Administration of anti–integrin α5 or -VEGF antibody inhibited TM4SF5-mediated vessel formation and tumor growth in nude mice. (A) When tumors sized to approximately 100 mm3, normal IgG, anti–integrin α5 or -VEGF (2.5 mg/kg body weight) was injected directly to the tumors twice a week (n = 3) with measurements of weights and tumor sizes to calculate tumor volumes (mean ± SD). (B-D) Tumors in the mice were dissected out and paraffin-blocked for immunohistochemistry for CD31 or integrin α5 (original magnification × 100) (B) or frozen immediately using liquid N2 for immunoblots of the indicated molecules (C,D). *P < .05 was considered significant. (E) The working model: Cooperation between TM4SF5 and integrin α5 results in c-Src activation, which plays roles in intracellular signaling as a complex with FAK.8 Integrin-mediated FAK activation appears to be synergistic with TM4SF5-mediated c-Src activation, but inactivation of FAK does not block the TM4SF5-mediated c-Src activation and VEGF induction. Physical association between TM4SF5 and the cytoplasmic tail of integrin α5 may be a potential underlying mechanism. Activation of the c-Src/FAK complex can lead to activation of STAT3 by phosphorylation at Tyr705, which can in turn promote transcriptional activity of the VEGF promoter. VEGF induced by cooperation between TM4SF5 and integrin α5 is secreted from epithelial tumor cells. The secreted VEGFs can bind to VEGF receptor on neighboring endothelial cells, consequently promoting angiogenic activities, including proliferation, viability/survival, and sustained vessel-like tube formation.
