Hypoxic gene regulation and oncogenic pathways in neuroblastoma

Neuroblastoma patients show remarkable clinical heterogeneity, with courses ranging from spontaneous regression to fatal tumor progression despite intense multi-modal treatment. Previous studies have shown that hypoxia pushes neuroblastoma cells towards a more immature phenotype, which correlates to aggressive disease. Here we define a role for the hypoxia inducible factor-2α in neuroblastoma tumor progression. While HIF-1α was transiently stabilized at hypoxia (1% oxygen), HIF-2α was induced and regulated HIF-specific target genes, such as VEGF, at later time points. Furthermore, HIF-2α was stabilized and transcriptionally active in cells grown at physiological oxygen levels (5% O2). Subsequent microarray analysis showed that HIF-2α induced genes, previously identified as hypoxia regulated, at this physiological oxygen level. Several of these genes have been implicated in tumorigenic processes and correlated to adverse patient outcome in various tumor forms. Indeed, siRNA mediated knock-down of HIF-2α in neuroblastoma cells significantly reduced xenograft tumor growth, as compared to siHIF1-α treated or wild-type cells. Moreover, immunohistochemical analyses of a large neuroblastoma tumor material arranged in a tissue microarray showed that HIF-2α expression correlated to VEGF, and that high HIF-2α levels was predictive of poor patient prognosis. Prognostic markers of neuroblastoma patient adverse prognosis include amplification of the MYCN oncogene and an undifferentiated morphology. While these features discriminate high- from low-risk patients with precision, identification of poor outcome low- and intermediate-risk patients is more challenging. We analyze two large neuroblastoma microarray data sets by using a priori-defined gene expression signatures. The results show that differential overexpression of Myc transcriptional targets and low expression of genes involved in sympathetic neuronal differentiation predict relapse and death from disease. This was evident not only for high-risk patients, but also was robust in identifying groups of poor prognosis patients otherwise judged to be at low- or intermediate-risk for adverse outcome. These data suggest that pathway-specific gene expression profiling might be useful in the clinic to adjust treatment strategies for children with neuroblastoma