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

To better understand whether KRAS and BRAF mutations prevalent in CRC engender the same phenotype, we created a set of isogenic cell lines by introducing different mutant KRAS alleles in a genetic background where the mutant BRAF V600E allele had been removed by knock-out. Parental RKO cells [27] have two BRAF V600E mutant alleles and one wild-type allele, where the two BRAF V600E mutant alleles have been removed in RKO BRAF WT cells [5] whereas parental HCT116 cells [28] have a single KRAS G13D allele removed in HCT116 KRAS WT cells [24]. We used genome editing by recombinant adeno-associated virus (rAAV) technology [29] to knock in mutant KRAS and BRAF alleles in RKO BRAF WT and HCT116 KRAS WT cells, respectively. Gene targeting constructs were generated by amplifying homology arms from the respective targeted CRC cells followed by introduction the mutations by overlapping PCR [30] (Supplementary Fig. 1 A, Additional File 1). The KRAS mutant constructs were then used to target RKO BRAF WT cells, resulting in three independent edited clones of each KRAS genotype (Table 1, Supplementary Fig. 1B, Additional File 1). Presence of the desired KRAS mutation in targeted cells was demonstrated by sequencing the targeted KRAS exon and the expression of wild type and mutant alleles of KRAS and BRAF was confirmed by Sanger sequencing of the RT-PCR products (Supplementary Fig. 2 A-B, Additional File 1; Supplementary Tables 1, Additional File 2). Finally, expression of the desired wild-type and mutant transcripts of KRAS and BRAF genes was confirmed by transcriptome sequencing (Supplementary Fig. 3 A-G, Additional File 1). Thus, a set of cell models where BRAF V600E and the KRAS codon 12 and 13 mutations can be studied in the same genetic background was generated and validated.

To find and understand common and distinct phenotypes of different Ras pathway mutations in CRC, we characterized the transcriptomes, proteomes and metabolomes of RKO and HCT116 cells with BRAF V600E mutation, KRAS mutations, or no Ras pathway mutation. At the transcriptome level, principal component analysis (PCA) of differentially expressed transcripts in the RNA sequencing data showed clear separation by genetic background between HCT116 and RKO cell lines and the derived cell clones. Surprisingly, no clear separation by Ras pathway mutation was observed (Fig. 1a). Similarly, PCA analysis of ~ 4,500 detected proteins separated the cell clones by genetic background but not by KRAS/BRAF mutation status (Fig. 1b). However, the metabolomics PCA analysis separated RKO as well as HCT116 cell clones by their Ras pathway genotype (Fig. 1c). The KRAS mutant cell lines clustered by mutation, separating mainly along the first principal component, which supports that the introduced mutations are responsible for more variation than any other variable. Taken together, under normal cell culture conditions the overall impact of the Ras pathway mutations at the transcriptome and proteome levels appeared limited whereas stronger effects were observed at the metabolome level.

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.

We next sought to determine whether the different KRAS and BRAF mutations alter the expression of specific genes or proteins. Because of the availability of multiple independent clones per genotype, we focused these analyses on the RKO genetic background. To find differentially expressed genes (DEGs) and proteins (DEPs) regulated by any or all of the different Ras pathway mutations, we compared their gene and protein expression data to the corresponding RKO isogenic controls (Supplementary Fig. 4 A-E and 6 A-D, Additional File 1; Supplementary table 2 A-J and 4 A-I, Additional File 2). We first identified 181 and 766 DEGs between all KRAS mutant clones and WT or BRAF V600E cells, respectively (Fig. 2a-b). For each KRAS mutation studied, the ratio of DEGs between comparisons to BRAF V600E or wildtype was in the range 1.90–2.49 (Supplementary Tables 2, Additional File 2). The notion that the transcriptional response differs more between KRAS and BRAF mutant cells than between KRAS mutants and cells lacking Ras pathway mutation was supported by analyses of HCT116 cells, where the ratio was 1.56 (Supplementary Fig. 5, Additional File 1). We hypothesized that the 22–34 % of DEGs found both in comparisons with BRAF mutant and wild-type cells and having the same direction of expression change could be specific to a particular KRAS mutation (Supplementary Fig. 5 A-D and F, Additional File 1). We identified 35, 70, 26 and 39 such KRAS G12C/D/V/G13D mutation specific DEGs, respectively (Supplementary Fig. 5 A-D and F; Supplementary Fig. 5E, Additional File 1; Supplementary Tables 3, Additional File 2). We next sought to determine whether these DEGs were also controlled by KRAS or BRAF mutations in clinical samples. Several DEGs were differentially expressed with the same direction of gene regulation in a KRAS G12 specific manner also in TCGA COAD data, including BCHE, BEST3, EXO1 [31], FCER2, FGF19, GPM6A, HOTAIR [31], KCNIP3, NTSR1, PRKAA2, SMC4, TMEM71, and TUBAL3 (Supplementary table 4 A-J, Additional File 2) where EXO1 and HOTAIR have previously been linked to Ras signaling. The expression of EXO1 and SMC4 is regulated by the DREAM complex [32], and they interact during DNA replication in yeast. Collectively, common and KRAS mutation specific DEGs with known as well as previously unknown links to the Ras pathway were identified.

