Purpose: Hypoxia is considered to be a major driving force behind tumor angiogenesis.The stabilization and activation at hypoxia of the hypoxia-inducible factors HIF-1α and HIF-2α and the concomitant induction of expression of vascular endothelial growth factor (VEGF) and other proangiogenic factors provide a molecular frame for hypoxiadriven tumor angiogenesis. This study has investigated how HIF and VEGF protein levels relate to each other with regard to vascularization, tumor stage, and overall survival in neuroblastoma. Experimental Design: Tissue cores taken from tumor specimens representing 93 children with neuroblastoma were arranged on a microarray and stained for HIF-1α, HIF-2α, VEGF, and CD31 proteins. Both fraction of positive cells and staining intensity were evaluated and protein levels were correlated with each other and with clinical variables. Results: Although high levels of both HIF-1α (P < 0.001) and HIF-2α (P < 0.001) correlated positively to VEGF expression, they did not fully correlate with each other. Moreover, HIF-1α (P = 0.002) and VEGF (P < 0.001), but not HIF-2α, correlated negatively to vascularization as determined by CD31 staining abundance. VEGF expression or degree of vascularization did not correlate with tumor stage or overall survival. High HIF-1α levels correlated with low tumor stage (P < 0.001) and were associated with a favorable patient prognosis (P = 0.08).
Conclusions:The discordant results on expression of HIF-1α and HIF-2α suggest that these two proteins are differentially regulated in vivo, thus reflecting distinctive protein expression/stabilization mechanisms. The association between HIF-1α and favorable outcome stresses the importance of discriminating HIF-2α from HIF-1α expression and has implications for using HIFs as treatment targets. (Clin Cancer Res 2009; 15(23):7130-6) Oxygen shortage, hypoxia, is a common state in solid tumors and a hallmark of aggressive tumor cells in that they have acquired the capacity to both survive and proliferate at conditions of low oxygen tensions (1). Tumor cells seem to use the same adaptation mechanisms to hypoxia as nontransformed cells in which stabilization and activation of the hypoxia-inducible factor-1 (HIF-1) and HIF-2 are central components. Each of these two heterodimeric transcription factors consist of one oxygen-sensitive α-subunit (HIF-1α and HIF-2α, respectively) in complex with one β-subunit (ARNT), which is expressed and transcribed independent of the cellular oxygen status (2). Once stabilized and activated, the HIFs transcribe genes that are involved in cellular and physiologic adaptation to hypoxia, which include shifts toward anaerobic metabolism with increased glycolysis and downregulation of energyconsuming processes such as DNA repair. An important effect of HIF activation is the increased transcription of genes encoding