The overall goal of this application is to test a hypothesis that the clinical translation of angiogenesis inhibitors for treating cancer may be facilitated by categorizing them into 'direct' and 'indirect' angiogenesis inhibitors. 'Direct' angiogenesis inhibitors block vascular endothelial cell proliferation and/or migration in response to a wide spectrum of pro-angiogenic proteins. Endothelial cells are genetically stable targets. 'Indirect' angiogenesis inhibitors down-regulate expression of an oncogene by tumor cells (e.g., EGF receptor tyrosine kinase), or block a product of that oncogene, (e.g., VEGF), or block a receptor for that product, (VEGF receptor). These are genetically unstable targets, and as a result 'indirect' angiogenesis inhibitors may eventually lose their efficacy due to the emergence of tumor clones which produce pro-angiogenic factors not targeted by the inhibitor. Toward this goal, we will characterize four 'direct' angiogenesis inhibitors in an attempt to modify the structure of two of them already in clinical trials, endostatin and TNP-470, to improve efficacy. A third 'direct' angiogenesis inhibitor recently discovered in human thyroid carcinoma will be purified and sequenced for its potential application in the clinic, and because it should enlarge our understanding of how the body normally employs endogenous angiogenesis inhibitors as tumor suppressor proteins. A fourth 'direct' angiogenesis inhibitor, thrombospondin-1, is also in clinical trials. It behaves as a major tumor suppressor gene by virtue of the fact that many different tumors repress expression of thrombospondin as a pre-requisite to switching to the angiogenic phenotype from a dormant in situ, non-angiogenic tumor. The molecular pathways by which thombospondin-1 expression is repressed during tumorigenesis of carcinomas vs sarcomas will be elucidated. The Specific Aims in this study contribute to a long-term future goal of the Folkman lab, to employ 'direct' angiogenesis inhibitors to prevent the angiogenic switch. Currently, all angiogenesis inhibitors in clinical application are employed only after the angiogenic switch. [unreadable] [unreadable]