Abstract
BackgroundLong-read sequencing of full-length cDNAs enables the detection of structures of aberrant splicing isoforms in cancer cells. These isoforms are occasionally translated, presented by HLA molecules, and recognized as neoantigens. This study used a long-read sequencer (MinION) to construct a comprehensive catalog of aberrant splicing isoforms in non-small-cell lung cancers, by which novel isoforms and potential neoantigens are identified.ResultsFull-length cDNA sequencing is performed using 22 cell lines, and a total of 2021 novel splicing isoforms are identified. The protein expression of some of these isoforms is then validated by proteome analysis. Ablations of a nonsense-mediated mRNA decay (NMD) factor, UPF1, and a splicing factor, SF3B1, are found to increase the proportion of aberrant transcripts. NetMHC evaluation of the binding affinities to each type of HLA molecule reveals that some of the isoforms potentially generate neoantigen candidates. We also identify aberrant splicing isoforms in seven non-small-cell lung cancer specimens. An enzyme-linked immune absorbent spot assay indicates that approximately half the peptide candidates have the potential to activate T cell responses through their interaction with HLA molecules. Finally, we estimate the number of isoforms in The Cancer Genome Atlas (TCGA) datasets by referring to the constructed catalog and found that disruption of NMD factors is significantly correlated with the number of splicing isoforms found in the TCGA-Lung Adenocarcinoma data collection.ConclusionsOur results indicate that long-read sequencing of full-length cDNAs is essential for the precise identification of aberrant transcript structures in cancer cells.
Highlights
Long-read sequencing of full-length cDNAs enables the detection of structures of aberrant splicing isoforms in cancer cells
Cataloging full-length transcript isoforms of lung cancer cell lines MinION full-length DNA sequencing was performed for 22 non-small cell lung cancer (NSCLC) cell lines (Additional file 1: Table S1), in which characteristic genomic driver mutation patterns and transcriptomic profiles of lung cancers were collectively represented
The obtained splicing patterns were further compared to the current transcript models of the Reference Sequence (RefSeq) database
Summary
Long-read sequencing of full-length cDNAs enables the detection of structures of aberrant splicing isoforms in cancer cells. These isoforms are occasionally translated, presented by HLA molecules, and recognized as neoantigens. Transcript regulations are disrupted at various steps, resulting in the accumulation of substantial numbers of aberrant transcripts [1]. In normal cells, these aberrant transcripts, even if transcribed, are subsequently degraded by the mRNA quality control system in a process known as nonsense-mediated mRNA decay (NMD). The NMD regulation mechanism is frequently disrupted in cancer cells [3,4,5], allowing the aberrant transcripts to escape degradation. The relationship between NMD and tumor immunity has been reported [8] and considered to be a therapeutic target for cancers in some conditions [9]
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