The growth of many malignant solid tumors is critically dependent on the proliferation of new vessels to supply the rapidly dividing cells with oxygen and nutrients. Recognition that inhibition of tumor angiogenesis leads to regression of these malignancies and controls metastatic spread in animals has led to a major effort to control cancer by targeting its vasculature. The urokinase receptor (uPAR) is not only highly expressed on malignant cells, but plays a critical role in regulating cell migration and adhesion of cell-associated proteolytic activity, which are all essential for angiogenesis. Recently, we have identified a new anti-angiogenic molecule, cleaved high molecular weight kininogen (HKa). We have shown that HKa binds to domain 2/3 of uPAR on endothelial cells and competes for occupancy with a major adhesive protein, vitronectin. HKa, when bound, also facilitates the binding and activation of PK to kallikrein, which cleaves pro-urokinase to urokinase by plasma kallikrein, and the subsequent enhancement of the formation of cell-associated plasmin from plasminogen. We now hypothesize that HKa-uPAR interactions may be critical in the control of angiogenesis, and that specific peptide sequences would be inhibitors of tumor angiogenesis. We will therefore study the binding of HKa to uPAR using both transfected cells and surface plasmon resonance with an emphasis on establishing which domains and subdomains of HKa are the sites of binding. We will use monoclonal antibodies, recombinant fragments (deletion mutants) and synthetic peptides derived from molecular homology models of domains 3 and 5 of HKa based on the crystal and NMR structures to identify the exact sequences responsible for HKa binding to urokinase. We will then test the effects of these polypeptides on four of the in vitro components of angiogenesis, namely, endothelial adhesion, migration, proliferation and cell surface-mediated proteolysis. Preliminary studies show that at nanomolar concentrations, deletion mutants or synthetic peptides derived from HKa inhibit migration of endothelial cells to vitronectin and endothelial cell proliferation. These studies will be extended using site-directed mutagenesis and optimized cyclic peptides to identify the minimum sequences. We will select the most potent peptides and test them in vivo on basic fibroblast growth factor stimulated chicken egg chorioallantoic membrane (CAM), where domain 5 at 30 nM inhibits angiogenesis, as well as tumor cells growing on the CAM. We will then test the polypeptides on human tumor cells which can grow in immunologically deficient mice. Finally, we will test these new anti-angiogenic peptides for their ability to prevent cancer growth metastasis in the Lewis lung tumor model. We expect that these studies will identify biologically active peptides which can serve as lead compounds in designing peptidomimetic angiogenic inhibitors.
Showing the most recent 10 out of 41 publications
