Common and mutation specific phenotypes of KRAS and BRAF mutations in colorectal cancer cells revealed by integrative -omics analysis

Abstract

Background: Genes in the Ras pathway have somatic mutations in at least 60 % of colorectal cancers. Despite activating the same pathway, the BRAF V600E mutation and the prevalent mutations in codon 12 and 13 of KRAS have all been linked to different clinical outcomes, but the molecular mechanisms behind these differences largely remain to be clarified.

Methods: To characterize the similarities and differences between common activating KRAS mutations and between KRAS and BRAF mutations, we used genome editing to engineer KRAS G12C/D/V and G13D mutations in colorectal cancer cells that had their mutant BRAF V600E allele removed and subjected them to transcriptome sequencing, global proteomics and metabolomics analyses.

Results: By intersecting differentially expressed genes, proteins and metabolites, we uncovered (i) two-fold more regulated genes and proteins when comparing KRAS to BRAF mutant cells to those lacking Ras pathway mutation, (ii) five differentially expressed proteins in KRAS mutants compared to cells lacking Ras pathway mutation (IFI16, S100A10, CD44, GLRX and AHNAK2) and 6 (CRABP2, FLNA, NXN, LCP1, S100A10 and S100A2) compared to BRAF mutant cells, (iii) 19 proteins expressed differentially in a KRAS mutation specific manner versus BRAF V600E cells, (iv) regulation of the Integrin Linked Kinase pathway by KRAS but not BRAF mutation, (v) regulation of amino acid metabolism, particularly of the tyrosine, histidine, arginine and proline pathways, the urea cycle and purine metabolism by Ras pathway mutations, (vi) increased free carnitine in KRAS and BRAF mutant RKO cells.

Conclusions: This comprehensive integrative -omics analysis confirms known and adds novel genes, proteins and metabolic pathways regulated by mutant KRAS and BRAF signaling in colorectal cancer. The results from the new model systems presented here can inform future development of diagnostic and therapeutic approaches targeting tumors with KRAS and BRAF mutations.

Keywords: BRAF; Colorectal cancer; Integrative -omics analysis; Isogenic cell models; KRAS; Ras pathway.

Fig. 1

Integrative analysis of transcriptomes, proteomes and metabolomes of KRAS G12C/D/V and G13D mutant RKO colorectal cancer cells. a Principal Component Analysis (PCA) of transcriptome sequencing data  from ~25M reads/sample. b global proteomic data from ~4,500 proteins. c LC/MS metabolomics data from average 700 metabolites from knock-ins of different mutant KRAS alleles in RKO CRC cells deprived of their mutant BRAF allele.

Fig. 2

Transcriptomic and proteomic analyses reveal joint and mutation-specific regulation of gene and protein expression by KRAS G12C/D/V and G13D mutations. a and b Differentially expressed genes. c Differentially expressed proteins. All having ½log2 FC½ > 1 and adjusted P < 0.05 between A KRAS mutant and WT or b and c BRAF V600E RKO cells.

Fig. 3

Differentially expressed proteins are primarily differentially expressed genes, but not vice versa, in KRAS mutants compared to BRAF V600E cells. Differentially expressed proteins (DEPs) were intersected with   differentially expressed genes from RNA sequencing data, comparing isogenic RKO BRAF V600E cells with KRAS G12C (a), G12D (b), G12V (c) and G13D (d). Differential expression was defined as ½log2 FC½ > 1 and adjusted P < 0.05 (hypergeometric distribution). Intersecting DEGs and DEPs are listed with genes common to all four comparisons in bold.

Fig. 4

Joint and mutation specific regulation of molecular pathways by KRAS mutations. Binary heatmaps from Ingenuity Pathway Analysis (IPA) of KRAS mutant cells compared to wild-type or BRAF V600E mutant cells. IPA analysis of transcriptome comparisons of (a) KRAS G12C/D/V/G13D versus wild-type, (b) KRAS G12C/D/V and G13D vs BRAF V600E. Proteome DEPs from (c) KRAS G12D/13D vs BRAF V600E comparisons; only these two comparisons had sufficient differentially expressed proteins (DEPs) for IPA analysis. The IPA analysis included DEGs or DEPs fulfillingㅣlog2 FCㅣ > 1, adjusted P value <0.05 and Z-score ≥ 2 or ≤ - 2.

Fig. 5

Metabolic pathway analysis reveals regulation of amino acid metabolism and carnitine biosynthesis by Ras pathway mutations. LC-MS metabolomics analysis was performed on KRAS knock-ins and isogenic controls followed by pathway analysis comparing (a) G12C, (b) G12D, (c) G12V and (d) G13D vs BRAF V600E (upper panel) and wildtype (lower panel). Pathways with P < 0.001 and 0.05 were designated with orange and blue bars, respectively. e The KRAS mutants showed increases in free Carnitine and decreases in acetyl and butyrylcarnitines compared to the isogenic BRAF wild-type control cell line. Average values were from six biological replicates with SD.