Project

Identification of candidate therapeutic long non-coding RNA genes in the pathway TP53

Duration
01 January 2014 → 31 December 2017
Funding
Regional and community funding: IWT/VLAIO
Research disciplines
  • Medical and health sciences
    • Laboratory medicine
    • Medical systems biology
    • Laboratory medicine
    • Medical systems biology
    • Molecular and cell biology
    • Laboratory medicine
    • Medical systems biology
    • Molecular and cell biology
Keywords
TP53 pathway neuroblastoma pediatric cancer
 
Project description

Neuroblastoma is a pediatric extracranial tumor, derived from neural crest progenitor cells normally giving rise to the sympathetic nervous system. This enigmatic malignancy shows clinical outcomes ranging from spontaneous regression and long-term survival, to aggressive high-risk tumors resulting in death in a high percentage of cases. Although patients having the worst prognosis are subjected to multimodal therapy, the survival rate still remains poor. To cater to the specific needs of the patients, a more targeted approach is preferred. However, targeted strategies require insight into the genetic composition of the cancer in order to identify new therapeutic targets. Neuroblastoma is classified as a copy number disease as copy number variations are frequently observed and contribute to the disease progression and prognosis. The most common copy number variations include 1p deletions, 11q deletions and 17q gains. Although these structural alterations show a high prevalence in neuroblastoma, the mutational incidence is considerably smaller. ALK is the most frequently mutated gene, with mutations observed in 8-10% of neuroblastoma patients. Other key driver genes that have been identified are MYCN, PHOX2B and TERT. MYCN is amplified in around 25% of cases, PHOX2B is a gene instrumental in normal development and TERT has been shown to be upregulated in high-risk neuroblastoma tumors leading to telomere elongation. However, these oncogenic drivers are protein coding genes, the non-coding part of the genome remains rather unexplored in neuroblastoma. To identify long intergenic non-coding RNAs (lincRNAs) with an oncogenic or tumor suppressive potential, we focused on lincRNAs having a link with MYCN, ALK or PHOX2B. By combining RNA-seq data from primary tumor samples with model systems for MYCN, ALK and PHOX2B, we could demonstrate that each of these driver genes regulates a distinct lincRNA set. Several of these lincRNAs were found to be associated with patient survival and disease stage. LincRNAs located in a super-enhancer or expressed specifically in neuroblastoma cells were further prioritized, leading to a core set of genes with potential implications in neuroblastoma tumorigenesis. In addition, we examined the data to find lincRNAs in control of MYCN or PHOX2B activity or expression, resulting in the identification of several lincRNAs that are known to be associated with c-MYC. Examples include TSIX and MEG3, that we classified as modulators of MYCN activity in neuroblastoma. Targets established by means of this computational workflow were enriched in several cancer hallmark gene sets. An example of such a hallmark is the IL6-JAK-STAT3 pathway, a network often hyperactivated in several cancer types leading to a poor outcome.

However, these results are predictions and still need to be experimentally validated. To this end, we aimed to unravel the function of our top candidate in neuroblastoma pathogenesis. NESPR, located in the vicinity of PHOX2B, is the most specific lincRNA in this pediatric malignancy, associated with survival and MYCN amplification. Antisense oligonucleotide (ASO) mediated gene perturbation resulted in a downregulation of PHOX2B expression and altered expression patterns for 780 other genes. Loss of NESPR expression reduced cell growth and colony formation capacity, while an increase in caspase activity suggests an induction of apoptosis. Although the precise mechanism of NESPR is not yet determined, we suspect a role in PHOX2B gene expression regulation through chromatin structure modification. However, analysis of NESPR binding sites also suggests a PHOX2B independent function. NESPR binding sites were shown to be enriched for GATA3 and ISL1 motif sequences, two super-enhancer associated transcription factors contained in the noradrenergic core regulatory circuit in charge of neuroblastoma cell identity.

In conclusion, by investigating the non-coding transcriptome of neuroblastoma we have provided a prioritized core set of lincRNAs associated with key driver genes in neuroblastoma. We experimentally validated one of the top candidate genes, verifying its functional activity in this pediatric malignancy.