Cancer Letters

Cancer Letters

Volume 371, Issue 2, 28 February 2016, Pages 214-224
Cancer Letters

Original Articles
MYCN controls an alternative RNA splicing program in high-risk metastatic neuroblastoma

https://doi.org/10.1016/j.canlet.2015.11.045Get rights and content

Highlights

  • A characteristic isoform expression pattern is detected in MYCN-amplified NBL tumors.

  • Several splicing factors control the splicing program in NBL tumors.

  • MYCN directly controls the expression of splicing factors PTBP1 and HNRNPA1.

  • Splicing factors PTBP1 and HNRNPA1 and their downstream splicing target PKM2 affect MYCN-amplified NBL cell proliferation.

Abstract

The molecular mechanisms underlying the aggressive behavior of MYCN driven neuroblastoma (NBL) is under intense investigation; however, little is known about the impact of this family of transcription factors on the splicing program. Here we used high-throughput RNA sequencing to systematically study the expression of RNA isoforms in stage 4 MYCN-amplified NBL, an aggressive subtype of metastatic NBL. We show that MYCN-amplified NBL tumors display a distinct gene splicing pattern affecting multiple cancer hallmark functions. Six splicing factors displayed unique differential expression patterns in MYCN-amplified tumors and cell lines, and the binding motifs for some of these splicing factors are significantly enriched in differentially-spliced genes. Direct binding of MYCN to promoter regions of the splicing factors PTBP1 and HNRNPA1 detected by ChIP-seq demonstrates that MYCN controls the splicing pattern by direct regulation of the expression of these key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage4 NBL patients (p ≤ 0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, an isoform of pro-tumor-growth, result in repressed growth of NBL cells. Therefore, our study reveals a novel role of MYCN in controlling global splicing program through regulation of splicing factors in addition to its well-known role in the transcription program. These findings suggest a therapeutically potential to target the key splicing factors or gene isoforms in high-risk NBL with MYCN-amplification.

Introduction

Pre-messenger RNA (pre-mRNA) splicing is the process of modifying primary messenger RNA into mRNA transcripts through intron removal and alternative exon usage. It is a universal phenomenon essential for regulating isoform expression in spliceosomes of all eukaryotic cells [1], [2], [3], [4], and is controlled by splicing factors [5]. The expression of different isoforms of a gene is determined by the transcription, together with the splicing program [6]. Splicing factors regulate gene splicing in a tissue- and/or disease-specific manner, and some of them such as FOX and hnRNP family members are implicated in tumor initiation, progression and metastasis [7], [8], [9], [10], [11]. Using exon expression arrays and other high-throughput methods, several splicing profiles have been reported in human cancers including glioblastoma, colon, bladder, prostate, and lung cancers [7], [12], [13], [14]. With the advent of massively parallel sequencing technologies, it is now readily to deconvolute the control mechanisms of RNA isoform expression through analyses of gene transcription and the splicing patterns of these genes in tumor samples.

Metastatic or stage 4 neuroblastoma (NBL) is characterized by heterogeneous clinical outcomes. Less than half of the patients older than 18 months with stage 4 disease survive, whereas those with MYCN-amplification (MYCN-A; 20–25% of all NBL) have an even worse outcome [15]. Stage 4S patients (2–5% of all NBL [16]), who are younger than 12 months of age, have a characteristic pattern of metastasis with rapid tumor growth followed by spontaneous regression and a good overall survival rate of >90% [17], [18]. Recent large-scale sequencing studies by our group and others [19], [20], [21], [22] have discovered only a few novel recurrent somatic mutations in NBL, with MYCN-A and ALK mutations being the most recurrent gene alteration events [23]. Despite MYCN's well-studied roles in transcription, the effects of MYCN on RNA splicing have not been extensively studied. Previous expression studies by our group using exon microarrays demonstrated that tumors with and without MYCN-A have different spicing patterns [24]. However, the mechanisms underlying the MYCN-associated splicing signature have not been investigated.

In this study, we took advantage of massively parallel RNA sequencing to investigate the pattern of transcription and splicing of genes controlled by MYCN. We identified six splicing factors important in MYCN-A tumors and demonstrated that MYCN is responsible for a gene splicing pattern by directly controlling these splicing factors especially PTBP1 and HNRNPA1 in MYCN-A samples. Furthermore, we observed that high PTBP1 and HNRNPA1 expression is associated with poor survival in high-risk NBL patients (p ≤ 0.05). Finally we demonstrated that knocking down of PTBP1 and HNRNPA1, or their downstream target, the pro-tumor-growth isoform PKM2, resulted in suppressed proliferation in a MYCN-A neuroblastoma cell line.

Section snippets

RNA isolation and whole transcriptome library construction

Clinical features for the 29 pre-treated primary tumors are shown in Table S1. Total RNA was extracted, RNA-seq libraries were made and sequenced as previously described [25], [26]. The integrity of total RNA was evaluated using an Agilent BioAnalyzer 2100 (Agilent, Palo Alto, CA), and only RNAs with RIN greater than 6.0 were used in this study.

RNA-seq reads alignment, differential expression and splicing analysis

First, 50 base nucleotide reads were filtered against a database of rRNA, tRNA, repetitive regions, and adapter sequences. The remaining reads were

RNAseq of NBL samples

In order to investigate the role of MYCN on transcription and splicing control of transcripts, we performed massively parallel RNA sequencing (RNA-seq) on 29 stage 4 NBL tumors (10 MYCN amplified (MYCN-A), 10 MYCN-not-amplified (MYCN-NA), and 9 4S; Table S1). The medium number of reads mapped to the genome was 103.7 million per sample (Fig. S1A), with 35.5% to the coding bases and UTRs, and 1.5% to the exon-exon junctions (Fig. S1B). We used random priming for construction of RNA-seq libraries

Discussion

Differential isoform expression had been difficult to study prior to the advent of massively parallel sequencing technologies. Single nucleotide resolution of RNA-seq reads allow mapping to specific transcription start sites and splicing boundaries, making it feasible to deconvolute transcriptional control and splicing regulation for individual transcripts on a genome wide scale. In this study, we examined the transcriptome of stage 4 NBL tumors for the aberrations that would alter isoform

Data access

Sequence data used for this study are available at dbGaP under the accession number phs000868.v1.p1.

Conflicts of interest

None.

Acknowledgements

We thank the Children's Oncology Group for the collection of samples for this study. This study utilized the high-performance computational capabilities of the Biowulf Linux cluster at the National Institutes of Health (https://hpc.nih.gov/).

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