Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer

Abstract

Metabolic reprogramming, in which altered utilization of glucose and glutamine supports rapid growth, is a hallmark of most cancers. Mutations in the oncogenes KRAS and BRAF drive metabolic reprogramming through enhanced glucose uptake, but the broader impact of these mutations on pathways of carbon metabolism is unknown. Global shotgun proteomic analysis of isogenic DLD-1 and RKO colon cancer cell lines expressing mutant and wild type KRAS or BRAF, respectively, failed to identify significant differences (at least 2-fold) in metabolic protein abundance. However, a multiplexed parallel reaction monitoring (PRM) strategy targeting 73 metabolic proteins identified significant protein abundance increases of 1.25-twofold in glycolysis, the nonoxidative pentose phosphate pathway, glutamine metabolism, and the phosphoserine biosynthetic pathway in cells with KRAS G13D mutations or BRAF V600E mutations. These alterations corresponded to mutant KRAS and BRAF-dependent increases in glucose uptake and lactate production. Metabolic reprogramming and glucose conversion to lactate in RKO cells were proportional to levels of BRAF V600E protein.In DLD-1 cells, these effects were independent of the ratio of KRAS G13D to KRAS wild type protein. 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 mutant status. Metabolic reprogramming is driven largely by modest (<2-fold) alterations in protein expression, which are not readily detected by the global profiling methods most commonly employed in proteomic studies. The results indicate the superiority of more precise, multiplexed, pathway-targeted analyses to study functional proteome systems. Data are available through MassIVE Accession MSV000079486 at

Comparative proteomics of a model MCF10A-KRasG12V cell line reveals a distinct molecular signature of the KRasG12V cell surface

“…PRM has been successfully used for multiplex quantitation of cytosolic [90] or soluble serum proteins [91]. Towards that goal, we selected xenotropic and polytropic retrovirus receptor 1 (XPR1) as a model surface protein that was found upregulated on the MCF10A-KRas G12V surface via SGM proteomics (Supplementary Table S7B).…”

WITHDRAWN: Quantitative mass spectrometry analysis of PD-L1 protein expression, N-glycosylation and expression stoichiometry with PD-1 and PD-L2 in human melanoma

“…Moreover, targeted MS is ideal for the analysis of clinical specimens, including formalin-fixed, paraffin-embedded (FFPE) sections (34). PRM provides a platform to systematically configure multiplexed, targeted assays for simultaneous analysis of dozens of proteins, thereby enabling focused quantitative analysis of biological systems and pathways (35,36).…”

“…Moreover, targeted MS is ideal for the analysis of clinical specimens, including formalin-fixed, paraffin-embedded (FFPE) sections (34). PRM provides a platform to systematically configure multiplexed, targeted assays for simultaneous analysis of dozens of proteins, thereby enabling focused quantitative analysis of biological systems and pathways (35,36).Here we describe the development of an addressable fractionation-PRM platform, which provides measurements of PD-1, PD-L1 and PD-L2 at femtomole per microgram tissue protein levels in FFPE sections of human melanoma biopsies. We compared our analyses to IHC measurements of PD-L1 in adjacent sections and further characterized the effects of variable cellular composition on measured PD-1, PD-L1 and PD-L2.…”

Mitochondrial Respiration in KRAS and BRAF Mutated Colorectal Tumors and Polyps

“…Although transcriptome profiles are not associated with specific mutations, the frequency of KRAS mutation varies among the CRC subtypes (23% in CMS1, 28% in CMS2, 68% in CMS3, and 38% in CMS4), these data suggest mutations may drive distinct programs of metabolism gene expression [7]. Mutations in KRAS or BRAF genes appear to play an important role in the regulation of metabolic reprogramming in multiple cancers, including CRC [8][9][10][11]. In this study, two established and common prognostic biomarkers in CRC were investigated: KRAS and BRAF mutation status.…”

“…Thus, the main goal of our study was to characterize the functional activity of mitochondrial OXPHOS among premalignant polyps and CRC, taking into account their KRAS and BRAF mutation status. To date, it has been shown that KRAS and BRAF mutations increase the glycolytic capacity of tumor cells and their glutaminolysis [8,35]. In our work, the function of the OXPHOS system was analyzed by means of high-resolution respirometry using freshly prepared postoperative tissue samples.…”