Inhibition of hypoxia inducible factors combined with all-trans retinoic acid treatment enhances glial transdifferentiation of neuroblastoma cells.

Neuroblastoma (NBL) is a heterogeneous tumor characterized by a wide range of clinical manifestations. A high tumor cell differentiation grade correlates to a favorable stage and positive outcome. Expression of the hypoxia inducible factors HIF1-α (HIF1A gene) and HIF2-α (EPAS1 gene) and/or hypoxia-regulated pathways has been shown to promote the undifferentiated phenotype of NBL cells. Our hypothesis is that HIF1A and EPAS1 expression represent one of the mechanisms responsible for the lack of responsiveness of NBL to differentiation therapy. Clinically, high levels of HIF1A and EPAS1 expression were associated with inferior survival in two NBL microarray datasets, and patient subgroups with lower expression of HIF1A and EPAS1 showed significant enrichment of pathways related to neuronal differentiation. In NBL 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, our data suggest that HIF inhibition coupled with ATRA treatment promotes differentiation into a more benign phenotype and cell senescence in vitro. These findings open the way for additional lines of attack in the treatment of NBL minimal residue disease.

Common genetic variants in NEFL influence gene expression and neuroblastoma risk.

The genetic etiology of sporadic neuroblastoma is still largely obscure. In a genome-wide association study, we identified single-nucleotide polymorphisms (SNP) associated with neuroblastoma at the CASC15, BARD1, LMO1, DUSP12, HSD17B12, HACE1, and LIN28B gene loci, but these explain only a small fraction of neuroblastoma heritability. Other neuroblastoma susceptibility genes are likely hidden among signals discarded by the multiple testing corrections. In this study, we evaluated eight additional genes selected as candidates for further study based on proven involvement in neuroblastoma differentiation. SNPs at these candidate genes were tested for association with disease susceptibility in 2,101 cases and 4,202 controls, with the associations found replicated in an independent cohort of 459 cases and 809 controls. Replicated associations were further studied for cis-effect using gene expression, transient overexpression, silencing, and cellular differentiation assays. The neurofilament gene NEFL harbored three SNPs associated with neuroblastoma (rs11994014: Pcombined = 0.0050; OR, 0.88; rs2979704: Pcombined = 0.0072; OR, 0.87; rs1059111: Pcombined = 0.0049; OR, 0.86). The protective allele of rs1059111 correlated with increased NEFL expression. Biologic investigations showed that ectopic overexpression of NEFL inhibited cell growth specifically in neuroblastoma cells carrying the protective allele. NEFL overexpression also enhanced differentiation and impaired the proliferation and anchorage-independent growth of cells with protective allele and basal NEFL expression, while impairing invasiveness and proliferation of cells homozygous for the risk genotype. Clinically, high levels of NEFL expression in primary neuroblastoma specimens were associated with better overall survival (P = 0.03; HR, 0.68). Our results show that common variants of NEFL influence neuroblastoma susceptibility and they establish that NEFL expression influences disease initiation and progression.

Impact of interleukin-6 -174 G>C gene promoter polymorphism on neuroblastoma.

BACKGROUND: Common variants in DNA may predispose to onset and progression of neuroblastoma (NB). The genotype GG of single nucleotide polymorphism (SNP) rs1800795 (-174 G>C) in interleukin (IL)-6 promoter has been associated with lower survival of high-risk NB. RESULT: To evaluate the impact of IL-6 SNP rs1800795 on disease risk and phenotype, we analyzed 326 Italian NB patients and 511 controls. Moreover, we performed in silico and quantitative Real Time (qRT)-PCR analyses to evaluate the influence of the SNP on gene expression in 198 lymphoblastoid cell lines (LCLs) and in 31 NB tumors, respectively. Kaplan-Meier analysis was used to verify the association between IL-6 gene expression and patient survival. We found that IL-6 SNP is not involved in susceptibility to NB development. However, our results show that a low frequency of genotype CC is significantly associated with a low overall survival, advanced stage, and high-risk phenotype. The in silico (p = 2.61 × 10(-5)) and qRT-PCR (p = 0.03) analyses showed similar trend indicating that the CC genotype is correlated with increased level of IL-6 expression. In report gene assay, we showed that the -174 C variant had a significantly increased transcriptional activity compared with G allele (p = 0.0006). Moreover, Kaplan-Meier analysis demonstrated that high levels of IL-6 are associated with poor outcome in children with NB in two independent gene expression array datasets. CONCLUSIONS: The biological effect of SNP IL-6-174 G>C in relation to promotion of cancer progression is consistent with the observed decreased survival time. The present study suggests that SNP IL-6-174 G>C may be a useful marker for NB prognosis.