Aberrant splicing isoforms detected by full-length transcriptome sequencing as transcripts of potential neoantigens in non-small cell lung cancer

Abstract

Background 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. 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. Results Full-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. Conclusions Our results indicate that long-read sequencing of full-length cDNAs is essential for the precise identification of aberrant transcript structures in cancer cells.

Alternative RNA Splicing—The Trojan Horse of Cancer Cells in Chemotherapy

“…It is responsible for aberrant transcripts degradation, which is enforced by a constant scan of each transcript by the NMD factors for mRNAs harboring premature translation termination codons. In cancer cells, the NMD regulation is also frequently disrupted, conferring aberrant transcripts accumulation [ 20 , 21 ]. Invasion and metastasis are the hallmarks of malignant tumors and depend on the interactions between cancer cells and the tumor microenvironment.…”

“…Recent combined long and short sequencing of 22 lung cancer cell lines and patients’ tissue specimens showed several aberrant transcript isoforms. They include unannotated exon, exon skipping, exon shuffling, intron retention, alternative first exon, alternative last exon, alternative 5′ splice site, and alternative 3′ splice site [ 20 ]. Additionally, novel combinations of several splicing events are also frequently found on a single full-length transcript.…”

“…These new patterns account for 13.6% of the unannotated cancer de novo isoforms, with characteristic NSCLC aberrant splicing events being fairly distinct among cancer specimens. Many genes produce multiple isoforms, each having different AS [ 20 ]. On the other hand, repetitive element loci in splicing sites, which can potentially lead to misalignment of the reads, were not found linked to novel splice sites [ 20 ].…”

“…Many genes produce multiple isoforms, each having different AS [ 20 ]. On the other hand, repetitive element loci in splicing sites, which can potentially lead to misalignment of the reads, were not found linked to novel splice sites [ 20 ]. 5.…”

“…Gene-fusion events were also reported to function as driver genes contributing to tumorigenicity in many cancers [ 32 , 33 ], such as newly identified in lung cancer fusion transcripts EML4-ALK, and ERGIC2-CHRNA6. The latter is a drastic frameshift fusion event causing an amino acid change that had actually four alternative splice isoforms, consisting of an unannotated exon and an alternative last exon [ 20 ]. NMD is a key mechanism for elimination of aberrant splicing isoforms.…”