Activation of a prometastatic gene expression program in hypoxic neuroblastoma cells.

The hypoxia inducible factor-1alpha (HIF1alpha) is a key regulator of oxygen homeostasis, modulating cell survival, and growth in cells exposed to hypoxia. In this study, neuroblastoma (NB) cells SH-SY5Y and SK-N-MC were employed to determine the mechanisms regulating adaptation to hypoxia. NB cells were cultured in a serum-free medium in the presence or absence of CoCl(2) (100 muM, hypoxia mimic) for up to 48 h. SH-SY5Y and SK-N-MC cell numbers were not affected by CoCl(2) treatment, while mitochondrial activity was reduced by approximately 50% in SH-SY5Y cells and by approximately 70% in SK-N-MC cells. Intracellular accumulation of HIF1alpha protein was detected as early as 30 min of post-hypoxia, followed by the increase of mRNA for vascular endothelial growth factor (VEGF) and nuclear accumulation of the ID1-2 transcription factors by 4 h. In hypoxic SH-SY5Y NB cells, real-time PCR analysis showed that the genes involved in maintenance of cell-cell and cell-matrix interactions (i.e. adenomatosis polyposis coli, E-cadherin, catenin, EphB2, fibronectin-1, HTATIP2, tissue inhibitor of metalloprotease-4) were down-regulated by up to 90%, while genes involved in enhancement of metastatic behavior (integrin a7b1, hepatocyte growth factor receptor, transforming growth factor-beta1, VEGF, kisspeptin, interleukin-1beta) were dramatically up-regulated above 200%. These changes were all consistent with the induction of epithelial-mesenchymal transition. We have thus demonstrated that NB cell adaptation to hypoxia, in addition to the modulation of HIF1alpha and VEGF expression and nuclear translocation of ID1 and ID2 transcription factors, involve in the activation of a gene expression program consistent with the pro-metastatic events. These processes are probably responsible for the NB cell transition from an adherent phenotype to a highly migratory, invasive and aggressive NB cell type.