EIF4EBP1 is transcriptionally upregulated by MYCN and associates with poor prognosis in neuroblastoma

Abstract

BackgroundNeuroblastoma (NB) accounts for 15% of cancer-related deaths in childhood despite considerable therapeutic improvements. While several risk factors, including MYCN amplification and alterations in RAS and p53 pathway genes, have been defined in NB, the clinical outcome is very variable and difficult to predict. Since genes of the mTOR pathway are up-regulated in MYCN-amplified NB, we aimed to define the predictive value of the mTOR substrate-encoding gene eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) expression in NB patients. MethodsSeveral independent NB patient cohorts with corresponding mRNA expression data were analyzed for EIF4EBP1 expression. An institutional NB cohort consisting of 69 prospectively collected tumors was employed to immunohistochemically analyze expression of EIF4EBP1-encoded protein (4EBP1). In addition, we performed an in vitro luciferase reporter gene assay with an episomal EIF4EBP1 promoter and genetically modulated MYCN expression in NB cells. FindingsEIF4EBP1 mRNA expression was positively correlated with MYCN expression and elevated in stage 4 and high-risk NB patients. High EIF4EBP1 mRNA expression was associated with reduced overall and event-free survival in the entire group of NB patients in three cohorts, as well as in stage 4 and high-risk patients. High levels of 4EBP1 were significantly associated with prognostically unfavorable NB histology. Functional analyses in vitro revealed that EIF4EBP1 expression is transcriptionally controlled by MYCN binding to the EIF4EBP1 promoter. InterpretationHigh EIF4EBP1 expression is associated with poor prognosis in NB patients and may serve to stratify patients with high-risk NB. FundingG.L. was supported by funding from the Elterninitiative Dusseldorf e.V., the Research Commission of the Medical Faculty of Heinrich Heine University, the Deutsche Forschungsgemeinschaft (Grant LE 3751/2-1), and the German Cancer Aid (Grant 70112624). The laboratory of T.G.P.G. is supported by the Barbara und Wilfried Mohr Foundation. BR is supported by the Israel Science Foundation (grant No. 1436/19). RESEARCH IN CONTEXT Evidence before this studyNB represents a particularly heterogeneous cancer entity, with 5-year event-free survival rate ranging from 50% to 98% depending on the patients risk group. While genes of the nutrient-sensing mTOR pathway were found to be up-regulated in MYCN-amplified NB tumors, their clinical relevance and prognostic value in NB patients remain unclear. In particular, the mTOR substrate-encoding gene EIF4EBP1 was studied in NB by three different groups and high EIF4EBP1 mRNA expression was observed in MYCN-amplified or contradictorily in more favorable stages 1 and 2 patients. Also, EIF4EBP1 was included in a prognostic gene signature for poor overall survival in NB. However, the prognostic value of EIF4EBP1 alone was not determined in NB and the expression of EIF4EBP1 encoded protein, 4EBP1, was not analyzed in NB tumor tissues and not correlated with clinicopathological features such as histological subtypes. Additionally, the transcriptional regulation of the EIF4EBP1 promoter by MYCN was not characterized. Added value of this studyThis study uncovers the prognostic potential of EIF4EBP1 at the mRNA and protein levels in NB patients. We report that high EIF4EBP1 expression is correlated with poor survival in three independent cohorts and that high 4EBP1 levels is associated with a prognostically unfavorable histological subtype. High EIF4EBP1 expression is also a factor of poor prognosis in stage 4 and high-risk patient groups. Finally, we found that MYCN activates the human EIF4EBP1 promoter through binding at three binding motifs. Implications of all the available evidenceEIF4EBP1 mRNA and 4EBP1 protein expression have prognostic value in NB, especially to stratify patients with advanced and more aggressive NB, such as patients with stage 4 disease and high-risk patients including those with unfavorable histological subtype NB. Enhanced EIF4EBP1 mRNA and 4EBP1 protein expression in NB are driven by direct transcriptional activation of EIF4EBP1 by MYCN.