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A Hypoxia-Driven Vascular Endothelial Growth Factor/Flt1 Autocrine Loop Interacts with Hypoxia-Inducible Factor-1 through Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase 1/2 Pathway in Neuroblastoma

¹ New Agent and Innovative Therapy Program, Division of Hematology and Oncology, Department of Pediatrics and ² Department of Pediatric Laboratory Medicine and Pathobiology, Hospital for Sick Children; ³ Institute of Medical Science, ⁴ Department of Laboratory Medicine and Pathobiology, and ⁵ Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; and ⁶ Department of Genetics, Institute of Medical Sciences, University of Tokyo, Japan

Requests for reprints: Sylvain Baruchel, New Agent and Innovative Therapy Program, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G1X8. Phone: 416-813-7795; Fax: 416-813-5327; E-mail: sylvain.baruchel{at}sickkids.on.ca.

Flt1, an "fms-like tyrosine kinase" receptor, has been suggested to play an active role in vascular endothelial growth factor (VEGF)–mediated autocrine signaling of tumor growth and angiogenesis. Here, we used a neuroblastoma model to investigate the role of VEGF/Flt1 signaling in hypoxia-mediated tumor cell survival, drug resistance, and in vivo angiogenesis. SK-N-BE(2), a highly malignant neuroblastoma cell line resistant to hypoxia-induced apoptosis expresses active Flt1 but lacks VEGFR2 expression. We found that 24-hour hypoxia (<0.1% O₂) alone (no serum deprivation) showed sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) associated with bcl-2 up-regulation and resistance to etoposide-induced (5 µmol/L) apoptosis. Treatment with anti-VEGF and anti-Flt1 antibodies inhibited ERK1/2 activation, down-regulated bcl-2, and reversed the hypoxia-mediated drug resistance to etoposide. Similar results were obtained with U0126 and ursolic acid, specific and nonspecific inhibitors of ERK1/2, respectively. We confirmed the protective role of Flt1 receptor by small interfering RNA knockout and Flt1 overexpression studies. Subsequently, we found that inhibition of VEGF/Flt1 autocrine signaling led to reduced hypoxia-inducible factor-1 (HIF-1) phosphorylation. Furthermore, the reduced phosphorylation was associated with down-regulation of basic fibroblast growth factor, a downstream target of the HIF-1 and VEGF pathways. Our findings suggested an expanded autocrine loop between VEGF/Flt1 signaling and HIF-1. We investigated the angiogenic activity of the loop in an in vivo Matrigel plug assay. The hypoxia-treated conditioned medium induced a strong angiogenic response, as well as the cooption of surrounding vessels into the plugs; ursolic acid inhibited the angiogenesis process. We also found that three other Flt1-expressing neuroblastoma cell lines show hypoxia-mediated drug resistance to etoposide, melphalan, doxorubicin, and cyclophosphamide. Taken together, we conclude that a hypoxia-driven VEGF/Flt1 autocrine loop interacts with HIF-1 through a mitogen-activated protein kinase/ERK1/2 pathway in neuroblastoma. The interaction, in the form of an autocrine loop, is required for the hypoxia-driven cell survival, drug resistance, and angiogenesis in neuroblastoma.

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