The BRAF(V600E) causes widespread alterations in gene methylation in the genome of melanoma cells

Abstract

Although BRAF(V600E) is well known to play an important role in the tumorigenesis of melanoma, its molecular mechanism, particularly the epigenetic aspect, has been incompletely understood. Here, we investigated the role of BRAF(V600E) signaling in altering gene methylation in the genome of melanoma cells using a methylated CpG island amplification/CpG island microarray system and searched for genes coupled to the BRAF(V600E) signaling through methylation aberrations. The results indicated that a wide range of genes with broad functions were linked to BRAF(V600E) signaling through their hyper- or hypomethylation. Expression of 59 genes hypermethylated upon BRAF knockdown was selectively tested and found to be largely correspondingly underexpressed, suggesting that these genes were naturally hypomethylated, and overexpressed with BRAF(V600E) in melanoma. This BRAF(V600E)-promoted hypomethylation was confirmed on genes selectively examined in primary melanoma tumors. Some of these genes were functionally tested and demonstrated to play a role in melanoma cell proliferation and invasion. As a mechanism of aberrant gene methylation driven by BRAF(V600E), expression of the DNA methyltransferase 1 and histone methyltransferase EZH2 was profoundly affected by BRAF(V600E). We have thus uncovered a previously unrecognized prominent epigenetic mechanism in the tumorigenesis of melanoma driven by BRAF(V600E). Many of the functionally important genes controlled by the BRAF(V600E) signaling through aberrant methylation may prove to be novel therapeutic targets for melanoma.

Figure 1

Procedures of the global identification of BRAF^V600E signaling-mediated DNA methylation targets in melanoma cells using MCA/CpG island microarray. (A) Stable shRNA knockdown of BRAF and suppression of the signaling of the MAP kinase pathway in BRAF^V600E-positive melanoma cells UACC62, A375 and M14. Cells were infected with lentivirus expressing BRAF shRNA, and stable populations were selected with 2 µg/ml puromycin. Transfection with the empty vector was used as a control. After a 2-week selection, cells were lysed and immunoblotted with BRAF and phosphorylated ERK (p-ERK) antibodies. The antibody against β-actin was used for quality control of western blotting. (B) Schematic diagram of the MCA/CpG island microarray procedure. Enrichment of methylated DNA and reduction of genome complexity were achieved by serial digestion with SmaI (methylation sensitive) and XmaI (methylation insensitive) restriction enzymes. The resulting amplicons, representative of the methylated fraction from cells stably transfected with BRAF shRNA and vector, were labeled with appropriate dyes and co-hybridized in a microarray platform as detailed in the Materials and Methods.

Figure 2

Detection of hyper- and hypomethylation DNA sites by the MCA/CpG island microarray approach and identification of specific genes with altered methylation status. (A) Reproducibility of the MCA/CpG island microarray analysis reflected by the close overall correlation among three independent experiments. (B) Results of examination of global DNA methylation in UACC62 cells by the MCA/CpG island microarray analysis after BRAF knockdown. Normalized log2 ratios of the methylation intensities of BRAF shRNA/vector of ≥ 0.6 and ≤ −1 were used as the cutoff values for hypermethylation and hypomethylation, respectively. Data are presented as mean values of three experiments, with red color representing high log₂ ratios (hypermethylation); black color, no change and green color, low log2 ratios (hypomethylation). The matched CpG microarray database (University Health Network Human CpG Microarray Database, http://data.microarrays.ca/cpg) was used to identify specific genes whose promoter methylation was altered after BRAF^V600E knockdown in UACC62 cells. The upper box frame in (B) illustrates 59 representative hypermethylated genes with high methylation intensity ratios (red) and the lower box frame in (B) illustrates 10 representative hypomethylated genes with low methylation intensity ratios (green). These genes contain two or more SmaI/XmaI sites in their promoters to maximize the chance of their promoter methylation identified by the MCA/CpG island microarray approach. The detailed experimental and technical descriptions are presented in Materials and Methods.

Figure 3

Analysis of the expression of genes hypermethylated upon BRAF^V600E knockdown and quantitative methylation-specific PCR (QMSP) validation of the MCA/CpG island microarray results on selected genes in melanoma cells. (A) Quantitative RT-PCR analysis of the expression of the 59 genes hypermethylated upon BRAF^V600E knockdown from Figure 2B in UACC62 cells in comparison with A375 cells. The genes presented in green are underexpressed. (B) QMSP validation of the hypermethylation status of genes revealed by the MCA/CpG island microarray analysis by similarly showing increased methylation upon BRAF^V600E knockdown. Selected eight genes were tested that showed decreased expression in both UACC62 and A375 cells, which are indicted with a blue asterisk (*) in (A). (C) Quantitative RT-PCR analysis of the expression of the 59 genes hypermethylated upon BRAF^V600E knockdown from Figure 2B in UACC62 cells in comparison with M14 cells. Shown are many underexpressed genes (green color), many of which are seen in both cells. (D) QMSP validation of the hypermethylation status of selected four genes revealed by the MCA/CpG island microarray analysis as in (B). These genes were underexpressed in both UACC62 and M14 cells as indicated with a blue asterisk (*) in (C). Details are described in the Materials and Methods. Significance of statistical analyses in (B and D): *p < 0.05; **p < 0.01.

Figure 4

Analysis of the methylation status of selected eight genes in primary melanoma tumors that became hypermethylated as revealed by MCA/CpG island microarray analysis upon BRAF knockdown in melanoma cells. Methylation of genomic DNA isolated from 60 primary melanoma tumors with known BRAF^T1799A mutation status was analyzed using QMSP. Details are described in the Materials and Methods. Results are presented for seven genes indicated at the top of each graph. The result for the HLX1 gene is not shown, as no methylation of this gene was found in any of these tumors. WT, wild-type BRAF; V600E, BRAF^T1799A (V600E) mutation; the horizontal lines represents 95% confidence interval; *p < 0.05.

Figure 5

Effects of silencing genes that were hypomethylated and overexpressed by the BRAF^V600E signaling on the proliferation and invasion of melanoma cells. (A) The expression of six selected genes, as indicated, was knocked down in melanoma A375 cells using vector-based RNA interference. Cells were infected with lentiviruses expressing shRNA targeting FGD1, HLX1, HMGB2, KLHL14, NR4A2 and ZBTB10. Stable cell pools obtained after selection with puromycin were lysed and immunoblotted with corresponding antibodies for the six proteins. Lentiviruses packaged with the empty vector were used as control. V, vector control; S, specific shRNA expression. (B) Cell proliferation assay of A375 cell pools with stable knockdown of the indicated genes. The proliferation rate was examined using MTT assay, and the OD values were measured daily over a 5-d time course. (C) Cell invasion assay of A375 cell pools with stable knockdown of the indicated genes. Cells were plated and cultured in Matrigel-coated transwell for 22 h, followed by examination and quantification of the invasive cells, as described in the Materials and Methods. The upper part shows representative results of invasive A375 cells with knockdown of each indicated gene. The bar graph in the lower part, corresponding to the upper part, shows the numbers of invasive cells.

Figure 6

Suppression of the expression of DNMT1 and EZH2 by BRAF^V600E knockdown in melanoma cells. Quantitative RT-PCR was performed to analyze the expression of DNMT1 and EZH2 genes in the three indicated melanoma cells stably transfected with the control empty vector (Vector) or BRAF-specific shRNA (BRAF KD). Relative gene expression in the BRAF-knockdown group against the control vector group is presented. The RT-PCR primer sequences for the two genes are presented in Table S4. The experimental details are described in the Materials and Methods.