Abstract 3161: Massively parallel transcriptome sequencing in neuroblastoma reveals specific mRNA splicing patterns controlled by RNA binding splicing factors.

Neuroblastoma (NB) is the most common solid extracranial tumor in children. Stage 4 NB is characterized with its clinical heterogynous outcome. The special category, stage 4S subgroup (2-5% of all NB) has a cure rate of above 90%. On the other hand, MYCN amplification (25-30% of all NB) is associated with poor outcome of 26% event free survival rate. Alternative splicing events have crucial roles in tumorigenesis and progression, and are nearly universal in human genes. However, biomarkers and drug designs typically focus on the main transcript of coding genes. High-throughput RNA sequencing (RNA-seq) experiments allow us to quantify all genes and their isoforms across samples. The purposes of this study are to systematically exam and compare the splicing programs in stage 4 NB subgroups, to evaluate the effects of splicing program, to identify key factors controlling the splicing program, and also to report isoform candidates that play important roles in dramatic different outcomes of advanced-stage NB subtypes, which represents potential diagnostic, prognostics and therapeutic targets for advanced-stage NB. In this study, we analyzed RNA-seq data of 29 stage 4 NB tumors and discovered an alternative splicing signature for MYCN amplified stage 4 tumors compared to MYCN non-amplified 4S tumors. In summary, 460 genes were alternatively spliced between MYCN non-amplified 4S and MYCN amplified stage 4 NB tumors. Pathway analysis with Ingenuity IPA demonstrated that alternatively spliced genes held enriched roles in cancer hallmarks biological functions including cell death, cell proliferation, apoptosis, cell movement and immune response. In particular, tissue-specific splicing factors such as RBFOX, CELF, and hnRNP families were differentially expressed between tumor subgroups. Regulatory motif sequences of these splicing factors were enriched in adjacent introns of differentially spliced exons. In addition, we used SHEP neuroblastoma Tet21N system and siRNA on IMR32 NB cell line to either induce or knockdown MYCN expression. RNA-seq analysis of MYCN induction and knockdown NB cell lines showed evidence of MYCN regulation of some of the splicing factors. This study systematically examined and compared the splicing programs in stage 4 NB subgroups. Moreover, it demonstrated that in addition to MYCN9s well characterized role in transcriptional regulation, it regulates splicing events by controlling the expression of splicing factors. Citation Format: Shile Zhang, Tom Badgett, Jun Wei, Young K. Song, Dominik Bogen, Peter Johansson, Catherine Tolman, Jianbin He, Xinyu Wen, Zhihui Liu, Carol Thiele, Javed Khan. Massively parallel transcriptome sequencing in neuroblastoma reveals specific mRNA splicing patterns controlled by RNA binding splicing factors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3161. doi:10.1158/1538-7445.AM2013-3161