Thrombospondin (TSP) is among the matrix molecules that have been implicated in angiogenesis. We recently reported that the extracellular matrix of breast cancer contains large amounts of TSP and that matrix- bound TSP promotes angiogenesis from vascular explants. TSP also induces and stabilizes capillary-like tubes formed by endothelial cells. In addition, we have shown that TSP promotes tumor cell invasion and up- regulates breast cancer cell production of plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) through a mechanism involving transforming growth factor beta-1 (TGF-beta). In this grant we will test the hypothesis that stromal cell-derived TSP promotes both breast cancer invasion and angiogenesis by modulating the activity of TGF-beta which in turn up-regulates production of PAI-1 and uPA. We will determine which stromal cells produce TSP in response to breast cancer cells by measuring TSP produced in tumor/stromal cell co- culture assays and by immunolocalizing TSP in tumor biopsies at the ultrastructural level. TSP will be measured by immunochemistry and Northern blot analysis. Functional involvement of stromal-derived TSP in tumor cell invasion will be assessed with specific antibodies against TSP and its CSVTCG-specific receptor using two different tumor/stromal cell co-culture assays. We will then determine the role of PAI-1 and uPA in TSP-induced tumor cell invasion and angiogenesis by evaluating the effect of anti-PAI-1 and uPA antibodies in our co-culture invasion and angiogenesis assays. We will also evaluate the effect of TSP on endothelial production of PAI-1 and uPA. To investigate how the TSP effects relate to TGF-beta, we will determine whether TSP up-regulates activation and/or production of TGF-beta by tumor and endothelial cells and whether TGF-beta in turn up-regulates TSP. These studies will be accomplished both at the protein and mRNA levels. We will also test the effect of anti-TGF-beta antibody on invasion and angiogenesis in TSP- treated cultures. Finally, we will design synthetic TSP antagonist peptides based on the TSP-adhesive amino acid sequences CSVTCG and CSTSCG and test their inhibitory activity in our angiogenesis and invasion assays. These studies will contribute to our long-term goal of developing new treatment for breast cancer based on the mechanisms whereby TSP modulates tumor cell invasion and angiogenesis.
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