Methylation of nonessential genes in cutaneous melanoma - Rule Out hypothesis

Abstract

Differential methylation plays an important role in melanoma development and is associated with survival, progression and response to treatment. However, the mechanisms by which methylation promotes melanoma development are poorly understood. The traditional explanation of selective advantage provided by differential methylation postulates that hypermethylation of regulatory 5'-cytosine-phosphate-guanine-3' dinucleotides (CpGs) downregulates the expression of tumor suppressor genes and therefore promotes tumorigenesis. We believe that other (not necessarily alternative) explanations of the selective advantages of methylation are also possible. Here, we hypothesize that melanoma cells use methylation to shut down transcription of nonessential genes - those not required for cell survival and proliferation. Suppression of nonessential genes allows tumor cells to be more efficient in terms of energy and resource usage, providing them with a selective advantage over the tumor cells that transcribe and subsequently translate genes they do not need. We named the hypothesis the Rule Out (RO) hypothesis. The RO hypothesis predicts higher methylation of CpGs located in regulatory regions (CpG islands) of nonessential genes. It also predicts the higher methylation of regulatory CpGs linked to nonessential genes in melanomas compared to nevi and lower expression of nonessential genes in malignant (derived from melanoma) versus normal (derived from nonaffected skin) melanocytes. The analyses conducted using in-house and publicly available data found that all predictions derived from the RO hypothesis hold, providing observational support for the hypothesis.

Figure 1.

Methylation of CpGs linked to essential and nonessential genes. All gene-linked CpGs were used as a reference group. Each panel includes violin plots of betas (left) and relative methylation of CpGs linked to melanoma essential and nonessential genes (right). Diamonds on violin plots indicate median, circles mean, vertical rectangles 25% and 75% of all data, and vertical lines min and max.

Figure 2.

Methylation of island, shores/shelves and open sea CpGs. Left parts of each panel show violin plots for island (top), shores/shelves (middle) and open sea CpGs (bottom). Diamonds indicate median, circles mean, vertical rectangles 25% and 75% of all data, and vertical lines min and max. On the right of each panel are the mean relative methylation of CpGs linked to essential and nonessential genes. As a reference group we used all gene-linked CpGs.

Figure 3.

The distribution of essential and nonessential genes by expression level (upper panel), and the distribution of CpGs linked to essential and nonessential genes by methylation level (lower panel) in TCGA SKCM samples.

Figure 4.

Relative methylation levels in melanomas versus nevi. The Y-axis shows the natural logarithms of the ratio of the mean methylation level expressed as beta in melanomas to the mean methylation level in nevi. Dots correspond to individual CpG sites. The blue horizontal line shows the median and black square, the mean methylation. Whiskers indicate outliers, and diamonds, 25% to 75% of the data. The numbers on the right show the mean relative methylation.

Figure 5.

Relative gene expression levels in melanoma versus nevi. The Y-axis shows the natural logarithm of the ratio of the mean expression level in melanomas to the mean expression level in nevi for each transcript. Individual transcripts are represented by dots. Blue horizontal lines show median and black square, mean expression. Whiskers indicate outliers and diamonds, 25% to 75% of the data. The numbers on the right indicate the mean relative expression levels in melanoma.

Figure 6.

A comparison of expression of nonessential genes in malignant and normal melanocytes from the study by Fujiwara et al. (upper panels) and Tremante et al. (lower panels). Left panels show mean expressions in malignant and normal melanocytes and right panels show mean differences in expression levels – malignant minus normal. Vertical bars show standard errors (SE) of mean. Black ovals on right panels show mean difference and bars SE of the difference.