Mechanisms of thymidine phosphorylase angiogenesis

DESCRIPTION provided by applicant): There is an increasing appreciation of the role of angiogenesis in the growth and metastasis of solid tumors, and data suggest that antiangiogenesis therapy could be a viable approach to treatment of advanced disease. Thymidine phosphorylase (TP) has been recently found to be an angiogenic factor, an observation that was based on its identity to a previously described angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF). TP is expressed at high levels in many human solid tumors including colorectal cancers, and the level of its expression has been correlated in numerous clinical studies, including one we conducted, with neovascularization, tumor aggressiveness, and poor patient prognosis. Immunohistochemical evaluation of TP by us and others has demonstrated that elevated TP expression in many, but not all, colon tumors occurs mainly in tumor-associated macrophages (TAMs), rather than in the tumor cells. We have found that a human monocytic cell line (THP1) expressed a higher level of TP than human colon carcinoma cell lines. We also found that TNF-alpha stimulated TP expression in THP1 cells and in WiDr colon carcinoma cells, and we will use these two cell lines as well as primary cultures of TAMs to determine the molecular basis for the regulation of TP expression (i.e. transcriptional, post-transcriptional). TP's angiogenic activity appears to be dependent on its catalytic activity, with 2-D-deoxyribose (2dR) serving as the putative mediator of endothelial cell activation. We have established an in vitro model in which human cancer cells or monocytes can be co-cultured with normal human endothelial cells (HUVEC), and can induce their migration in a TP-dependent manner. We will use this model to begin to determine the mechanisms by which TP stimulates HUVEC and human microvascular endothelial cell (HMEC) migration, using analogs of thymidine and 2dR we will synthesize to test the hypothesis that migration is solely related to 2dR formation, and by using a TP inhibitor we have made and a neutralizing TP antibody to test the hypothesis that TP's intracellular actions are sufficient to stimulate HUVEC migration. Little further is known of the mechanism of action of TP. Our studies showing differences in the specific integrins which mediate TP vs. VEGF-induced HUVEC migration are the first to explore the cellular actions of TP on endothelial cells. We have also found that TP induces tyrosine phosphorylation of FAK (focal adhesion kinase), a protein that plays a central role in cell attachment, migration, and signaling. We will continue our studies to define the integrin-associated signal transduction pathways that are activated by TP in HUVEC and HMEC, testing the hypothesis that these differ initially from those occurring in response to angiogenic factors such as VEGF and bFGF, which have clearly defined cell-surface receptors. In addition to providing a basic understanding of the mechanisms of angiogenic action of TP colon cancers, these studies could provide insights to optimize the use of current and future anti-angiogenic agents which target integrins.