Topotecan Blocks Hypoxia-Inducible Factor-1A and Vascular Endothelial Growth Factor Expression Induced by Insulin-Like Growth Factor-I in Neuroblastoma Cells Kiichiro Beppu, 1 Katsuya Nakamura, 1 W. Marston Linehan, 2 Annamaria Rapisarda, 3 and Carol J. Thiele 1 1 Pediatric Oncology Branch and 2 Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland and 3 Science Applications International Corp.-Frederick, Inc., National Cancer Institute, Frederick, Maryland Abstract 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 thera- peutic options to control neuroblastoma tumor growth. VEGF mRNA is controlled by growth factors and hypoxia via the transcription factor hypoxia-inducible factor (HIF-1A). HIF- 1A protein levels are regulated by the von Hippel Lindau tumor suppressor gene, VHL , which targets HIF-1A degrada- tion. To determine whether the levels of VEGF in neuroblas- tomas 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-1A protein that temporally paralleled increases in VEGF mRNA, whereas HIF-1B levels were unaffected. VEGF and HIF-1A levels were blocked by inhibitors of phosphatidylinositol 3- kinase and mammalian target of rapamycin. Furthermore, we confirmed that HIF-1A mediates f40% of the growth factor activity stimulating VEGF protein expression. Topotecan blocked the IGF-I-stimulated increase in HIF-1A but not HIF-1B, 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-1A have the potential to inhibit VEGF expression and limit neuroblastoma tumor growth. (Cancer Res 2005; 65(11): 4775-81) Introduction Angiogenesis, the growth of new capillary blood vessels, is es- sential for tumor development and metastasis. To initiate angio- genesis, tumor cells make an angiogenic switch by perturbing the local balance of proangiogenic and antiangiogenic factors (1). Vascular endothelial growth factor (VEGF) is a strong proangio- genic factor and an attractive target for antiangiogenic therapies. A major regulator of VEGF is the hypoxia-inducible factor (HIF)-1a. The importance of this factor in the regulation of VEGF levels was revealed with the finding that the von Hippel Lindau gene (VHL ) tumor suppressor gene in kidney cancer encoded a protein that targets HIF-1a protein to the proteosome for degradation (2). Mutations in VHL lead to increased levels of HIF-1a and, in turn, constitutively high VEGF levels. Although hypoxia via HIF-1a is a major regulator of VEGF levels, recent in vitro studies reveal that insulin-like growth factor-I (IGF-I) induces VEGF expression in colon and prostate cancer through a phosphatidylinositol 3-kinase (PI3K)–dependent mechanism either directly and/or indirectly mediated by HIF-1a (3, 4). Neuroblastoma is the most common extracranial solid tumor in childhood (5). The extent of angiogenesis or VEGF expression in neuroblastoma tumor tissue is correlated with metastatic disease, N-myc amplification, and poor clinical outcome (6, 7). Together with frequent expression of VEGF in neuroblastoma cell lines and tumors (8), VEGF targeted therapy may be effective against neuroblastoma. In fact, blockade of VEGF function inhibits neuroblastoma tumor growth (9). In neuroblastoma cells exposed to hypoxic conditions, there is evidence of coexpression of IGFs and VEGF in vitro (10) and in vivo (11). Moreover, IGFs play a role in neuroblastoma tumor progression by stimulating N-myc expression (12), cell survival/proliferation (13), and motility (14); enhancing cell survival (15); affecting chemosensitivity (16); and mediating resistance to retinoids (17). Furthermore, blockade of IGF-I receptor (IGF-IR) ameliorates retinoid resistance (17) and results in suppression of neuroblastoma xenograft tumor growth (18). Little is known about the regulation of VEGF in neuroblastoma under normoxic conditions. Here, we study the regulation of VEGF expression in neuroblastoma cell lines by serum and the growth factor IGF-I. We find that serum and serum-derived growth factors, especially IGF-I, induce VEGF expression and secretion in neuro- blastoma cells through induction of HIF-1a expression via both PI3K/Akt and mitogen-activated protein kinase (MAPK) pathways. Additionally, the VHL gene, a major negative regulator of HIF-1a, is not mutated in neuroblastoma cell lines. Targeted decreases in HIF-1a expression using a HIF1-a small interfering RNA (siRNA) decrease VEGF expression, and treatment with topotecan, a drug under clinical evaluation in neuroblastoma, inhibits HIF-1a and recombinant VEGF expression in neuroblastoma cells. Materials and Methods Cell culture and reagents. One million neuroblastoma cells (SMS- KCNR, SK-N-BE2, SH-SY5Y, LAN-1, and SK-N-AS cells) per well were Note: K. Beppu and K. Nakamura contributed equally to this work. Requests for reprints: Carol J. Thiele, Cell and Molecular Biology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, 1-3940 MSC-1105, Bethesda, MD 20892. Phone: 301-496-1543; Fax: 301-451-7052; E-mail: ct47a@nih.gov. I2005 American Association for Cancer Research. www.aacrjournals.org 4775 Cancer Res 2005; 65: (11). June 1, 2005 Research Article