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

Abstract

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 hypoxiamediated 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% O 2 ) 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 Mmol/L) apoptosis. Treatment with anti-VEGF and anti-Flt1 antibodies inhibited ERK1/ 2 activation, down-regulated bcl-2, and reversed the hypoxiamediated 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-1A (HIF-1A) phosphorylation. Furthermore, the reduced phosphorylation was associated with down-regulation of basic fibroblast growth factor, a downstream target of the HIF-1A and VEGF pathways. Our findings suggested an expanded autocrine loop between VEGF/Flt1 signaling and HIF-1A. We investigated the angiogenic activity of the loop in an in vivo Matrigel plug assay. The hypoxiatreated 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-1A 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. (Cancer Res 2005; 65(16): 7267-75)

Effects of Inflammatory Factors on Mesenchymal Stem Cells and Their Role in the Promotion of Tumor Angiogenesis in Colon Cancer

“…In the tumor microenvironment, hypoxia can cause some effector cells to produce HIF-1␣, which is the upstream regulatory gene of VEGF (42). VEGF also has been demonstrated to interact with the HIF-1␣ via an autocrine loop (43,44). The level of VEGF and HIF-1␣ could directly and indirectly indicate the ability of a tumor to cause angiogenesis.…”

ZFAT is a critical molecule for cell survival in mouse embryonic fibroblasts

“…Recent studies of Flt1 in several tumor cells suggest that VEGF/Flt1 signaling has autocrine survival activity [15][16][17][18]. Treatment with anti-VEGF and anti-Flt1 antibodies inhibits ERK1/2 activation and downregulates Bcl-2 expression in the highly malignant neuroblastoma cell line SK-N-BE(2) [19]. Furthermore, reduced endogenous VEGF or VEGFR1 expressions induce apoptosis in MDA-MB-231 and MCF-7 breast cancer cells [20].…”

Multiple receptor tyrosine kinases regulate HIF-1α and HIF-2α in normoxia and hypoxia in neuroblastoma: implications for antiangiogenic mechanisms of multikinase inhibitors

“…In particular, epidermal growth factor receptor (EGFR) modulates HIF-1a levels in prostate, lung and breast cancer cells (Zhong et al, 2000;Phillips et al, 2005;Peng et al, 2006). Studies in neuroblastoma cells have indicated the presence of a hypoxia-driven VEGF/ VEGFR-1 autocrine loop modulating HIF-1a (Das et al, 2005). Whether other RTKs are capable of modulating HIF-1a, and whether RTKs can modulate HIF-2a levels is unknown.…”