Tumor escape from endogenous, extracellular matrix-associated angiogenesis inhibitors by up-regulation of multiple proangiogenic factors

Purpose: Thrombospondin-1 (Tsp1), endostatin, and tumstatin are extracellular matrix-associated proteins that inhibit angiogenesis. We examined the mechanisms by which tumor cells may bypass the antiangiogenic effects of these endogenous regulators. Experimental Design: CT26 colon and RenCa renal carcinoma cells were stably transfected with Tsp1, endostatin, or tumstatin cDNA. Subcutaneous and metastatic tumor growth in syngeneic mice was analyzed. Expression of proangiogenic factors in resulting tumors was measured by quantitative real-time PCR. The combination of Tsp1 and vascular endothelial growth factor (VEGF) receptor-2 inhibition was also examined. Results: There was significant suppression of angiogenesis in flank tumors and liver metastases formed from cells overexpressing Tsp1, endostatin, or tumstatin. However, all tumors ultimately escaped angiogenesis inhibition. The combination of all three angiogenesis inhibitors had no additive effect beyond overexpression of a single inhibitor. Using quantitative real-time PCR, we found that VEGF and platelet-derived growth factor (PDGF) -A levels were routinely up-regulated at least 5-fold in all CT26 tumors overexpressing any antiangiogenic protein, and there were variable increases in angiopoietin 2 (Ang2), basic fibroblast growth factor, and PDGF-B. In contrast, RenCa tumors, which have high baseline levels of VEGF and PDGF-B, relied on basic fibroblast growth factor, Ang1, and PDGF-A up-regulation to counteract Tsp1 overexpression. Growth of CT26 cells with Tsp1 overexpression was suppressed when anti-VEGFR-2 treatment was added. Conclusions: Cancer cells with overexpression of three different endogenous angiogenesis inhibitor eventually escape angiogenesis inhibition by up-regulation of various proangiogenic factors. Tsp1, endostatin, and tumstatin may be functionally redundant in this system. These endogenous angiogenesis inhibitors are likely best used in combination with the blockade of proangiogenic pathways or with traditional chemotherapy or radiation therapy.