Next, we proceeded to identify DEPs regulated by Ras pathway mutations. The ratio of DEPs identified in comparisons of KRAS mutants to BRAF V600E or wild-type was in the range 7.4–24 (Supplementary Tables 5, Additional File 2). As compared to RKO cells with no Ras pathway mutation, known Ras regulated or interacting proteins were identified as DEPs: LGALS1 [33] was a DEP in KRAS G12D and G13D mutants, whereas IFI16 [34], S100A10 [35], CD44 [36], GLRX [37] and AHNAK2 [38] were DEPs in one of the KRAS mutants (Supplementary table 5B, D, F and H, Additional File 2). From the proteins highlighted in [7], AKAP12 was a DEG in comparisons of KRAS mutant to BRAF V600E as well as wild-type cells but not a DEP. Interestingly, 6 DEPs were common to all four KRAS mutations when comparing to BRAF V600E, of which 3 were upregulated (LCP1, S100A10 and S100A2 [39]) and 3 downregulated (CRABP2 [40], FLNA [38] and NXN) more than 10-fold in KRAS mutant clones (Fig. 2c) (Supplementary Fig. 6 A-D, Additional File 1; Supplementary table 5 A, C, E and G, Additional File 2). When identifying KRAS G12C/D/V/13D mutation specific DEPs versus BRAF V600E, we identified 2 (OCRL and VAMP8), 3 (OCIAD2, H1-0 and S100A13), 7 (ANXA2 [41], GNG12, METTL7B, PROCR, CGB1, CD44 and CA9) and 7 (PHGDH [42], AHNAK2 [38], ASMTL, CPT1A, FASTKD5, HMGA1 [43] and FTH1), respectively. Notably, CD44, PROCR and HMGA1 have previously been found upregulated by KRAS G12V [44]. To assess the link between transcription and translation, we identified 11, 14, 17 and 21 DEGs, respectively, as regulated at both levels by KRAS G12C/D/V/G13D mutation as compared to BRAF V600E (p ≤ 3.58E-18, hypergeometric distribution; Fig. 3a-d). Of these, CRABP2, FLNA, LCP1, NXN, S100A2, and S100A10 were regulated at both levels in KRAS mutant cells. While the majority (61–78 %) of DEPs in all four mutants were also DEGs, only 0.2–0.5 % of the DEGs were also DEPs; the vast majority of transcriptional regulation was not reflected in altered protein expression while more than half of DEPs were regulated through altered gene expression. Thus, 6/6 of DEPs identified in comparisons of mutant KRAS clones to isogenic cells with no Ras pathway mutation have previously known roles in Ras signaling, whereas 6 common and 19 KRAS mutation specific DEPs of which 8 were previously known were identified in comparison to BRAF V600E isogenic cells.

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.

Next, we sought to investigate common and unique pathways controlled by different KRAS mutations and whether different pathways are involved in KRAS and BRAF signaling through Ingenuity Pathway Analysis (IPA) of transcriptomes (Supplementary table 6 A-D, Additional File 2) and proteomes (Supplementary table 7 A-B, Additional File 2). In comparison to cells lacking Ras pathway mutation, BRCA1-DNA damage signaling, mismatch repair, and cell-cycle checkpoint signaling was enriched in KRAS G12D mutant cells (Supplementary table 6B, Additional File 2) and actin-based motility, RhoGTPase signaling, and axonal guidance signaling in KRAS G13D (Supplementary table 6D, Additional File 2) (Fig. 4a). Compared to BRAF V600E cells, the Integrin-Linked Kinase (ILK) pathway was significantly altered in KRAS G13D cells at the transcriptome and protein levels, as well as in KRAS G12D at the proteome level (Fig. 4b and c; Supplementary table 6D and 7B, Additional File 2). The Molecule Activation Prediction (MAP) tool predicted ILK signal activation based on the protein expression changes in KRAS G12D and G13D, which is in turn predicted to activate downstream genes related to cell proliferation, adhesion, motility, cancer progression EMT, cancer stem cell markers and chemoresistance [45, 46] (Supplementary Fig. 7 A-C, Additional File 1). Additionally, a canonical pathway analysis of DEPs versus BRAF V600E showed enrichment of the Wnt/beta-catenin signaling pathway in KRAS G13D clones and of the serine biosynthetic pathway in G12D clones (Supplementary table 7 A-B, Additional File 2). Taken together, known (MMR, ILK, cell cycle checkpoint, actin-based motility, RhoGTPase signaling, axonal guidance, serine and glycine biosynthetic pathways) as well as novel (BRCA1-DNA) pathways were significantly regulated as a consequence of Ras mutation and the ILK pathway emerged as regulated by KRAS but not BRAF mutation.

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.

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