Abstract
Abstract
Background
Alternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.
Methods
We recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.
Results
Mass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRASG12V and KRASQ61H cells, respectively, 997 of which were shared (p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its variant RBM45M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS.
Conclusion
Proteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.
Funder
National Cancer Institute
National Science Foundation
Publisher
Springer Science and Business Media LLC
Subject
Cancer Research,Genetics,Oncology
Reference67 articles.
1. American Cancer Society. Cancer Facts & Figures 2021. 2021. https://www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2021.html.
2. Campbell JD, Alexandrov A, Kim J, Wala J, Berger AH, Pedamallu CS, et al. Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas. Nat Genet. 2016;48:607–16.
3. Kahles A, Lehmann K-V, Toussaint NC, Hüser M, Stark SG, Sachsenberg T, et al. Comprehensive analysis of alternative splicing across tumors from 8,705 patients. Cancer Cell 2018;0.
4. Jayasinghe RG, Cao S, Gao Q, Wendl MC, Vo NS, Reynolds SM, et al. Systematic Analysis of Splice-Site-Creating Mutations in Cancer. Cell Rep. 2018;23:270–281.e3.
5. Lu X, Peled N, Greer J, Wu W, Choi P, Berger AH, et al. MET exon 14 mutation encodes an actionable therapeutic target in lung adenocarcinoma. Cancer Res. 2017;77:4498–505.
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