Oncogenic Mutation BRAF V600E Changes Phenotypic Behavior of THLE-2 Liver Cells through Alteration of Gene Expression

Abstract

The accumulation of mutations in cancer driver genes, such as tumor suppressors or proto-oncogenes, affects cellular homeostasis. Disturbances in the mechanism controlling proliferation cause significant augmentation of cell growth and division due to the loss of sensitivity to the regulatory signals. Nowadays, an increasing number of cases of liver cancer are observed worldwide. Data provided by the International Cancer Genome Consortium (ICGC) have indicated many alterations within gene sequences, whose roles in tumor development are not well understood. A comprehensive analysis of liver cancer (virus-associated hepatocellular carcinoma) samples has identified new and rare mutations in B-Raf proto-oncogene (BRAF) in Japanese HCC patients, as well as BRAF V600E mutations in French HCC patients. However, their function in liver cancer has never been investigated. Here, using functional analysis and next generation sequencing, we demonstrate the tumorigenic effect of BRAF V600E on hepatocytes (THLE-2 cell line). Moreover, we identified genes such as BMP6, CXCL11, IL1B, TBX21, RSAD2, MMP10, and SERPIND1, which are possibly regulated by the BRAF V600E-mediated, mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) signaling pathway. Through several functional assays, we demonstrate that BRAF L537M, D594A, and E648G mutations alone are not pathogenic in liver cancer. The investigation of genome mutations and the determination of their impact on cellular processes and functions is crucial to unraveling the molecular mechanisms of liver cancer development.

Keywords: BRAF mutation; MAPK/ERK; hepatocellular carcinoma; liver cancer.

Figure 1

Alignment of BRAF amino acid sequences from different species with mutation sites indicated as *. Sequence alignment was performed between species—Homo sapiens, Mus musculus, Bos taurus, Danio rerio, and Xenopus tropicalis—by PRALINE software. Results are shown as a color-coded pattern. The scoring scheme ranges from 0 (for the least conserved alignment position) (in blue) up to 10 (for the most conserved alignment position) (in red). BRAF mutations selected for study have exhibited the highest conservation score between species.

Figure 2

Protein expression measured by Western blot. (a) Expression of ERK-phosphorylated (P-ERK), FLAG (BRAF), and ERK proteins in THLE-2 cells overexpressed with p3XFLAG-CMV empty plasmid (EM), or plasmid containing BRAF WT (WT) and BRAF mutations (V600E, E648G, L537M, D594A). The density of each band was quantified by ImageJ software (b) The protein expression level was normalized to FLAG (BRAF) and presented as a fold change (±SD) of band intensity in the presented blot. The experiment was conducted in four independent replicates (n = 4) and analyzed using a one-way analysis of variance (ANOVA) with Bonferroni correction, *** p  <  0.001. The expression of ERK proteins was equal for all samples. M—protein marker 10–245 kDa.

Figure 3

Cell migration assay on THLE-2 cells with overexpression of BRAF mutations. (a) Scratch assay picture of non-transfected (CTR) THLE-2 cells, transfected with BRAF wild type (WT), mutants (D594A, V600E, L537M, E648G), and empty plasmid (EM). Dark dashed lines indicate the trace of the wound front. Scale bar 200 μm. (b) Bar chart with the number of migrated cells (±SEM) for all experimental groups, n = 3, *** p  <  0.001. The statistical analysis was carried out using ANOVA with Bonferroni correction.

Figure 4

Effect of BRAF mutant overexpression in THLE-2 cells on cell proliferation. A statistically significant augmentation in hepatocyte proliferation, when compared with control, was observed in BRAF V600E-transfected cells. The results are presented as a fold change (±SEM) normalized to BRAF WT-transfected cells. The statistical analysis was carried out using ANOVA with Bonferroni correction, n = 3, *** p  <  0.001.

Figure 5

Effect of BRAF V600E overexpression on the THLE-2 cells invasion. THLE-2 cells exhibited augmented invasion capacity after overexpression of BRAF V600E compared with control cells. Quantitative data of invasion assay are presented as the mean ± SD, *** p  <  0.001. One-way ANOVA with Bonferroni corrections was applied for statistical analysis.

Figure 6

Principal component analysis (PCA) plot and the number of DEGs obtained from BGI RNA-Seq (transcriptome) sequencing. (A) The clustering of color-coded dots indicates high consistency between replicate samples. Dots refer to RNA samples extracted from (1) THLE-2 cells transfected with empty plasmid (EM) are marked in red, (2) BRAF WT-transfected THLE-2 cells (WT) are presented as green, (3) BRAF V600E-transfected THLE-2 cells (V600E) are marked in blue. (B) Venn diagram of DEGs. Three comparisons were analyzed, as follows: WT vs. EM, V600E vs. EM, V600E vs. WT. The overlapping part of the different circles indicates the number of DEGs common to respective groups. (C) The results indicated down-regulated and up-regulated genes in comparisons, as follows: WT vs. EM, V600E vs. EM, and V600E vs. WT. A p-value cut-off of 0.1 after Benjamini–Hochberg multiple-testing correction was used to fulfill the criteria of significance.

Figure 7

Heatmap of all significantly differentially expressed genes. Rows represent genes and each column is a contrast. The bars on the left display significantly expressed genes. In the main plot, the scale runs from red (negative fold change) to blue (positive fold change). The blue bar near the top of the plot is BRAF and BRAFP1 (BRAF Pseudogene 1), where the fold change is highest compared with the empty plasmid-transfected cells.

Figure 8

Validation of RNA-seq data by quantitative RT-qPCR (qPCR). The analysis was performed for 8 differentially expressed genes. Black bars represent results obtained from RNA-seq and gray bars indicate qPCR data. B2M was applied for the normalization of gene expression. All data are mean (±SEM), n = 2.

Figure 9

Top 10 significantly dysregulated canonical pathways. The bars present the various pathways in comparisons (WT vs. EM, V600E vs. EM, V600E vs. WT) based on a significant enrichment score (–log(p-value)).

Figure 10

Cytoscape-ClueGo gene network interaction analysis, up- (blue) and down-regulated (red) genes. (a) Overexpression of BRAF V600E in THLE-2 cells influences the interferon- and metalloproteinase-dependent signaling pathways. (b) Analysis pointed out dysregulation of genes involved in the endothelium development and viral response. (c) An enhancement of IL-17 signaling pathway with attenuation of gene expression in response to viral infection and cytokine production.

Figure 11

Effects of BRAF mutations on the selected gene expression. A significant upregulation (fold change) of BMP6, IL1B, TBX21, MMP10, and SERPIND1 was seen in BRAF V600E-transfected THLE-2 cells. Results are presented as a fold change in contrast to the expression of BRAF WT-transfected cells (±SEM). B2M was subjected as a reference gene. The statistical analysis was performed using a one-way ANOVA with Bonferroni correction, n = 3, ** p < 0.01, *** p  <  0.001.