Topotecan blocks hypoxia-inducible factor-1alpha 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-1alpha). HIF-1alpha protein levels are regulated by the von Hippel Lindau tumor suppressor gene, VHL, which targets HIF-1alpha 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-1alpha protein that temporally paralleled increases in VEGF mRNA, whereas HIF-1beta levels were unaffected. VEGF and HIF-1alpha levels were blocked by inhibitors of phosphatidylinositol 3-kinase and mammalian target of rapamycin. Furthermore, we confirmed that HIF-1alpha mediates approximately 40% of the growth factor activity stimulating VEGF protein expression. Topotecan blocked the IGF-I-stimulated increase in HIF-1alpha but not HIF-1beta, 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-1alpha have the potential to inhibit VEGF expression and limit neuroblastoma tumor growth.
Beppu
Kiichiro
K
Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Nakamura
Katsuya
K
Linehan
W Marston
WM
Rapisarda
Annamaria
A
Thiele
Carol J
CJ
D000970:Antineoplastic Agents / Q000494:pharmacology; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007334:Insulin-Like Growth Factor I / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D020935:MAP Kinase Signaling System; D009447:Neuroblastoma / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering / Q000235:genetics; D018336:Receptors, Aryl Hydrocarbon / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D019772:Topotecan / Q000494:pharmacology*; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D000970:Antineoplastic Agents; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D017077:Culture Media, Conditioned; D004268:DNA-Binding Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D018336:Receptors, Aryl Hydrocarbon; D019772:Topotecan; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D042461:Vascular Endothelial Growth Factor A; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497396:ARNT protein, human; C497442:HIF1A protein, human; C497446:VHL protein, human