International Journal of

Molecular Sciences

Review
Epigenetics in the Diagnosis and Therapy of
Malignant Melanoma
Simeon Santourlidis 1 , Wolfgang A. Schulz 2 , Marcos J. Araúzo-Bravo 3,4 , Daniela Gerovska 3 , Pauline Ott 1 ,
Marcelo L. Bendhack 5 , Mohamed Hassan 6,7 and Lars Erichsen 8, *

1 Epigenetics Core Laboratory, Institute of Transplantation Diagnostics and Cell Therapeutics, Medical Faculty,
Heinrich-Heine University Duesseldorf, 40225 Duesseldorf, Germany;
[email protected] (S.S.); [email protected] (P.O.)
2 Department of Urology, Medical Faculty, Heinrich-Heine University Duesseldorf,
40225 Duesseldorf, Germany; [email protected]
3 Group of Computational Biology and Systems Biomedicine, Biodonostia Health Research Institute,
20014 San Sebastián, Spain; [email protected] (M.J.A.-B.); [email protected] (D.G.)
4 IKERBASQUE, Basque Foundation for Science, 48009 Bilbao, Spain
5 Department of Urology, University Hospital, Positivo University, Curitiba 80030-200, Brazil;
[email protected]
6 Department of Surgery, Tulane University School of Medicine, New Orleans, LA 70112, USA;
[email protected]
7 Institut National de la Santé et de la Recherché Médicale, University of Strasbourg, 67000 Strasbourg, France
8 Institute for Stem Cell Research and Regenerative Medicine, Medical Faculty, Heinrich-Heine University
Düsseldorf, 40225 Duesseldorf, Germany
* Correspondence: [email protected]; Tel.: +49-0211-81-16905

Abstract: Epigenetic mechanisms are fundamentally important for cancer initiation and development.



However, a survey of the literature reveals that, to date, they appear less comprehensively investi-


gated in melanoma than in many other cancers, e.g., prostate, breast, and colon carcinoma. The aim of
Citation: Santourlidis, S.; Schulz,
this review is to provide a short summary of epigenetic aspects of functional relevance for melanoma
W.A.; Araúzo-Bravo, M.J.; Gerovska,
pathogenesis. In addition, some new perspectives from epigenetic research in other cancers with
D.; Ott, P.; Bendhack, M.L.; Hassan,
potential for melanoma diagnosis and therapy are introduced. For example, the PrimeEpiHit hy-
M.; Erichsen, L. Epigenetics in the
Diagnosis and Therapy of Malignant
pothesis in urothelial carcinoma, which, similarly to malignant melanoma, can also be triggered by
Melanoma. Int. J. Mol. Sci. 2022, 23, a single exogenous noxa, states that one of the first steps for cancer initiation could be epigenetic
1531. https://doi.org/10.3390/ changes in key genes of one-carbon metabolism. The application of such insights may contribute to
ijms23031531 further progress in the diagnosis and therapy of melanoma, a deadly type of cancer.

Academic Editor: Paola Ghiorzo

Keywords: melanoma; DNA methylation; epigenetics
Received: 11 January 2022
Accepted: 26 January 2022
Published: 28 January 2022

Publisher’s Note: MDPI stays neutral

1. Introduction
with regard to jurisdictional claims in One early starting point of an organism’s life is the pluripotent stem cell of the inner
published maps and institutional affil- cell mass, which already harbors all needed inherited, evolutionary-shaped genetic, and
iations. epigenetic information. During later development, depending on which differentiation
path is taken, only an adequate part of this information will come into practice, to fulfill the
specialized demands on cellular function requested by the environment of each specifically
differentiated cell. Thus, firstly, it is fascinating that all environmentally imposed infor-
Copyright: © 2022 by the authors.
mation is burned within one undifferentiated cell, and secondly, that later it is selectively
Licensee MDPI, Basel, Switzerland.
unleashed in a programmed and preparatory manner to determine differentiation and
This article is an open access article
function of a cell in full compliance with the upcoming, multifaceted environmental needs.
distributed under the terms and
The precision of differentiation is the key to life.
conditions of the Creative Commons
During ontogeny, owing to the efforts of C. H. Waddington, we started to understand
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
that this occurs through the gradual establishment of various epigenetic mechanisms, which
4.0/).
build up a mediating bond between the genome and the environment and are heritable

