Gene expression deregulation by KRAS G12D and G12V in a BRAF V600E context

Abstract

Background: KRAS and BRAF mutations appear of relevance in the genesis and progression of several solid tumor types but the co-occurrence and interaction of these mutations have not yet been fully elucidated. Using a microsatellite stable (MSS) colorectal cancer (CRC) cell line (Colo741) having mutated BRAF and KRASWT, we also aimed to investigate the KRAS-BRAF interaction. Gene expression profiles for control KRASWT, KRAS G12V and KRAS G12D transfected cells were obtained after cell clone selection and RT-PCR screening. Extensive qPCR was performed to confirm microarray data.

Results: We found that the KRAS G12V state deregulated several genes associated to cell cycle, apoptosis and nitrogen metabolism. These findings indicated a reduced survival and proliferation with respect to the KRASWT state. The KRAS G12D state was, instead, characterized by several other distinct functional changes as for example those related to chromatin organization and cell-cell adhesion without affecting apoptosis related genes.

Conclusion: These data predict that the G12D mutation may be more likely selected in a BRAF mutated context. At the same time, the presence of the KRAS G12V mutation in the cells escaping apoptosis and inducing angiogenesis via IL8 may confer a more aggressive phenotype. The present results get along with the observations that CRCs with G12V are associated with a worse prognosis with respect to the WT and G12D states and may help identifying novel CRC pathways and biomarkers of clinical relevance.

Figure 1

Venn diagrams obtained by SAM analysis. Global comparison among the genes regulated by the KRAS^G12D, KRAS^G12V and KRAS^WT isoforms expressed by transfection in Colo741 cells. The number of probe sets associated to the co-regulated genes is reported in the overlapping areas.

Figure 2

Microarray analysis performed with TIGR MeV program: principal component analysis. Microarray analysis of Colo741 cell clones transfected with constructs expressing the KRAS^G12D (G12D), KRAS^G12V (G12V) and KRAS^WT (WT) isoforms. Probe sets associated to dysregulation of gene expression levels among the three groups were identified using SAM (see Materials and Methods). The corresponding values from two independent microarray analysis were averaged. Principal component analysis (PCA) is shown to provide the 2D projections onto the plane spanned by the two principal components for the three different KRAS profiling data sets.

Figure 3

Microarray analysis performed with TIGR MeV program: hierarchical clustering. Heat map visualization obtained by hierarchical clustering (HCL). Ratios for each probe relative to the mean value (calculated from the two independent microarray analysis for each condition) were used to rearrange the gene list on the basis of their expression pattern. Probes corresponding to genes with similar regulation trend were placed close to each other. The color-ratio bar indicates intensity of gene up-regulation (red), down-regulation (green) and no change (black).

Figure 4

Real-Time RT-PCR validation of microarray data. Real-Time RT-PCR analysis performed on Colo741 cell clones transfected with constructs expressing the KRAS^G12D (G12D), KRAS^G12V (G12V) and KRAS^WT (WT) isoforms to validate the microarray data. This was accomplished on randomly selected genes from Table 1 and showed, in arbitrary units, KRAS isoform-dependent regulation of cell cycle arrest genes (A), of cellular component organization and biogenesis genes (B), of immune system process genes (C) or sterol metabolic process genes (D). Other KRAS isoform-regulated genes associated to miscellaneous functions and randomly selected from Tables S1 and S2, are shown in (E). Real-Time RT-PCR and microarray data are respectively indicated by gray and black bars. Expression levels are relative to the expression of the housekeeping Ribosomal protein L19 gene (RPL19). Standard deviations of Real-Time RT-PCR data are indicated as vertical bars. Gene name symbols used are those approved by the Human Genome Organisation Gene Nomenclature Committee .