Our laboratory recently cloned angiocidin, a protein capable of binding to thrombospondin-1(TSP-1). TSP-1 is a matrix protein that has been shown to have numerous and complex roles in cancer growth and metastasis. Angiocidin is over-expressed in many solid tumor types and tumor capillary endothelial cells. The exogenous protein has been thought to function as an endogenous inhibitor of tumor growth and angiogenesis. The anti-tumor growth effects of exogenously added angiocidin include the induction of apoptosis in both endothelial and tumor cells, the inhibition of tumor cell invasion, the inhibition of endothelial tube formation, and the inhibition of tumor growth in Lewis Lung carcinoma implants. In this study, we sought to evaluate the function of endogenously expressed angiocidin in endothelial cells. We transfected anti-angiocidin siRNA in human umbilical vein endothelial cells (HUVEC). We observed a 90% reduction of the target mRNA levels after 24 hours, as evaluated by RT-PCR. Endogenous angiocidin expression was reduced by 80% after three days, as evaluated by western blot analysis. Anti-angiocidin siRNA also down-regulated 50% of the activity and 90% of the protein expression of matrix metalloproteinase 2 (MMP-2) in HUVEC. Reduction of endogenous angiocidin inhibited endothelial tube formation in Matrigel. Cells expressing low levels of angiocidin grew more slowly and were less invasive than control cells. We also found that the expression of polyubiquitinated proteins was higher in anti-angiocidin siRNA treated cells as compared to normal and control siRNA treated cells. These results suggest that endogenous angiocidin in contrast to exogenous angiocidin promotes endothelial cell invasion and angiogenesis primarily through its ability to mediate polyubiquitin- dependent protein degradation. Additionally, the lack of the MMP-2 expression in the siRNA treated endothelial cells may also contribute to their anti-angiogenic phenotype. Furthmore, our results suggest that endogenous angiocidin is an important factor in the regulation of MMP-2 expression.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]