Topotecan blocks hypoxia-inducible factor-1α, and vascular endothelial growth factor expression induced by insulin-like growth factor-I in neuroblastoma cells

The extent of angiogenesis and/or vascular endothelial growth factor (VEGF) expression in neuroblastoma tumors correlates with metastases, N-myc amplification, and poor clinical outcome. Understanding the mechanisms regulating VEGF expression in neuroblastoma cells provides additional therapeutic options to control neuroblastoma tumor growth. VEGF mRNA is controlled by growth factors and hypoxia via the transcription factor hypoxia-inducible factor (HIF-1 alpha). HIF-1 alpha protein levels are regulated by the von Hippel Lindau tumor suppressor gene, VHL, which targets HIF-1 alpha degradation. To determine whether the levels of VEGF in neuroblastomas are due to mutations in VHL, we evaluated genomic DNA from 15 neuroblastoma cell lines using PCR. We found no mutations in exons 1, 2, or 3 of the VHL gene. VEGF mRNA levels in neuroblastoma cells cultured in serum-free medium increased after 8 to 16 hours in serum, insulin-like growth factor-I (IGF-I), epidermal growth factor, or platelet-derived growth factor. Serum/IGF-I induced increases in HIF-1 alpha protein that temporally paralleled increases in VEGF mRNA, whereas HIF-1 beta levels were unaffected. VEGF and HIF-1 alpha levels were blocked by inhibitors of phosphatidylinositol 3-kinase and mammalian target of rapamycin. Furthermore, we confirmed that HIF-1 alpha mediates similar to 40% of the growth factor activity stimulating VEGF protein expression. Topotecan blocked the IGF-I-stimulated increase in HIF-1 alpha. but not HIF-1 beta, and this resulted in a decrease in VEGF in four neuroblastoma cell lines tested. These data indicate that growth factors in an autocrine or paracrine manner play a major role in regulating VEGF levels in neuroblastoma cells and that targeted therapies to phosphatidylinositol 3-kinase, mammalian target of rapamycin, and/or HIF-1 alpha have the potential to inhibit VEGF expression and limit neuroblastoma tumor growth.