Epigenetic genes regulated by the BRAFV600E signaling are associated with alterations in the methylation and expression of tumor suppressor genes and patient survival in melanoma

Abstract

We have previously reported that the BRAFV600E signaling causes genome-wide aberrations in gene methylation in melanoma cells. To explore the potential molecular mechanisms for this epigenetic effect of BRAFV600E, in this in silico study we analyzed 11 microarray datasets retrieved from NCBI GEO database and examined the relationship of the expression of the epigenetic genes (genes involved in epigenetic regulation) with BRAFV600E signaling, methylation and expression of tumor-suppressor genes (TSGs) in melanoma, and patient survival with this cancer. Among 273 epigenetic genes examined, 12 genes were down-regulated (named DD genes) and 16 were up-regulated (UU genes) by suppression of the BRAFV600E signaling using inhibitors. While the expression of 245 non-DD/UU genes overall had no correlation with the expression and methylation of a set of potential TSGs, the expression of DD genes was significantly correlated negatively with the TSG expression and positively with TSG methylation. Expression of UU genes was positively, albeit weakly, associated with the TSG expression. Overall, no correlation was found between UU gene expression and TSG methylation. Importantly, the expression of DD genes, but not UU genes, was significantly associated with decreased survival of patients with melanoma. Interestingly, the promoters of DD genes contain more binding motifs of c-fos and myc, two BRAFV600E signaling-related transcription factors, than those of UU and non-DD/UU genes. Thus, these results link epigenetic genes to methylation and suppression of tumor suppressor genes as a mechanism involved in BRAFV600E-promoted melanoma tumorigenesis and uncover a novel molecular signature that predicts a poor prognosis of melanoma.

Fig. 1

Differentially expressed epigenetic genes in melanoma cells after inhibition of BRAFV600 signaling. The differently expressed genes were identified by SAM procedure, and the heatmaps was generated by R program.

Fig. 2

Pearson correlation analysis of the expression of epigenetic genes to the expression of the 11 potential TSGs in melanoma. (A) Box-Whisker plots of Pearson’s r. The Pearson’s r between the expression of 11 potential TSGs themselves were used as control of positive correlation. The box-plot shows the five statistics (lower whisker is 5% minimum, lower box part is the 25th percentile, solid line in box presents the median, upper box part is 75th percentile, and upper whisker is 95% maximum). Welch t-test was used to analyze the difference of Pearson’s r between the three groups of epigenetic genes. (B) Individual Pearson’s r of the expression of each DD and UU gene to the expression of 11 potential TSGs. Each row represents Pearson’s r of the DD and UU genes to a TSG from one microarray dataset. The DD or UU genes whose Pearson’s r values are statistically significant from the whole epigenetic genes were indicated with symbol ^*(Randomization test, Supp. Fig. 3A). The missing values were shown with white color.

Fig. 3

Pearson correlation analysis of the expression of epigenetic genes to the methylation level of the 31 potential TSGs. (A) Box-Whisker plots of Pearson’s r. The Pearson’s r of the expression of the 31 methylated genes to their own methylation level were used as control of negative correlation. The five statistics of the box-plot and statistic method were described in legend. (B) Individual Pearson’s r of the expression of each DD and UU gene to the methylation of the 31 methylated genes. The DD or UU genes whose Pearson’s r values are statistically significant from the whole epigenetic genes were indicated with symbol ^*(Randomization test, Supp. Fig. 3B).

Fig. 4

Survival analysis in patients with melanoma. Kaplan–Meier analysis were performed for groups based on the sum of the relative expression level of DD (A, C) or UU (B, D) genes. The survival information was retrieved from dataset GSE19234 (A, B) and GSE22153 (C, D).