Oncogene-driven post-transcriptional regulation in lung cancer

Thesis (Ph.D.)--University of Washington, 2022Lung cancer is often caused by genetic mutations which alter the activity of proteins in the RAS family, with somatic mutation of the KRAS gene occurring in ~30% of lung adenocarcinoma tumors. A gene of the same family, RIT1, is mutated or amplified in 10-15% of lung adenocarcinomas. Although therapies targeting some KRAS-mutated tumors have begun to enter the clinic, a comprehensive understanding of the Ras family and their regulatory functions continues to elude us, preventing effective treatments and complete cures for all Ras-driven lung cancers. I employed high-throughput multi-omic methods to interrogate the regulation of gene expression and protein abundance in oncogenic KRAS and RIT1 cells. Quantitative proteomic and transcriptomic profiling revealed that both mutant KRAS and mutant RIT1 promoted canonical Ras signaling, and overexpression of wild-type RIT1 resulted in phenotypes similar to those of oncogenic RIT1 and KRAS, including induction of epithelial-to-mesenchymal transition. Thus, RIT1 amplifications which result in increased gene expression may be tumorigenic. Then, with quantitative phosphoproteomics, I found that KRAS variant cells differentially phosphorylated SR protein phosphosites. To determine how KRAS may regulate alternative splicing, I analyzed unique RNA sequencing profiles of lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer. Mutant KRAS induced the greatest number of differential alternative splicing events when compared to the wild-type gene, second only to the differential splicing between RNA binding protein RBM45 and its mutant RBM45M126I. These data suggest that in addition to widespread transcriptional changes, Ras signaling pathways in cancer promote post-transcriptional splicing changes that may contribute to oncogenic processes. In sum, I provided a baseline for how oncogenic KRAS and RIT1 affect the proteome and transcriptome of lung cancer cells, and exposed potential oncogenic mechanisms and therapeutic vulnerabilities in RIT1 or KRAS altered cells