English

Abstract

Introduction Src kinase activation has been rep orted in unrelated human cancers and considered a possible target of anti-invasive therapies. Our aim here is to identify the relationship of Src kinase with CD44-signalling. Although CD44 is implicated in the invasion of cancer cells, its role in invadopodia formation needs further elucidation. Materials and Methods To study the role of Src, PC3 cells were transfected with Src constructs by adenoviral-mediated delivery. Gelatin degradation assay was performed to determine the invasive property of PC3 cells. Immunoblotting and immunostaining analyses were used to determine complex interaction of proteins with CD44. PC3 cells knockdown of CD44 were used to characterize the function of CD44 in invadopodia formation and invasion. Results In this study, we show that expression of constitutively-active Src in prostate cancer 3 (PC3) cells significantly increased the invasiveness of PC3 cells via increasing the number of invadopodia as a result of CD44-associated complex (Src-Cortactin-WiskottAldrich Syndrome Protein WASP) forma tion. The inhibition of invadopodial structures in CD44 knockdown PC3 cells resulted in reduced invasion. Conclusion Based on these results, we conclude that CD44-Src-cortactin-WASP is an invasion promoting complex. Proteins in this complex are relevant targets for intervening invadopodia formation and migration/invasion processes in prostate cancer cells. Introduction Prostate cancer is a disease of extensive metastases, with secondary lesions in lymph nodes, bones and sometimes in visceral organs, including liver, lungs and even the brain. Actin dynamics is a critical process that modulates cellular activity via regulation of signalling pathways through receptors and cell adhesion to the extracellular matrix (ECM). CD44 is a cell surface receptor and regulator of cell migration and tumour metastasis1–5. We have shown pre viously in prostate cancer cells (PC3) that surface expression of CD44 and formation of CD44-Matrix metalloproteinase 9 (MMP9) complex generates motility enhancing signals through the degradation of ECM proteins6,7. These cells use invadopodia-like structures for invasion into ECM. Localisation of MMP9 in the invadopodia assists in the focal degradation of matrix during invasion7. Signalling and actin-binding proteins (e.g. Src, cortactin, WASP and Arp2/3) have been shown to contribute to the formation of invadopodia8–12. CD44 receptor has the potential to inte grate adhesive and signalling activities to modulate migration/invasion processes during cancer progression. CD44 shares some properties with the integrin receptor. Similar to integrin, the cytoplasmic domain of CD44 associates with several proteins involved in signalling and actin dynamics13,14. However, the role of CD44 in the form ation of invadopodia is not clear. PC3 cells knockdown of MMP9 (PC3/MMP9-/-) are highly adhesive which is due to the expression of highly glycosylated CD44 variant isoform v6. These cells are less invasive due to a deficiency in the expression of standard CD44 (CD44s) and failure in the formation invadopodia2. Pertinent to proteins that CD44 interacts with, it is highly possible that CD44 might regulate the formation of invadopodia. Therefore, we hypothesise that similar to integrins, CD44 associates with proteins involved in signalling, and these, in turn, are associated with actin and actin-binding proteins. We show here that Src has a direct role in the formation of invadopodia through its interaction with CD44. CD44-associated signalling complex (Src, WASP, cortactin and Arp2/3) regulates the process of actin polymerisation involved in invadopodia formation. Materials and Methods The protocol of this study has been approved by the relevant ethical committee related to our institution in which it was performed. Antibodies towards CD44, MMP9, Src, WASP, cortactin and cortactin (Y421) were bought from Santo Cruz Biotechnology, Inc. (Santa Cruz, CA). An antibody towards phosphoSrc Y418 was obtained from Cell Signalling (Beverly, MA). Rhodamine phalloidin and other chemicals were purchased from Sigma. CY2 and CY3 conjugated secondary antibodies were purchased from Jackson Immuno Research Laboratories, Inc. (West Grove, PA).