Original ArticlesMYCN controls an alternative RNA splicing program in high-risk metastatic neuroblastoma
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/).
References (64)
- et al.
Function of alternative splicing
Gene
(2013) - et al.
Pre-mRNA splicing in disease and therapeutics
Trends Mol. Med
(2012) Splicing of pre-mRNA: mechanism, regulation and role in development
Curr. Opin. Genet. Dev
(1993)- et al.
Alternative splicing in colon, bladder, and prostate cancer identified by exon array analysis
Mol Cell Proteomics
(2008) - et al.
MYCN-non-amplified metastatic neuroblastoma with good prognosis and spontaneous regression: a molecular portrait of stage 4S
Mol Oncol
(2008) - et al.
Advances in the understanding of constitutional and somatic genomic alterations in neuroblastoma
Cancer Genet
(2011) - et al.
Extracting regulatory sites from the upstream region of yeast genes by computational analysis of oligonucleotide frequencies
J. Mol. Biol
(1998) - et al.
PKM2 contributes to cancer metabolism
Cancer Lett
(2015) - et al.
The long noncoding RNA Pnky regulates neuronal differentiation of embryonic and postnatal neural stem cells
Cell Stem Cell
(2015) - et al.
Direct conversion of fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits
Cell
(2013)
Pyruvate kinase type M2 and its role in tumor growth and spreading
Semin. Cancer Biol
PKM2 isoform-specific deletion reveals a differential requirement for pyruvate kinase in tumor cells
Cell
Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing
Nat. Genet
Alternative isoform regulation in human tissue transcriptomes
Nature
Splicing programs and cancer
J Nucleic Acids
Global analysis of aberrant pre-mRNA splicing in glioblastoma using exon expression arrays
BMC Genomics
Exon array analysis using re-defined probe sets results in reliable identification of alternatively spliced genes in non-small cell lung cancer
BMC Genomics
Small interfering RNA-mediated reduction in heterogeneous nuclear ribonucleoparticule A1/A2 proteins induces apoptosis in human cancer cells but not in normal mortal cell lines
Cancer Res
Transcriptome instability in colorectal cancer identified by exon microarray analyses: associations with splicing factor expression levels and patient survival
Genome Med
Cancer-associated regulation of alternative splicing
Nat. Struct. Mol. Biol
Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array
BMC Genomics
Whole genome exon arrays identify differential expression of alternatively spliced, cancer-related genes in lung cancer
Nucleic Acids Res
The International Neuroblastoma Risk Group (INRG) classification system: an INRG Task Force report
J. Clin. Oncol
Recent advances in neuroblastoma
N. Engl. J. Med
Principles and Practice of Pediatric Oncology
Association of age at diagnosis and genetic mutations in patients with neuroblastoma
JAMA
Sequencing of neuroblastoma identifies chromothripsis and defects in neuritogenesis genes
Nature
The genetic landscape of high-risk neuroblastoma
Nat. Genet
Integrated genomic analyses identify ARID1A and ARID1B alterations in the childhood cancer neuroblastoma
Nat. Genet
Exon array analysis reveals neuroblastoma tumors have distinct alternative splicing patterns according to stage and MYCN amplification status
BMC Med Genomics
Khan, Massively parallel sequencing reveals an accumulation of de novo mutations and an activating mutation of LPAR1 in a patient with metastatic neuroblastoma
PLoS ONE
Purification of total RNA from mammalian cells and tissues
Cited by (40)
Alternative RNA splicing defects in pediatric cancers: new insights in tumorigenesis and potential therapeutic vulnerabilities
2022, Annals of OncologyCitation Excerpt :In neuroblastoma, the most common extracranial solid tumor in children, the splicing factors hnRNPA1 and PTBP1 are up-regulated due to N-Myc overexpression.113,114 Both splicing factors increase neuroblastoma cell proliferation and the differential splicing of PKM2 through the inclusion of exon 10.114 Like INSR, the PKM2 splice variant is primarily expressed during fetal development and in several cancers due to its favorable effect on cell metabolism.115
The role of RNA processing and regulation in metastatic dormancy
2022, Seminars in Cancer BiologyThe role of alternative splicing in cancer: From oncogenesis to drug resistance
2020, Drug Resistance UpdatesCitation Excerpt :The molecular mechanism underlying this effect was proposed to be dependent on the PHF5A protein, which in glioblastoma cells as opposed to non-malignant cells, facilitated recognition of specific (C-rich) splice sites in essential genes. Similarly, expression of N-MYC-induced glycolysis-promoting splice variant PKM2 was suggested to contribute to this synthetic lethality effect in metastatic neuroblastoma (S. Zhang et al., 2016; T. Zhang et al., 2016). A more recent report demonstrated that a CLK inhibitor T-025 exhibited increased cytotoxicity against tumor cells with MYC-activation and/or high CLK2 expression (Iwai et al., 2018).
Prognostic value and oncogene function of heterogeneous nuclear ribonucleoprotein A1 overexpression in HBV-related hepatocellular carcinoma
2019, International Journal of Biological MacromoleculesCitation Excerpt :Alternative splicing of HNRNPA1 is regulated throughout the human transcriptome and uses several mechanisms in conjunction with several splicing factors [30]. Knockdown of HNRNPA1 and its downstream target PKM2 can inhibits cancer cell growth [31]. In addition, HNRNPA1 may prevent tumor cells from entering senescence by binding to telomeric DNA sequences [32].
Alternative splicing and related RNA binding proteins in human health and disease
2024, Signal Transduction and Targeted Therapy
- 1
These authors contributed equally.