Membrane and actin dynamics in the control of migratory and invasive strategies
Giorgio Scita Ph.D.
c/o IFOM-IEO Campus
Via Adamello, 16 - 20139 Milan, Italy
Tel: +39-02-574303277 - Fax: +39-02-574303231
Tumor cells utilize highly versatile strategies and processes to invade into surrounding tissue. Cell migration, a hallmark of metastasis, is a typical case in point. Recent studies using in vitro surrogate assays to model 3D cell migration and in vivo analysis of invading tumors by multi-photon-microscopy, have revealed how tumor cells use different motility modes to disseminate. Each of these modes is driven and controlled by distinct molecular pathways [2,3]. The plasticity and versatility of tumor cell migration is a result of cells being able to switch between these migratory modes . These adaptive responses provide migratory ‘escape’ strategies after pharmacotherapeutic intervention, by prompting alternative mechanisms of cancer cell dissemination in tissues that overcome single-pathway-hitting pharmacological weapons [4,5]. It is therefore essential to identify the various critical pathways and cellular processes that enable tumor cells to plastically adapt their motility modes during migration and invasion of surrounding tissues. Only after this knowledge has been gleaned, can pharmacological strategies targeted against multiple, deregulated tumor-specific migratory mechanisms, be devised and clinically tested with any hope of success.
An emerging critical process that regulates cell migratory modes is trafficking of plasma membrane components, such as Receptor Tyrosine Kinases (RTK) and integrins, and signaling molecules controlling actin dynamics-based motility and the localized production of intracellular-confined signaling cues . However, little is known as to how membrane trafficking and actin-based, motility machineries are co-opted in invasive cancer cells, and which are the endocytic players and genetic programs implicated in regulation of tumor migratory modes and invasion. Similarly, the pathways controlling directional migration are incompletely defined.
The goal of our laboratory is, therefore, to identify and elucidate how endocytic/recyling and signaling components control the dynamic location of critical regulators of actin reorganization, and control the expression of genes needed for the correct execution and mantainance of migration programs and the acquisition of metastatic properties, particularly in response to stimulation of RTKs.
Two lines of research are implemented within this context:
1. Endo/exocytic pathways in tye control of migratory programs.
The first step of cell motility and invasion is the extension of polarized, actin-based migratory protrusions. These include canonical lamellipodia, apically-restricted circular dorsal ruffles (CDR) and invadopodia, metalloproteases-rich, matrix adhesive platforms. We previously demonstrated that endocytosis and recycling controlled by the small G protein Rab5 are essential for the formation of CDR and the acquisition of a mesenchymal mode of motility of various tumor cells. More recently, we have accumulated evidence that Rab5 is also involved in the formation of invadopodia in response to HGF stimulation, and promotes invasion into 3D. Finally, preliminary observations indicated that the plastic ability of tumor cells to switch from and maintain different modes of motility requires new gene expression. Thus, Rab5 appear to be a critical endocytic molecule that regulates through distinct signalling pathways, trafficking routes and presumably genetic programs the formation of both apical CDR and ventral invadopodia, promoting the invasion of cells into 3D extracellular matrices.
Based on this background the specific aims of our research are:
1.1) To identify the critical trafficking steps, endo/exocytic molecules and signaling pathways controlled by Rab5 and RTK capable of coordinating different type of membrane protrusions (namely: lamellipodia, CDR), cell shape changes and polarity with formation of invasive metalloproteases-dependent structures in three dimension (Emanuela Frittoli, Andrea palamidessi/Pier Pier Paolo Di Fiore).
1.2) Identify novel actin regulatory molecules and endocytic proteins specifically involved in 3D migration and invasion by a stepwise, RNAi-based functional genomic screening and gene profiling of tumors cells undergoing Rab5- and HGF-induced amoeboid to mesenchymal transition ((Emanuela Frittoli, Andrea palamidessi/Pier Pier Paolo Di Fiore).
2. Molecular characterization of the networks of proteins linking actin and membrane dynamics.
One additional, yet not fully understood aspect in the regulation of migratory and invasive protrusion is the mechanism that enables the force generating system of the actin polymerization machineries to be connected to the plasma membrane during the extension of polarized migratory protrusions, the essential first step in 2D and 3D migration. We have been characterizing two minimal networks of actin regulators and membrane binding proteins that are predicted to specifically mediate actin and membrane dynamics, thus controlling cell migratory protrusions and/or trafficking processes. The first of this network comprises the actin remodeler Eps8 and VASP and the actin and membrane binding and bending proteins IRSp53, and critical contributes to the formation of flipodia, finger like projection marking the acquisition of direction motility; the second network is centered around a family of biochemically well characterized, but with ill-defined biological functions, F-Bar containing proteins, the Toca family, that interact with WASP and WAVE (our unpublished observations), two critical regulators of actin polymerizations mediating actin-based internalization and trafficking prcesses and epithelial morphogenesis (in the nematodes-our unpublished observations).
Within this context the specific goals of these lines of reaseach are:
2.1) Define the molecular mechanisms at the bases of the dynamic formation of Eps8, VASP, IRSp53 complexes using a combination of in vitro biochemical and cell biological approaches, which will be corroborated by the use of cells and systems derived from mice genetically devoid of each individual members of this network. The knowledge of the mechanisms of regulation of this network will be than utilized to explore the mechanisms whereby elevated expression of Eps8 confers migratory and invasive advantages to a subset of tumor cells of Oral squamous cell carcinoma, colon and pancreatic carcinoma (Andrea Disanza-cell and mouse models-, Francesca Milanesi-in vitro approaches)
2.2) Using the nematode as a model system we have recently shown that Toca family proteins are essential for endocytosis, further controlling epithelial morphogenesis by regulating the proper location of cell-cell junctional proteins in an actin dependent manner. The aim of this research line is thus to explore the role of this family of proteins in mammary epithelial morphogenesis using two dimensional epithelial monolayers and three dimensional epithelial sphere formation assays (Chiara Giuliani-C. elegans; Flavia Troglio: mammalian cells)
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