Cancer Letters

Cancer Letters

Volume 393, 1 May 2017, Pages 40-51
Cancer Letters

Original Article
Prognostic alternative mRNA splicing signature in non-small cell lung cancer

https://doi.org/10.1016/j.canlet.2017.02.016Get rights and content

Highlights

  • Genome-wide prognostic alternative splicing signature was profiled in NSCLC.

  • Prognostic predictor for NSCLC patient was built with high performance.

  • Splicing networks uncovered the potential molecular mechanisms in LUAD and LUSC.

Abstract

Alternative splicing provides a major mechanism to generate protein diversity. Increasing evidence suggests a link of dysregulation of splicing associated with cancer. Genome-wide alternative splicing profiling in lung cancer remains largely unstudied. We generated alternative splicing profiles in 491 lung adenocarcinoma (LUAD) and 471 lung squamous cell carcinoma (LUSC) patients in TCGA using RNA-seq data, prognostic models and splicing networks were built by integrated bioinformatics analysis. A total of 3691 and 2403 alternative splicing events were significantly associated with patient survival in LUAD and LUSC, respectively, including EGFR, CD44, PIK3C3, RRAS2, MAPKAP1 and FGFR2. The area under the curve of the receiver-operator characteristic curve for prognostic predictor in NSCLC was 0.817 at 2000 days of overall survival which were also over 0.8 in LUAD and LUSC, separately. Interestingly, splicing correlation networks uncovered opposite roles of splicing factors in LUAD and LUSC. We created prognostic predictors based on alternative splicing events with high performances for risk stratification in NSCLC patients and uncovered interesting splicing networks in LUAD and LUSC which could be underlying mechanisms.

Introduction

Protein diversity is essential for generating remarkable regulatory and functional complexity in eukaryotic cells. Alternative splicing of pre-mRNA is a prevalent mechanism for producing mRNA isoforms with a finite set of genes [1]. Alternative splicing is a process during which introns of most human multi-exon genes are removed and specific exons are selectively included or excluded [2], [3], [4]. Beyond protein diversity, reduced translation of mRNA isoforms was also regulated by alternative splicing by introducing a premature stop codon leading to degradation [5]. Thus, alternative splicing is an essential process and changes in splicing patterns are closely related to protein functions.

In the last decade, splicing defects and generation of specific isoforms have been found to act as drivers of cancer by extensive genomic and functional studies [6], [7], [8], [9]. It is of particular interest to cancer researchers since growing evidences prove that alternative splicing events are involved in oncogenic processes including proliferation, apoptosis, hypoxia, angiogenesis, immune escape and metastasis [8], [10]. Moreover, Changes in expression of splicing factors may cause global alterations of alternative splicing behavior in cancer [11], [12], and in some cases, mutations of splicing factors can give rise to a specific cancer-promoting splicing isoforms [13].

Lung cancer is the most common incident cancer and the leading cause of cancer death in China [14] and worldwide [15]. Non-small cell lung cancer (NSCLC) is the most prevalent and heterogeneous subtype of lung cancer [16], including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). However, unlike other genomic data such as copy number variation, DNA methylation and gene expression, systematic analysis of alternative splicing is still lacking despite its important role in cancer [17], [18]. Limited efforts have been made in comprehensive genome-wide profiling of alternative splicing in cancer patients, especially in NSCLC.

Several RNA-seq studies of alternative splicing in cancer available now mainly focus on identifying “cancer-specific” alternative splicing events by comparing cancer tissues with normal controls. For example, an early transcriptome study revealed splicing signatures specific to breast cancer such as novel isoforms of CDK4, LARP1, ADD3, and PHLPP2 [19]. A recent analysis of TCGA RNA-seq data identified 163 recurrent differential alternative splicing events between three types of cancer and normal tissues [20].

With rapid accumulation of RNA-seq data in clinical samples, it is possible to study the role of alternative splicing in patients clinical outcomes in relative large populations [21], [22], [23]. Associations between alternative splicing events and patient survival have been reported in subtypes of glioblastoma and breast cancer [24], [25]. However, systematic cancer survival analyses of alternative splicing at individual exon resolution have been lacking, especially in NSCLC.

The Cancer Genome Atlas (TCGA) project provides a rich source for the investigation of alternative splicing patterns in cancer [23]. Exon, splice and transcript isoform expression levels are available from the TCGA Data Portal. We systematically profiled the genome-wide alternative splicing events in 491 LUAD and 471 LUSC cohort from TCGA, uncovering a number of survival associated alternative splicing events. More importantly, prognostic predictors for LUAD, LUSC and merged NSCLC patients were constructed with high performance and we uncovered interesting splicing networks in LUAD and LUSC which could be underlying mechanisms.

Section snippets

Alternative splicing events curation from TCGA RNA-seq data

RNA-seq data of TCGA LUAD and LUSC cohorts were downloaded from TCGA data portal (https://tcga-data.nci.nih.gov/tcga/). We used SpliceSeq tool, a java program application, to analyze the mRNA splicing patterns of TCGA samples [26]. SpliceSeq starts with a reference model for each gene constructed from all of the gene's protein-coding transcripts in the Ensemble gene database. The Percent Spliced In (PSI) value, a common, intuitive ration for quantifying splicing event from zero to one [26], was

Integrated mRNA splicing events profiles in the TCGA LUAD and LUSC cohort

Integrated mRNA splicing events profiles were analyzed in depth for 491 LUAD patients and 471 LUSC patients from the TCGA. In LUAD cohort, we detected 45,062 mRNA splicing events in 10,367 genes, which contains 17,903 ESs in 6075 genes, 2781 RIs in 1585 genes, 8993 APs in 3304 genes, 8549 ATs in 3407 genes, 3057 ADs in 1995 genes, 3559 AAs in 2302 genes and 220 MEs in 202 genes; In LUSC cohort, we detected 50,732 mRNA splicing events in 10,558 genes, which contains 22,737 ESs in 6638 genes,

Discussion

Alternative splicing, an important process which modifies mRNA isoforms, allows cells to generate a large number of mRNA and protein isoforms with diverse regulatory and functional properties. The plasticity of alternative splicing is often exploited by cancer cells to produce isoform switches that promote cancer cell survival, proliferation, metastasis and drug resistance [8], [31], [32]. For example, TAF6δ is a splice variant of the TAF6 protein that is a core subunit of the general RNA

Funding

This study is supported by The National Key Basic Research Development Plan 973 Plan (2014CB542006); CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-1-001); and International Science and Technology Corporation and Exchange Project (2015DFA31090).

Acknowledgments

Not applicable.

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