Of all cancers, melanoma has the highest mutation frequency, with up to 100 mutations per Mb ^[1][10]. Its broad mutation landscape includes prominently genes involved in MAPK signaling. In 40–60% of all cutaneous melanomas, the BRAF proto-oncogene, encoding a serine–threonine kinase, is affected by activating point mutations, most commonly V600E ^[1][10]. In 28–30% of all melanomas, somatic mutations activate members of the proto-oncogene RAS GTPase family, primarily NRAS, followed by KRAS and HRAS ^[2][11]. Moreover, 13–17% of all melanomas, with loss-of-function mutations, inactivate the tumor suppressor Neurofibromin 1 (NF1) ^[2][11], a negative regulator of RAS signal transduction. Further important mutations in melanomas inactivate CDKN2A (13.3%) and TP53 (15.1%), which are believed to allow oncogene-driven melanocytes to overcome senescence and evade apoptosis ^[3][12]. Interestingly, CDKN2A gene mutations inactivating the cell cycle inhibitor p16 have been reported to be more frequent in metastases (14%) than in primary tumors (7%) ^[4][13]. It is assumed that the high variability in the mutation frequency in melanoma is attributed to whether the external inducer ultraviolet radiation (UV) was involved or not. More than 70% of cutaneous melanomas are believed to be caused by UV radiation exposure ^[2][11].

The differentiated, functionally highly specialized, pigment-producing melanocyte in the basal layer of the skin epidermis is persistently exposed to an exogenous noxa—namely, UV radiation. UVB, with wavelengths between 290 nm and 320 nm, is most harmful to the genome, causing prominently the formation of dimeric photoproducts between adjacent pyrimidine bases on the same strand, but also inducing a much wider range of DNA damage, such as protein–DNA crosslinks, single-strand breaks, and thymine glycol formation ^[5][14]. As a result, ultimately, irreparable damage of the DNA may force a choice between programmed cell death, apoptosis, or the transformation to a new, more dedifferentiated cell status that remains viable but may lose some or all of its specialized functions. This transformation is accompanied by extensive genetic mutations and chromatin restructuring processes.

2. Histone Modifications and Significance for Melanoma Progression

Multiple histone modification changes have been reported in CM, and some of them have been linked to tumor behavior. First, an increase in the global levels of demethylated histone H3 at lysine 9 (H3K9me2) has been found in melanoma samples, compared with the normal peritumoral skin tissue ^[6][15]. H3K9me2 recruits the heterochromatin protein 1 (HP1) which directs DNA methyltransferase 1 (DNMT1)-dependent DNA methylation in vivo ^[7][16] and plays a key role in the formation of transcriptionally inactive heterochromatin ^[8][17]. This histone modification is established by the histone methyltransferase G9a ^[7][16], which has been found significantly upregulated in melanoma patients ^[9][18] who have a poorer outcome ^[10][19]. Conversely, G9a silencing elevates the self-renewal capability of differentiated melanoma cells in a Sox2-dependent manner ^[11][20]. Sox2 is a master regulator of pluripotency in embryonic stem cells. In addition, inhibition of G9a induced cell death in diverse melanoma cell types and diminished tumor growth in vivo ^[12][21]. The importance of chromatin-modifying enzymes in regulating tumorigenesis was underscored in a zebrafish melanoma model. Overexpression of either SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) or suppressor of variegation 3–9 homolog 1 (SUV39H1), both enzymes methylating histone H3 on lysine 9 (H3K9), significantly accelerates melanoma formation ^[13][22]. Independently, it was shown that the H3K9-specific methyltransferase SUV39H1 establishes trimethylation at H3K9 at the promoter of the tumor suppressor gene retinoblastoma 1 (RB1), and this recruits DNA methyltransferase 3A, which mediates DNA methylation of the promoter. Thus, RB1 expression becomes epigenetically repressed, and, in turn, E2F1, which is inhibited by RB1, becomes transcriptionally activated. The authors show that this promotes UV-induced skin tumorigenesis in vivo. Conversely, depletion of SUV39H1 in melanoma cells leads to RB1 activation and reduced E2F1 transcriptional activity, inhibiting melanoma development ^[14][23]. The RB protein exerts tumor suppressor function by negative control of the cell cycle and by binding to E2F family members and repressing their functions at the promoters of genes, which are important for S-phase progression and cell proliferation ^[15][24]. RB interacts with histone acetyltransferases (HATs) and -deacetylases (HDACs), supports repair of DNA double-strand breaks (DSB), chromosome condensation, and silencing of repetitive sequences. Through its chromatin regulatory functions, it affects genomic stability ^[15][24]. E2F1 promotes cell proliferation but only develops its oncogenic properties when pathways that mediate E2F1-induced apoptosis are disabled ^[16][25]. A variety of functional studies show that melanoma cells reprogram their survival pathways and expand their intrinsic resistance to apoptosis during melanoma progression ^[17][26]. Interestingly, the p16INK4A–Rb–E2F pathway, which is an important regulator of cell cycle and differentiation, and its dysfunction can lead to oncogenesis, is altered in more than 80% of human neoplasias ^[16][25]. p16INK4A arrests the cell cycle in G1 by inhibiting CDK4 and CKD6, thereby preventing the inactivation of pRB ^[18][27]. In 72 melanomas greater than 1.0 mm and 29 metastases, its expression has been found lost in 100% of the cases ^[19][28]. Straume et al. reported the loss of p16 protein expression by promoter hypermethylation in 19% of primary cutaneous melanomas and in 33% of metastases ^[20][29]. The RB1-target E2F1 positively regulates the enhancer of zeste homolog 2 (EZH2), which is a histone–lysine N-methyltransferase enzyme and a core subunit of the polycomb repressive complex PRC2, which is responsible for global changes of chromatin architecture and essential in early development but is downregulated in normal adult tissues. EZH2 is an important driver of melanoma progression ^[21][30], and its increased activity leads to increased global H3K27me3. Increased H3K27me3 is an indicator of poor prognosis and is associated with aggressive and metastatic forms of melanoma ^[22][31]. The 5-year survival rate of EZH2-high patients was 48%, compared with 71% in the EZH2-low group ^[23][32]. The EZH2-mediated elevation of H3K27me3 has been described to be involved in epigenetic silencing of the tumor suppressor genes RUNX3 and CDH1 in advanced-stage human melanoma tissues ^[24][33].

