We have cloned a novel high affinity thrombospondin-1 (TSP-1) binding protein from a prostate cancer cell library. The protein, termed angiocidin, inhibited endothelial cell viability and tube formation in vitro and prevented the growth of tumors in vivo. When injected intravenously into mice bearing Lewis Lung carcinoma, the protein inhibited the growth of the tumor by more than 500% as compared to the buffer control. As shown by immunohistochemical staining of human breast tumors angiocidin localized to malignant ductal epithelium while no significant staining of epithelium in normal and benign tissues was observed. In this proposal we will test the hypothesis that angiocidin exerts its anti-angiogenic activity by competing with TSP-1, its high affinity ligand, and regulating expression of other endothelial proteins important in angiogenesis. We will establish which steps in the angiogenic process are inhibited by angiocidin, what structural determinants of angiocidin mediate this activity, and what other proteins may be binding and regulating angiocidin activity. Our goal is to understand how angiocidin blocks angiogenesis. Cur long range goal is to develop angiocidin as a treatment for cancer. These goals will be accomplished by the following aims.
In aim 1, we will establish the capacity of angiocidin to modulate the following TSP-1 dependent biological steps important in angiogenesis: a) endothelial cell adhesion to extracellular matrix b) growth factor dependent endothelial cell proliferation c) growth factor dependent endothelial cell invasion.
In aim 2 we will map the structural domains of angiocidin that mediate its anti- angiogenic activity and TSP-1 binding activity with synthetic peptides and monoclonal antibodies whose binding epitopes will be mapped using peptide phage display.
In aim 3 we will determine the molecular basis of the anti-angiogenic activity of angiocidin by establishing that endothelial cells bind angiocidin through cell surface receptors, which will be identified by affinity chromatography and molecular cloning. To further unravel the anti-angiogenic activity of angiocidin, genes regulated by angiocidin will be identified by cDNA expression array analysis. Finally in aim 4 the in vivo activity of angiocidin in preventing tumor growth as well as its in vivo distribution will be evaluated in a syngeneic mouse and orthotopic xenograft mouse model of tumor growth. These models will be used to show that injected angiocidin as well as angiocidin produced by transfected tumor cells decreases tumor microvessel density. The role of TSP-1 in the activity of angiocidin will be further evaluated in tumor bearing TSP-1 null mice. The experiments outlined above should advance the field of angiogenesis research by filling the gap in our understanding of how angiogenesis is regulated by molecules like angiocidin and its ligand TSP- 1. Finally, these experiments should provide the basis for the development of angiocidin as an anti-cancer therapeutic.
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