Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer

Abstract

BIOCHEMISTRY

Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer

Josiah Ewing Hutton, III.

Dissertation under the direction of Professor Daniel C. Liebler Analysis of cancer cells that have undergone metabolic reprogramming, where glucose metabolism is altered to support rapid proliferation, is typically performed utilizing transcriptomic and metabolomic techniques. Transcriptomic data does not always accurately reflect protein expression levels, and metabolomic data alone does not sufficiently explain how these cancer cells have undergone metabolic reprogramming. Mutations in the oncogenes KRAS and BRAF induce metabolic reprogramming in colorectal cancers through enhanced glucose transport, but the broader impact of these oncogenes on metabolic pathways is unclear. I hypothesized that mutant KRAS and mutant BRAF drive metabolic reprogramming in colorectal cancer cells by diverting glucose and glutamine carbons to the production of biosynthetic precursors for rapid growth and that this metabolic reprogramming can be detected by mass spectrometry based proteomic methods. These methods were used, in conjunction with RNA-Seq and metabolite measurements, to determine if isogenic DLD-1 and isogenic RKO cell lines had undergone metabolic reprogramming driven by mutant KRAS G13D and mutant BRAF V600E, respectively. Glucose consumption and lactate production rates indicated that the isogenic cells expressing the mutant oncogenes had undergone metabolic reprogramming. The global transcriptomic and shotgun proteomic analyses did not reveal any metabolic reprogramming at the mRNA or protein levels. However, more sensitive, precise, targeted proteomic analysis of 73 metabolism proteins in these cell lines revealed biologically important protein expression fold changes in proteins involved in glucose metabolism, glutamine metabolism, and the serine biosynthesis pathways. A study of 8 KRAS wild type and 8 KRAS mutant human colon tumors confirmed the association of increased expression of glycolytic and glutamine metabolic proteins with KRAS mutations. These results demonstrate that mutant KRAS and mutant BRAF drive metabolic reprogramming through modest protein expression changes. Moreover, targeted proteomics was required to measure small protein expression differences that could not be detected by transcriptomics or shotgun proteomics. Measurement of the mutant and wild type KRAS and BRAF proteins indicated that the abundance of wild type and mutant BRAF proteins reflected the allelic composition of the RKO cells, whereas wild type and mutant KRAS protein levels did not in DLD-1 cells. Levels of mutant KRAS G13D protein did not reflect the degree of metabolic reprogramming in the DLD-1 cell lines, whereas levels of mutant BRAF V600E protein were proportional to the degree of metabolic reprogramming in the RKO cells. These results underscore the importance of performing targeted, quantitative protein measurements to evaluate the impact of oncogenic mutations.

Approved Date Daniel C. Liebler, Ph.D.