HIF-1α and HIF-2α Are Differentially Regulated In vivo in Neuroblastoma: High HIF-1α Correlates Negatively to Advanced Clinical Stage and Tumor Vascularization

Abstract

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

Opposite functions of HIF-α isoforms in VEGF induction by TGF-β1 under non-hypoxic conditions

“…A detectable increase of HIF-1a and HIF-2a transcript levels at 3 h after TGF-b1 treatment suggests that TGFb1 might directly activate the transcription of both genes in normoxia. We are exploring the molecular mechanism underlying TGF-b1 activation of HIF-a gene Despite of well-established HIF-1a role in tumor angiogenesis, several lines of evidences have supported differential or contrasting roles of HIF-1a and HIF-2a in VEGF expression (Lo¨fstedt et al, 2007;Noguera et al, 2009). It was demonstrated that HIF-1a and HIF2a are regulated differently in a time-and oxygendependent fashion, and that hypoxia-induced VEGF expression is primarily driven by HIF-2a at prolonged hypoxia, whereas HIF-1a functions as a major VEGF inducer during acute hypoxic conditions (Uchida et al, 2004;Holmquist-Mengelbier et al, 2006;Helczynska et al, 2008).…”

“…Furthermore, high level of HIF-2a, but not HIF-1a, associates with distant metastasis, an immature stem-cell-like phenotype and poor outcome in some tumor types (Giatromanolaki et al, 2003;Yoshimura et al, 2004;Pietras et al, 2008). HIF-2a is associated with breast-cancer-specific death and distant metastasis, whereas in neuroblastoma, HIF-1a correlates negatively to vascularization and shows an association with low tumor stage and favorable patient prognosis (Holmquist-Mengelbier et al, 2006;Helczynska et al, 2008;Noguera et al, 2009). These findings raise an important issue that if these HIF-2a-positive cells indeed are tumor-initiating or stem cells, the capacity to produce VEGF and attract blood vessel formation under physiologic oxygen tensions might be crucial during an early phase of establishment of solid tumors (Noguera et al, 2009).…”

Neuroblastoma aggressiveness in relation to sympathetic neuronal differentiation stage

“…At least in MYCNamplified tumors, we could verify that that the HIF-2α positive cells were tumor-derived by showing that they harbored the MYCN amplification. In neuroblastoma specimens, intense HIF-2α staining correlates with aggressive disease and poor outcome (64), and thus, we proposed that these immature and stem cell-like cells are putative neuroblastoma stem cells (64,93,94)…”

The HIF-2α-Driven Pseudo-Hypoxic Phenotype in Tumor Aggressiveness, Differentiation, and Vascularization

“…While HIF-1α is mainly restricted to perinecrotic zones, HIF-2α protein is also expressed at other locations, most notably in cells adjacent to blood vessels. Presence of tumor cells staining intensely for HIF-2α, more so than high number of HIF-2α + cells, correlates positively to distant metastasis and negative overall survival (HolmquistMengelbier et al 2006;Noguera et al 2009). In contrast, HIF-1α protein expression did not correlate to aggressive disease or negative outcome ).…”

“…Importantly, and the reason for the focus on HIF-2α in this review, HIF-1α and HIF-2α are differently regulated in tumors such as neuroblastoma, breast cancer, non-small cell lung carcinoma (NSCLC) and glioma and seem to have different impact on tumor behavior and patient outcome in these tumors (Holmquist-Mengelbier et al 2006;Helczynska et al 2008;Heddleston et al 2009;Kim et al 2009b;Li et al 2009;Noguera et al 2009). …”