TEK is a newly cloned orphan receptor tyrosine kinase that is expressed on endothelial precursors and on endothelial cells participating in vessel growth such as in the embryonic vasculature and the adult wound vasculature. This restricted pattern of expression suggests that TEK might be an important regulator of endothelial differentiation, blood vessel assembly, and vascular growth and repair. The long range goals of the present proposal are to determine the role of TEK in breast tumor angiogenesis and to develop rational approaches to inhibit tumor angiogenesis based on disrupting TEK signal transduction. Towards these goals, the specific aims of this proposal are to: 1) Identify the TEK ligand. Because TEK is expressed exclusively on endothelia of growing blood vessels the TEK ligand is likely to be an important mediator of angiogenesis. Identification of the TEK ligand and characterization of the structure function relationships involved in TEK ligand binding would allow rational development of TEK specific antagonists. For use in expression cloning systems, we have developed a versatile affinity reagent consisting of the TEK extracellular domain fused to molecular """"""""tags"""""""" which allow one step purification and high specific activity radioactive or fluorescent labeling. 2) Develop and test rational inhibitors of tumor angiogenesis based on mutated forms of TEK. Mutated forms of various receptor tyrosine kinases including soluble extracellular and kinase deficient forms, have been shown to be potent inhibitors of kinase activation and signal transduction. Considering the potential role of TEK in tumor vessel growth, we have developed both the soluble extracellular form and the dominant negative form of TEK for use as angiogenesis inhibitors. We will test delivery systems for these inhibitors in vitro in cultured endothelial cells and smooth muscle cells and ex vivo in excised carotid arteries. The most effective delivery systems will then be tested in vivo in a novel tumor window model of tumor angiogenesis. 3) Determine the expression of TEK relative to other endothelial receptors in human breast cancer specimens. Because TEK is expressed only on endothelial cells involved in angiogenesis, it holds special promise as a specific target for the tumor endothelium. However, other receptor tyrosine kinases such as the receptors for FGF and VEGF may also play a role in the development of the tumor vasculature. To determine the relative roles of these endothelial receptors in breast tumor angiogenesis, we will use in situ hybridization and immunohistochemistry to determine the relative expression of TEK, FGF receptor 1, and the VEGF receptors, flt and flk , in human breast cancer specimens. These studies will help to define the importance of TEK in the breast tumor vasculature and to identify populations of patients which may benefit from anti-TEK therapy.
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