TRAIL negatively regulates VEGF-induced angiogenesis via caspase-8-mediated enzymatic and non-enzymatic functions.

Solid tumors supply oxygen and nutrients required for angiogenesis by producing vascular endothelial growth factor (VEGF). Thus, inhibitors of VEGF signaling abrogate tumor angiogenesis, resulting in the suppression of tumor growth and metastasis. We here investigated the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on VEGF-induced angiogenesis. TRAIL inhibited VEGF-induced in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs) and in vivo neovascularization in chicken embryos and mice. TRAIL blocked VEGF-induced angiogenic signaling by inhibiting ERK, Src, FAK, paxillin, Akt, and eNOS. Further, TRAIL blocked intracellular Ca(2+) elevation and actin reorganization in HUVECs stimulated with VEGF, without inhibiting VEGF receptor-2 tyrosine phosphorylation. TRAIL increased caspase-8 activity, without inducing caspase-9/-3 activation and apoptosis. Moreover, TRAIL resulted in cleavage of FAK into FAK-related non-kinase-like fragments in VEGF-stimulated HUVECs, which was blocked by a caspase-8 inhibitor and cellular caspase-8-like inhibitory protein. Biochemical and pharmacological inhibition of caspase-8 and FAK blocked the inhibitory effects of TRAIL on VEGF-stimulated anti-angiogenic signaling and events. In addition, caspase-8 knockdown also suppressed VEGF-mediated signaling and angiogenesis, suggesting that procaspase-8 plays a role of a non-apoptotic modulator in VEGF-induced angiogenic signaling. These results suggest that TRAIL inhibits VEGF-induced angiogenesis by increasing caspase-8 activity and subsequently decreasing non-apoptotic signaling functions of procaspase-8, without inducing caspase-3 activation and endothelial cell cytotoxicity. These data indicate that caspase-8 may be used as an anti-angiogenic drug for solid tumors resistant to TRAIL and anti-tumor drugs.