The mechanism of integrin inactivation and cell signalling induced by c-erbB2, an oncogenic growth factor receptor

Abstract

Overexpression of the oncogenic growth factor receptor, c-erbB2, is linked with poor prognosis in breast carcinomas and is used as a target for therapeutic strategies. To elucidate the mechanism underlying the tumorigenic action of an elevated c-erbB2 level, leading to homodimerization and activation, we have used an inducible system where a hybrid receptor consisting of the cytoplasmic and transmembrane domains of c-erbB2 fused to the extracellular domain of the trkA NGF receptor has been stably transfected into an immortalized mammary epithelial cell line. In the transfectants, the homodimerization and activation of c-erbB2 is induced by adding NGF. Using this system, we have examined the intracellular signalling induced by c-erbB2 homodimerization with respect to changes in cell-matrix interaction and cell morphology, events that are essential for development of metastasis in tumours. Analyses of the spreading and adhesive ability of the cells on collagen revealed that an early effect of c-erbB2 homodimer signalling was to inhibit the function of the Ñ2 Ò1 integrin, a collagen-binding adhesion molecule. We could further demonstrate that the major mechanism of Ñ2 Ò1 integrin inactivation is due to extracellular conformational change, and that it was mediated by several parallel signalling pathways downstream of c-erbB2 when monitored using pharmacological inhibitors, transient transfections, and conformational sensitive antibodies. These signalling pathways were MEK ERK kinase 1/2 (MEK 1/2), Protein Kinase B (PKB) and Rho pathways, where PKB contributes to suppression of adhesion and inactivation of extracellular conformation of Ò1 integrin through cytoskeletal rearrangements. We also could show that another signal protein, integrin-linked kinase (ILK), inhibits adhesion upon c-erbB2 homodimer signalling. However in contrast to other signalling pathways involved, ILK was not able to change integrin conformation. ILK is implicated in regulation of several cellular processes such as cell adhesion, cell morphology and cell motility; moreover it can promote cell cycle progression and tumour formation. An approach to clarify the role of ILK in suppression of cell-matrix adhesion downstream of c-erbB2, is to elucidate the mechanism underlying ILK activation when c-erbB2 homodimerization is induced. Using in vitro kinase assays and RNAi technique we could demonstrate that c-erbB2 induced mode of ILK activation is regulated by a SH2/SH3 domain containing adaptor protein, Nck-2, that was further shown to interact with c-erbB2, when homodimerized. Nck-2 may be important in ILK mediated inhibition of adhesion, downstream of c-erbB2 and may act as a possible link between ILK and cerbB2 in carcinogenesis, upon long-term activation of this receptor. In summary, this thesis presents new findings about inside-out regulation of integrin-mediated adhesion, which gives in addition a better insight into the mechanism behind c-erbB2 induced suppression of cell-matrix interaction, presumably an early event in carcinogenesis induced by overexpression of this oncoprotein.