Characterization of Hypoxia Inducible Factor HIF-1α function and identification of its targets to improve therapeutic intervention in Neuroblastoma malignancy

Neuroblastoma (NB) is the most frequent malignant tumor in pediatric age derived from primitive cells of the sympathetic nervous system. It is composed of cells with different levels of neural differentiation and high tumor cell differentiation grade correlates with a positive outcome. Current NB treatments include surgery, the use of radio or cytotoxic therapies and the pro differentiating agents as retinoids to eradicate minimal residual disease (MRD). Nowadays there is no treatment that heals completely NB. Expression of the hypoxia inducible factors (HIFs) HIF1A and EPAS1 and/or hypoxia-regulated pathways has been shown to promote the undifferentiated phenotype of NB cells. The first hypothesis of this study is that HIF1A and EPAS1 expression represents one of the mechanisms responsible for the lack of responsiveness of NB to differentiation therapy. Clinically, high levels of HIF1A and EPAS1 expression were associated with inferior survival in two NB microarray datasets, and patient subgroups with lower expression of HIF1A and EPAS1 showed significant enrichment of pathways related to neuronal differentiation. In NB cell lines, the combination of all-trans retinoic acid (ATRA) with HIF1A or EPAS1 silencing led to an acquired glial-cell phenotype and enhanced expression of glial-cell differentiation markers. Furthermore, HIF1A or EPAS1 silencing might promote cell senescence independent of ATRA treatment. Taken together, this data suggest that HIFs inhibition with ATRA treatment promotes differentiation into a more benign phenotype and cell senescence in vitro. Between HIFs, HIF1A is the most promising factor to be silenced to induce differentiation in cotreatment with ATRA. HIF1A protein expression is changeble inside solid tumors because of oxygen levels. The second intent of this study has been to indentify HIF1A target genes whose expression is activated or repressed by HIF1A regardless microenviroment. The expression of these “HIF1A target genes” will be related to all tumor area and their silencing might lead solid tumor to be easily wiped out. Transcriptome analysis of HIF1A silenced NB cell lines and in silico analysis have suggested putative HIF1A targets to be used in combination with ATRA. These findings open the way for additional lines of attack in the treatment of NB minimal residue disease