Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer
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 unexp...
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| Format: | Article |
| Language: | English |
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BMC
2022-12-01
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| Series: | BMC Cancer |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12885-022-10311-1 |
| _version_ | 1811291747842523136 |
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| author | April Lo Maria McSharry Alice H. Berger |
| author_facet | April Lo Maria McSharry Alice H. Berger |
| author_sort | April Lo |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T04:34:00Z |
| format | Article |
| id | doaj.art-37b055328bd444adb4bdac801f188e35 |
| institution | Directory Open Access Journal |
| issn | 1471-2407 |
| language | English |
| last_indexed | 2024-04-13T04:34:00Z |
| publishDate | 2022-12-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Cancer |
| spelling | doaj.art-37b055328bd444adb4bdac801f188e352022-12-22T03:02:13ZengBMCBMC Cancer1471-24072022-12-0122111310.1186/s12885-022-10311-1Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancerApril Lo0Maria McSharry1Alice H. Berger2Human Biology Division, Fred Hutchinson Cancer CenterHuman Biology Division, Fred Hutchinson Cancer CenterHuman Biology Division, Fred Hutchinson Cancer CenterAbstract 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.https://doi.org/10.1186/s12885-022-10311-1KRASSplicingLung cancerGene expressionProteomics |
| spellingShingle | April Lo Maria McSharry Alice H. Berger Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer BMC Cancer KRAS Splicing Lung cancer Gene expression Proteomics |
| title | Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer |
| title_full | Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer |
| title_fullStr | Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer |
| title_full_unstemmed | Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer |
| title_short | Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer |
| title_sort | oncogenic kras alters splicing factor phosphorylation and alternative splicing in lung cancer |
| topic | KRAS Splicing Lung cancer Gene expression Proteomics |
| url | https://doi.org/10.1186/s12885-022-10311-1 |
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