VEGFR1, an “fms-like tyrosine kinase” receptor has been suggested to play active role in VEGF mediated autocrine signaling of tumor growth and angiogenesis. Here, we investigated the role of VEGF/VEGFR1 signaling in hypoxia-mediated tumor cell survival and angiogenesis. SK-N-BE-2, a highly malignant p53 mutated neuroblastoma cell line resistant to hypoxia-induced apoptosis expresses an active VEGFR-1 receptor in the absence of VEGFR-2. Cells were exposed to severe hypoxia (<0.1% O2) for 24hr alone or concurrent treatment with a neutralizing anti-VEGF antibody. Cellular survival and apoptosis was measured by alamar blue cytotoxicity and caspase-3/7 activation assay. The expression of hypoxia and cellular survival -related proteins HIF-1, bcl-2, Bax, XIAP, and survivin was studied by Western blot analysis. We found that SK-N-BE-2 cells survived 24hr of hypoxia and this was associated with the upregulation of HIF-1, VEGF, VEFR1 and bcl-2. Treatment with anti-VEGF antibody down-regulated bcl-2, and induced apoptosis during hypoxia. Further investigation revealed that anti-VEGF antibody treatment down-regulated hypoxia-induced sustained activation of ERK1/2. Interestingly, treatment with a specific MEK 1/2 inhibitor, U0126 resulted in the down-regulation of bcl-2, VEGF, and HIF-1 and induction of cellular apoptosis during hypoxia. We then investigated the expression of bFGF (basic fibroblast growth factor), a potent angiogenic peptide involved in tumor angiogenesis. We found that hypoxia significantly upregulated bFGF, while treatment with U0126 downregulated bFGF. Together, our results suggest that VEGF-VEGFR1 form an autocrine loop that co-operates with HIF-1 to maintain sustained activation of ERK1/2 during hypoxia which is then required for the bcl-2 and bFGF upregulation during hypoxia leading to cell survival and enhanced angiogenic potential.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]