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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.

SUBMITTER: Hou P

PROVIDER: S-EPMC3293379 | biostudies-other | 2012 Jan

REPOSITORIES: biostudies-other

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

Hou Peng P Liu Dingxie D Dong Jianli J Xing Mingzhao M

Cell cycle (Georgetown, Tex.) 20120115 2

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 ...[more]

PMID: 22189819

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Project description:BackgroundMost mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation.MethodsDNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation.ResultsThree cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines.ConclusionCell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.

Dataset Information

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

Publications

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

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