3. DNA Methylation Alterations in Melanoma

A study of 16 melanoma cell lines, an elevated methylation status was reported for the following gene promoters: ESR1 (50%), MGMT (50%), RARB2 (44%), RIL (88%), RASSF1A (69%), PAX7 (31%), PGRB (56%), PAX2 (38%), NKX2-3 (63%), OLIG2 (63%), HAND1(63%), ECAD (88%), CDH13 (44%), and CDKN2A/p16 (6%) ^[25][38]. Conversely, the genes CD2, EMR3, CARD15, EV12A, HLA-DP1, IFNG, IL2, ITK, KLK10, LAT, MPO, PSCA, PTHLH, PTHR1, RUNX3, and TNFSF8 have been found hypomethylated in 25 primary melanomas, compared with 29 benign nevi ^[26][39]. Interestingly, distinct hypermethylated genes have been found associated with genetic mutation subgroups, e.g., NF1 hypermethylation with NF1- and RAS-mutated melanomas, PTEN hypermethylation with BRAF-mutated, and CDK2A/B hypermethylation with BRAF-, RAS-, NF1-mutated and triple-WT melanomas ^[27][40]. Repetitive LINE-1 retrotransposon hypomethylation may result in their reactivation, LINE-1 RNA, and protein expression, and has been linked to apoptosis, DNA damage and repair, tumor progression, cellular plasticity, and stress response ^[28][41]. In 75% of 16 melanoma cell lines, significant hypomethylation of LINE-1 sequences was found ^[29][42].

4. One-Carbon Metabolism in the Etiology of Epigenomic Aberrations in Melanoma

While a plethora of DNA methylation alterations occur in melanoma, it remains elusive how they are caused. It has been proposed that either active processes, e.g., an aberrant activity or function of DNMT enzymes, or passive ones, for instance, changes in epigenetic modifications that regulate targeting of DNA methylation, may be involved ^[30][36].

A major risk factor for bladder cancer is persistent exposure to the harmful carcinogens of tobacco smoking, which is estimated to account for 50% of tumors ^[31][44] and contributes to the high mutational rate in that cancer. There is thus a parallel to the etiology of melanoma, where exposure to another exogenous carcinogen—harmful UVB radiation—is the major cause. The previously proposed PrimeEpiHit hypothesis for UC ^[31][32][44,45] may, therefore, explain methylation alterations in CM carcinogenesis. According to this modified hypothesis, chronic UVB radiation exposure may occasionally also hit genes with key functions in one-carbon-group metabolism. As a result, their transcription may become impaired, and subsequently, their epigenetic status may be altered. Epigenetic silencing because of gene disruption has been experimentally demonstrated ^[33][46]. Interestingly, analyzing a comprehensive mortality rate dataset for 30 types of cancer for 52 provinces in Spain, spanning 1978–1992, it has been found that melanoma correlated with bladder and lung cancer, suggesting common risk factors and mechanisms ^[34][47]. Key genes involved in one-carbon-group metabolism of course comprise a very small percentage of the whole genome, but due to the chronic carcinogen exposure, nonetheless, this may occur at some minor frequency, which appears in accordance with the low incidence.

Impairment of key genes of one-carbon-group metabolism causes imbalances in the involved methyl group metabolic pathways. This disturbs the delicate SAM:SAH ratio and, consequently, genome-wide DNA methylation alterations, including LINE-1 hypomethylation that contributes to genetic instabilities; thus, cellular transformation occurs. Notably, this process could be enhanced by the well-described deficiencies of one-carbon-group metabolism associated with aging, which are likewise characterized by accumulation of SAH and DNA hypomethylation ^[35][48]. Age is an important risk factor for UC, as well as CM. Figure 1 provides an overview illustration of the key metabolites and enzymes involved in methyl group and polyamine metabolism and their interactions.