal growth cones. RNA localization and local protein translation in these domains are important for various cellular processes. RNA transport and local synthesis have been implicated in cell migration and tumor cell metastasis as well as in neuronal plasticity in neurons. Characterization of the mRNAs present in these domains is key to understanding the functional role of mRNA translocation and local protein translation in cellular processes. We describe here a method to segregate pseudopodia of metastatic cancer cells from the cell body using porous polycarbonate filters. This approach enables the purification and identification of RNAs and proteins in these protrusive cellular domains.

Correction: Actin Cytoskeleton Regulation of Epithelial Mesenchymal Transition in Metastatic Cancer Cells

[This corrects the article DOI: 10.1371/journal.pone.0119954.].