Endostatin Suppresses Colorectal Tumor-Induced Lymphangiogenesis by Inhibiting Expression of Fibronectin Extra Domain A and Integrin α9

Endostatin is a natural occurring anti-angiogenic peptide and has been shown to inhibit tumor lymphangiogenesis by suppressing the expression of tumor-stimulating growth factors. We have previously shown that fibronectin alternative extra domain A (EDA) facilitates lymphangiogenesis of colorectal tumors. Since it is known that EDA interacts with integrin alpha 9 in the lymphatic endothelial cells (LECs), we hypothesized that endostatin may target EDA-integrin alpha 9 pathway to inhibit colorectal tumor-induced lymphangiogenesis. To test this hypothesis, we examined the effect of endostatin on EDA secreted by SW480 colorectal cancer cells and treated human LECs with different doses of endostatin in the presence of conditional medium from SW480 cells. We found that endostatin significantly reduced EDA secretion by SW480 cells and the expression of integrin alpha 9 in LECs. Immunofluorescence studies showed that EDA and integrin alpha 9 colocalized on the cell membrane of LECs and these colocalizations were dramatically reduced by endostatin. Co-immunoprecipitation studies demonstrated that EDA interacted with integrin alpha 9 in LECs, and showed that endostatin treatment inhibited the formation of EDA-integrin alpha 9 complex in LECs. Furthermore, we found that the arrangement and polarity of LEC cytoskeletons were destroyed by endostatin substantially, leading to a reduced formation of tube-like structures of LECs and a suppressed chemotaxis of LECs toward SW480 cells. Consistently, EDA and integrin alpha 9 expressions as well as lymphangiogenesis were significantly suppressed by endostatin in colorectal cancer xenografts. In conclusion, our results suggest that endostatin reduces colorectal tumor-induced lymphangiogenesis, at least in part, by inhibiting EDA-integrin alpha 9 pathway. J. Cell. Biochem. 112: 2106-2114, 2011. (C) 2011 Wiley-Liss, Inc.