Understanding the role of long non-coding RNAs in neuroblastoma development and progression

Neuroblastoma (NB), a common cancer of childhood, contributes to 15% of all pediatric cancer deaths. The improper neuronal differentiation of neural crest cells to mature neurons in the sympathetic nervous system leads to NB tumor formation. NB is an extremely heterogeneous disease and high-risk NB is very difficult to treat, with the incidence of relapse in 50% of cases despite of intensive chemotherapeutic treatment. Long non-coding RNAs (lncRNAs) are a class of biological molecules that are transcribed but not translated to any functional protein. The mechanism of functions for these lncRNAs are diverse and context-specific. De-regulation of lncRNAs has been proposed to play a critical role in cancer development and progression. The goal of the current thesis was to identify novel neuroblastoma-specific lncRNAs for better stratification of the disease and characterizing their functional role in greater detail. In the first study, we characterized differentially expressed lncRNAs between low-risk and high-risk NB tumors using transcriptome profiling. Among the differentially expressed lncRNAs, we chose a lncRNA, neuroblastoma associated transcript 1 (NBAT1), that maps to NB hotspot locus, 6p22.3, which has been shown to harbor several NB-specific risk-associated SNPs. We showed that NBAT1 is a tumor suppressor lncRNA and it carries out this tumor suppressor function through regulating cellular proliferation and differentiation. Consistent with its tumor suppressor properties, its higher expression in NB patients predicts a good prognosis. Mechanistically, NBAT1 controls NB cell growth through epigenetically silencing cell proliferating genes, as well as NB cell differentiation by repressing the neuron-restrictive silencer factor NRSF, also known as REST. In the second study, we sought to investigate the functional connection between NBAT1 and its sense partner CASC15 lncRNA in NB development and progression. Like NBAT1, CASC15 harbors NB-specific tumor suppressor properties and its higher expression in NB patients correlates with good clinical outcomes. We show that CASC15/NBAT1 (6p22lncRNAs) promote cell differentiation by the specific regulatory interactions with SOX9 and USP36 located on 17q, which is frequently gained in NB. We could show mechanistically that 6p22lncRNAs dictate SOX9 expression by controlling CHD7 stability via modulating cellular localization of USP36, which is a deubiquitinase. In the third and final study, we found that NBAT1 is a p53 responsive lncRNA and regulates p53 subcellular localization. We observed that a decrease in NBAT1 expression in NB cells results in resistance to genotoxic drugs, which in part occurs due to cytoplasmic p53 accumulation and concomitant loss of p53 dependent gene expression. Higher expression of the p53 exporter CRM1 in NBAT1 depleted cells contributes to p53 cytoplasmic localization, while CRM1 inhibition in these cells restores p53 localization. We observed that combined inhibition of CRM1 and MDM2 sensitized aggressive NB cells with cytoplasmic p53, suggesting that this drug combination could be a potential therapeutic strategy for high-risk NB patients. In summary, these findings highlight the regulatory role of lncRNAs in NB disease development

Subcellular Distribution of p53 by the p53-Responsive lncRNA NBAT1 Determines Chemotherapeutic Response in Neuroblastoma

Neuroblastoma has a low mutation rate for the p53 gene. Alternative ways of p53 inactivation have been proposed in neuroblastoma, such as abnormal cytoplasmic accumulation of wildtype p53. However, mechanisms leading to p53 inactivation via cytoplasmic accumulation are not well investigated. Here we show that the neuroblastoma risk-associated locus 6p22.3-derived tumor suppressor NBAT1 is a p53-responsive lncRNA that regulates p53 subcellular levels. Low expression of NBAT1 provided resistance to genotoxic drugs by promoting p53 accumulation in cytoplasm and loss from mitochondrial and nuclear compartments. Depletion of NBAT1 altered CRM1 function and contributed to the loss of p53-dependent nuclear gene expression during genotoxic drug treatment. CRM1 inhibition rescued p53-dependent nuclear functions and sensitized NBAT1-depleted cells to genotoxic drugs. Combined inhibition of CRM1 and MDM2 was even more effective in sensitizing aggressive neuroblastoma cells with p53 cytoplasmic accumulation. Thus, our mechanistic studies uncover an NBAT1-dependent CRM1/MDM2-based potential combination therapy for patients with high-risk neuroblastoma. Significance: This study shows how a p53-responsive lncRNA mediates chemotherapeutic response by modulating nuclear p53 pathways and identifies a potential treatment strategy for patients with high-risk neuroblastoma

The Risk-Associated Long Noncoding RNA NBAT-1 Controls Neuroblastoma Progression by Regulating Cell Proliferation and Neuronal Differentiation

Neuroblastoma is an embryonal tumor of the sympathetic nervous system and the most common extracranial tumor of childhood. By sequencing transcriptonnes of low- and high-risk neuroblastomas, we detected differentially expressed annotated and nonannotated long noncoding RNAs (lncRNAs). We identified a lncRNA neuroblastoma associated transcript-1 (NBAT-1) as a biomarker significantly predicting clinical outcome of neuroblastoma. CpG methylation and a high-risk neuroblastoma associated SNP on chromosome 6p22 functionally contribute to NBAT-1 differential expression. Loss of NBAT-1 increases cellular proliferation and invasion. It controls these processes via epigenetic silencing of target genes. NBAT-1 loss affects neuronal differentiation through activation of the neuronal-specific transcription factor NRSF/REST. Thus, loss of NBAT-1 contributes to aggressive neuroblastoma by increasing proliferation and impairing differentiation of neuronal precursors

Sense-Antisense lncRNA Pair Encoded by Locus 6p22.3 Determines Neuroblastoma Susceptibility via the USP36-CHD7-SOX9 Regulatory Axis

Trait-associated loci often map to genomic regions encoding long noncoding RNAs (lncRNAs), but the role of these lncRNAs in disease etiology is largely unexplored. We show that a pair of sense/antisense lncRNA (6p22lncRNAs) encoded by CASC15 and NBAT1 located at the neuroblastoma (NB) risk-associated 6p22.3 locus are tumor suppressors and show reduced expression in high-risk NBs. Loss of functional synergy between 6p22lncRNAs results in an undifferentiated state that is maintained by a gene-regulatory network, including SOX9 located on 17q, a region frequently gained in NB. 6p22lncRNAs regulate SOX9 expression by controlling CHD7 stability via modulating the cellular localization of USP36, encoded by another 17q gene. This regulatory nexus between 6p22.3 and 17q regions may lead to potential NB treatment strategies