HIF2A and IGF2 Expression Correlates in Human Neuroblastoma Cells and Normal Immature Sympathetic Neuroblasts

Abstract

During normal sympathetic nervous system (SNS) development, cells of the ganglionic lineage can malignantly transform and develop into the childhood tumor neuroblastoma. Hypoxia-inducible transcription factors (HIFs) mediate cellular responses during normal development and are central in the adaptation to oxygen shortage. HIFs are also implicated in the progression of several cancer forms, and high HIF-2α expression correlates with disseminated disease and poor outcome in neuroblastoma. During normal SNS development, HIF2A is transiently expressed in neuroblasts and chromaffin cells. SNS cells can, during development, be distinguished by distinct gene expression patterns, and insulin-like growth factor 2 (IGF2) is a marker of sympathetic chromaffin cells, whereas sympathetic neuroblasts lack IGF2 expression. Despite the neuronal derivation of neuroblastomas, we show that neuroblastoma cell lines and specimens express IGF2 and that expression of HIF2A and IGF2 correlates, with the strongest correlation in high-stage tumors. In neuroblastoma, both IGF2 and HIF2A are hypoxia-driven and knocking down IGF2 at hypoxia resulted in downregulated HIF2A levels. HIF-2α and IGF2 were strongly expressed in subsets of immature neuroblastoma cells, suggesting that these two genes could be co-expressed also at early stages of SNS development. We show that IGF2 is indeed expressed in sympathetic chain ganglia at embryonic week 6.5, a developmental stage when HIF-2α is present. These findings provide a rationale for the unexpected IGF2 expression in neuroblastomas and might suggest that IGF2 and HIF2A positive neuroblastoma cells are arrested at an embryonic differentiation stage corresponding to the stage when sympathetic chain ganglia begins to coalesce.

PI3K–mTORC2 but not PI3K–mTORC1 Regulates Transcription of HIF2A/EPAS1 and Vascularization in Neuroblastoma

“…(48). These results and our earlier findings (4,12,13,49) suggest that HIF2 inhibition, either directly or via the signaling pathways that activate HIF2a transcription and translation, is an attractive target for the treatment of aggressive neuroblastoma. Because the PI3K pathway appears to be a major activator of HIF2 activity and PI3K/mTOR inhibitors are in clinical use and late clinical trials (reviewed in ref.…”

“…Unlike canonical HIF posttranslational regulation, we show here that the hypoxia-induced increase in HIF2a protein expression in neuroblastoma cells is, to a large extent, explained by transcriptional upregulation of HIF2A. Interestingly, this mode of regulation mimics the situation during normal SNS development where HIF2a protein is transcriptionally regulated in immature neuroblasts and paraganglia cells (12,36).…”

“…cDNA synthesis and qRT-PCR was performed as described previously (12). Three reference genes were used to normalize gene-of-interest expression.…”

“…We recently showed that HIF2a and insulin-like growth factor (IGF)-II are coexpressed in SNS ganglia and paraganglia during distinct periods of normal human embryogenesis and fetal development. Expression of HIF2A and IGF2 thereto correlate in clinical neuroblastoma specimens, and IGFII regulates hypoxic expression of HIF2A (12).…”

“…Tumor hypoxia and HIF1a and HIF2a protein expression are associated with aggressive disease, metastasis, resistance to therapy, and thus poor clinical outcome for patients with various cancers (3)(4)(5)(6)(7)(8)(9). HIF1a and HIF2a share high sequence homology, but it is becoming increasingly evident that HIF1a and HIF2a have differential spatial and temporal regulation in response to hypoxia in human tumors and developing tissues (4,5,7,8,(10)(11)(12).…”