Role of N-WASP in skin cancer and role of N-WASP binding proteins in filopodia formation

The actin cytoskeleton, made of polymerized actin together with the actin associated proteins, plays a critical role in many actin-based structures, including cell adhesion and cell motility. N-WASP promotes the formation of actin-rich structures such as filopodia, which affect cell adhesion and motility. Although N-WASP regulates cell migration and adhesion, function of N-WASP in cancer and metastasis has not been well characterized. N-WASP is expressed in the non-tumorigenic HaCaT cells however the expression was found to be low in the tumorigenic, non-metastatic epithelial cell line, HSC5 and high in the metastatic cell line, A5-RT3. A qPCR analysis of the SCC patient samples revealed a consistently low expression of N-WASP. Analysis of the adhesion properties of epithelial cells in the presence and deficiency of N-WASP revealed that N-WASP is important for cell adhesion and spreading on fibronectin. The knockdown of N-WASP expression in the cell lines, HaCaT and A5-RT3, respectively led to decrease in cell-ECM adhesion and an increase in cell migration. The overexpression of N-WASP in a tumorigenic cell line HSC-5 resulted in an increase in the cell adhesion, spreading on fibronectin and a decrease in cell migration. Altered localization and expression level of paxillin and vinculin in cells deficient in N-WASP was observed in both tumorigenic and non-tumorgenic epithelial cell lines, HaCaT and A5-RT3, respectively. Thus, the data suggests that deficiency of N-WASP in both tumorigenic and non-tumorigenic cell lines leads to decrease in cell-ECM adhesion to fibronectin accompanied by an increase in cell motility. Also, the varied expression level observed in tumorigenic and metastatic cell lines indicate a role for N-WASP in metastasis. The role of the different domains of N-WASP in cell-ECM adhesion and migration were characterized using N-WASP-/- MEFs. A defect in the cell-ECM binding and enhanced migration was observed in the N-WASP-/- cells. The mutants, N-WASPΔWH1 and the N-WASPY256E failed to restore the cell-ECM adhesion in the N-WASP-/- (N-WASPΔWH1) and the N-WASP-/- (N-WASPY256E) cells and the mutants did no inhibit cell migration in both cell lines. An increase in the paxillin localization and a decrease in the vinculin localization were also observed in the N-WASP-/- (N-WASPΔWH1) and the N-WASP-/- (N-WASPY256E) cell lines. The above results hence suggest the importance of the WH1 domain and the site, N-WASPY256 in cell-ECM adhesion and migration. N-WASP interacts with the mammalian verprolins; WIP (WASP Interacting Protein), CR16 (Corticoid Regulated) and WIRE (WIp-RElated) in regulating the actin cytoskeleton. In order to characterize the function of CR16 independent of N-WASP, yeast two hybrid screen was carried out and IRSp53 (Insulin Receptor Substrate) was identified as a CR16 interacting protein. Expression of IRSp53 with CR16 in N-WASP−/− mouse embryonic fibroblasts induced filopodia. The induction of filopodia is dependent on CR16–IRSp53 interaction as mutation in the SH3 domain of IRSp53 caused the CR16–IRSp53 interaction to be abolished as well as the ability to induce filopodia. Similarly, the Verprolin (V)-domain of CR16 is critical for IRSp53–CR16 interaction and for filopodia formation. Cdc42 regulates the interaction between CR16 and IRSp53, as mutations, which abolish Cdc42-IRSp53 interaction, lead to loss of IRSp53–CR16 interaction. Expression of Cdc42G12V (active mutant) with CR16–IRSp53 caused significant increase in the number of filopodia per cell. Thus IRSp53 activity is controlled by CR16 by controlling the IRSp53–CR16 interaction to generate filopodiaDOCTOR OF PHILOSOPHY (SBS