Widespread Dysregulation of MiRNAs by MYCN Amplification and Chromosomal Imbalances in Neuroblastoma: Association of miRNA Expression with Survival

Abstract

MiRNAs regulate gene expression at a post-transcriptional level and their dysregulation can play major roles in the pathogenesis of many different forms of cancer, including neuroblastoma, an often fatal paediatric cancer originating from precursor cells of the sympathetic nervous system. We have analyzed a set of neuroblastoma (n = 145) that is broadly representative of the genetic subtypes of this disease for miRNA expression (430 loci by stem-loop RT qPCR) and for DNA copy number alterations (array CGH) to assess miRNA involvement in disease pathogenesis. The tumors were stratified and then randomly split into a training set (n = 96) and a validation set (n = 49) for data analysis. Thirty-seven miRNAs were significantly over- or under-expressed in MYCN amplified tumors relative to MYCN single copy tumors, indicating a potential role for the MYCN transcription factor in either the direct or indirect dysregulation of these loci. In addition, we also determined that there was a highly significant correlation between miRNA expression levels and DNA copy number, indicating a role for large-scale genomic imbalances in the dysregulation of miRNA expression. In order to directly assess whether miRNA expression was predictive of clinical outcome, we used the Random Forest classifier to identify miRNAs that were most significantly associated with poor overall patient survival and developed a 15 miRNA signature that was predictive of overall survival with 72.7% sensitivity and 86.5% specificity in the validation set of tumors. We conclude that there is widespread dysregulation of miRNA expression in neuroblastoma tumors caused by both over-expression of the MYCN transcription factor and by large-scale chromosomal imbalances. MiRNA expression patterns are also predicative of clinical outcome, highlighting the potential for miRNA mediated diagnostics and therapeutics.

N-myc and Noncoding RNAs in Neuroblastoma

“…Other studies have confirmed direct binding of N-myc to the mir-17-92 promoter (50, 55), as well as a positive correlation between expression of MYCN and members of the mir-17-92 cluster in primary tumors and/or neuroblastoma cell lines (49,51,(55)(56)(57)(58)(59)(60)(61). As miRNAs simultaneously target a variety of different mRNAs, it became clear that activation of the mir-17-92 cluster enables N-myc to turn multiple cellular processes toward malignant transformation.…”

“…In one of the largest miRNA profiling studies in neuroblastoma so far, Bray and colleagues (49) profiled 430 miRNAs in a total of 145 primary neuroblastoma tumors, including 36 with MYCN amplification. They found both up-and downregulated miRNAs (14 and 23, respectively) when MYCN-amplified tumors were compared with nonamplified tumors.…”

“…Although the expression of several miRNAs have been documented to correlate with MYCN expression, there is now growing evidence that N-myc predominantly acts repressive on the overall miRNA composition in MYCNamplified neuroblastoma cells (47,49,(59)(60)(61)71) and upon N-myc induction in nonamplified neuroblastoma cells (55). Lin and colleagues (71) used real-time RT-PCR to profile the expression of 162 miRNAs in 66 primary neuroblastoma tumors (including 13 with MYCN-amplification) and found a nearly global downregulation of miRNAs in high-risk tumors, especially in those with MYCN amplification.…”

Neuroblastoma therapy: what is in the pipeline?

“…Results show that the widespread deregulation of miRNAs expression in NB tumours is due to the chromosomal imbalances and overexpression of the MYCN (Bray et al 2009). MYCN might regulate C S Verissimo et al: Novel neuroblastoma targets www.endocrinology-journals.org the expression of miRNAs that play a role in NB proliferation (Chen & Stallings 2007).…”

“…There are indications that miRNA expression levels are predictive of clinical outcome. This suggests that miRNAs might be used as diagnostic markers and targets for NB therapy (Bray et al 2009). An overview of differently expressed miRNAs that have been proposed as NB therapeutic targets is provided in Table 3 and their expression in other tumours is given in Supplementary Table 2, see section on supplementary data given at the end of this article.…”