Angiogenesis, or the formation of new blood vessels, is believed to be required for growth of tumors of the breast beyond a maximum size of 1-2 mm. The factors that regulate tumor angiogenesis are incompletely understood but directed endothelial cell (EC) movement, followed by proliferation of trailing EC, appears to initiate the formation of capillaries during normal vessel development and tumor angiogenesis. Tumor-derived basic fibroblast growth factor (bFGF) may be an important initiator of tumor angiogenesis since the factor potently stimulates EC movement in vitro and since a significant fraction of breast cancer tumors exhibit amplification of FGF- or FGF receptor-related genes and expression of their protein products. The signal transduction pathway(s) responsible for bFGF-stimulated movement is not known; however, we have evidence that the activity of a novel, 56 kDa pertussis toxin-sensitive G-protein is required. This G-protein (designated G/m, or putative motility-related G- protein) is physically associated with the flg-type FGF receptor, FGFR-1 and is activated by bFGF to bind GTP. G/m is required for maximal activation of the cytosolic form of phospholipase A/2 (cPLA/2), and the arachidonate released (or a downstream metabolite) is necessary for bFGF- mediated EC movement. The long-term goal of our research is to understand this pathway in detail, and in particular, to elucidate the role of G m in angiogenic responses of EC. We propose as a hypothesis that breast tumor cells secrete bFGF which initiates migratory processes in EC of nearby blood vessels. We also propose that the interaction of bFGF with FGFR-1 activates G/m by increasing GTP-binding, and that G/m-GTP activates cPLA/2 to release arachidonate, the precursor of a promigratory eicosanoid(s). We will test this hypothesis by pursuing the following Specific Aims: (1) map the site on FGFR-1 that interacts with G/m, (2) purify G/m from fetal bovine brain, (3) characterize the functional and kinetic properties of purified G/m, and (4) investigate the presence and location of G/m in breast cancer tissues. Successful completion of these Specific Aims will provide important molecular information on the signaling pathways regulating bFGF-stimulated EC motility. The elucidation of the intracellular pathways that regulate EC movement, and therefore the initial steps of tumor angiogenesis, may provide a foundation for the development of therapies that interfere with these processes in vivo. Since the signaling events initiated by diverse growth factors may converge to a single critical pathway regulating cell movement, our approach may have particular significant in view of the ability of transformed cells able to secrete multiple growth- and motility-inducing agents. Identification of G/m as a factor involved in early breast cancer may also have diagnostic value in guiding the course of surgical adjuvant therapy to optimize patient outcome.
