Hypoxia and differentiation in human neuroblastoma cells

Abstract: The childhood tumour neuroblastoma is derived from immature cells of the sympathetic nervous system, which have become arrested at different maturation stages. Neuroblastoma is a malignancy with a high degree of heterogeneity, and there is a correlation between a poor differentiation status and a more aggressive phenotype. The MYCN gene is amplified in approximately 25% of neuroblastoma tumours and is correlated to an aggressive tumour phenotype. A role of MYCN in keeping these tumours at an immature stage has been suggested. However, we show that over-expression of MYCN in non-amplified neuroblastoma cells do not restrain their capacity to differentiate. Furthermore, in a panel of 28 neuroblastoma tumours and 27 cell lines, we do not see a correlation between the degree of MYCN expression and the expression of neuronal or neuroendocrine marker genes. Previously hypoxia and/or nutrient deprivation has been suggested to induce a neuroendocrine lineage shift in neuroblastoma tumours. However, when growing neuroblastoma cell lines at hypoxia and/or low glucose conditions, we observe a down-regulation of both neuronal and chromaffin marker genes. Instead, genes normally expressed in early neural crest cells are induced. We therefore propose that hypoxia and/or glucose deficiency induce a dedifferentiation of neuroblastoma cells, thereby rendering them a more aggressive phenotype. In addition, when growing neuroblastoma cells at hypoxia and/or without glucose, we find that hypoxia protects from glucose-deprivation induced cell death. This further adds to the malignant potential of hypoxic neuroblastoma cells. We have further investigated the hypoxia inducible transcription factors HIF-1a and HIF-2a in neuroblastoma cells and find that they have separate patterns of activation over time and in response to different oxygen levels.