Int. J. Mol. Sci. 2022, 23, 1531. https://doi.org/10.3390/ijms23031531 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022, 23, 1531 2 of 13

and plastic. This ensures the propagation of the unaltered cell functionality after division
and the ability to adequately adapt to fluctuant environmental pressures, in an evolved
space of tolerance, without losing the functional assignment.
In aging, we observe the opposite process, i.e., the gradual loss of the integrity of these
epigenetic mechanisms accompanied by loss of cell function and cellular deterioration,
followed by a substantial increase in the risk for cancer development. Obviously, a cancer
cell has been forced to give up the original differentiation status and functional specificity
and has reduced its existence to simple survival, often linked to an aberrant, out-of-joint
elevated, unlimited proliferation rate and dedifferentiation that is reminiscent of stem cells.
We see that this occurs because of pressuring harmful exogenous noxae. Since we can
trigger them, obviously we are able to willingly impact our own epigenetics, and thus, we
may, to some extent, master our fate.
The first epigenetic hallmark of cancer recognized since 1914, originally described
by Theodor Boveri [1], is the reconfiguration of chromatin providing dense “hyperchro-
matic” sites, widely exploited diagnostically later, e.g., by George Papanicolaou in the
1930s [2], to save millions of lives [3]. Meanwhile, the main mechanisms involved in this
are distinct combinations of histone modifications associated with active and non-active
chromatin compartments, restructuring the genome-wide chromatin compaction and DNA
methylation [4].
Firstly, described in 1950 [5], DNA methylation has been intensively investigated,
especially in the last 35 years, and newly developed and adjusted methods have meanwhile
reached a high standard of improvement; once concerning screening, their widespread ap-
plications now include histone modification-/DNA methylation array technology, bisulfite
sequencing, and especially DNA methylation-specific PCR (MSPCR) for fast and cost-
effective diagnosis, as well as the first FDA-approved epigenetic method for early detection
of colon cancer.

2. Incidence and Etiology of Cutaneous Melanoma

Cutaneous Melanoma (CM) is an aggressive and invasive cancer of the skin, with
324,635 new cases and 57,043 new deaths in 2020 worldwide, contributing 1.7% of all
cancers in the world population [6], with rising incidence [7]. Its incidence also increased
with age, with new cases most common in the 65–74 age group [8]. The median age of onset
is 64 years [8]. Melanoma causes severe morbidity since it often metastasizes at very early
stages and often defies therapy by pronounced chemoresistance. The median survival of
metastatic melanoma patients is 5–8 months [7]. The prognosis is even worse if melanoma
affects mucosal areas [9].
Of all cancers, melanoma has the highest mutation frequency, with up to 100 mu-
tations per Mb [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 com-
monly V600E [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 [11].
Moreover, 13–17% of all melanomas, with loss-of-function mutations, inactivate the tumor
suppressor Neurofibromin 1 (NF1) [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 [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%) [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 [11].
Int. J. Mol. Sci. 2022, 23, 1531 3 of 13

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 forma-
tion [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.

3. 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 [15]. H3K9me2 recruits the heterochromatin protein
1 (HP1) which directs DNA methyltransferase 1 (DNMT1)-dependent DNA methylation
in vivo [16] and plays a key role in the formation of transcriptionally inactive heterochro-
matin [17]. This histone modification is established by the histone methyltransferase
G9a [16], which has been found significantly upregulated in melanoma patients [18] who
have a poorer outcome [19]. Conversely, G9a silencing elevates the self-renewal capability
of differentiated melanoma cells in a Sox2-dependent manner [20]. Sox2 is a master regu-
lator 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 [21].
The importance of chromatin-modifying enzymes in regulating tumorigenesis was un-
derscored 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 ac-
celerates melanoma formation [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 transcrip-
tionally 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 [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 [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 [24].
E2F1 promotes cell proliferation but only develops its oncogenic properties when pathways
that mediate E2F1-induced apoptosis are disabled [25]. A variety of functional studies
show that melanoma cells reprogram their survival pathways and expand their intrinsic
resistance to apoptosis during melanoma progression [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 [25].
p16INK4A arrests the cell cycle in G1 by inhibiting CDK4 and CKD6, thereby preventing
the inactivation of pRB [27]. In 72 melanomas greater than 1.0 mm and 29 metastases, its
expression has been found lost in 100% of the cases [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 [29].
Int. J. Mol. Sci. 2022, 23, 1531 4 of 13

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 [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 [31]. The 5-year survival
rate of EZH2-high patients was 48%, compared with 71% in the EZH2-low group [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 [33].
Notably, it has been proposed that abnormally high levels of EZH2 found in can-
cer cells may shift expression profiles toward a stem-cell-like state [30]. In melanoma,
elevated EZH2 contributes to a shift to a more invasive and metastasizing phenotype.
EZH2 inhibition was suggested as a promising approach for preventing the transition to
advanced cancer stages [34]. Accordingly, pharmacological inhibition of EZH2 by GSK503
decreased melanoma progression in melanoma-bearing mice in vivo and doubled the
survival time [35].

4. DNA Methylation Alterations in Melanoma

A further epigenetic hallmark of melanoma—namely, DNA methylation alterations,
has been recently comprehensively reviewed [36]. In addition to focal DNA hypermethy-
lation of the well-investigated tumor suppressor genes PTEN, CDKN2A (p16/p14), and
RASSF1A, with promoter hypermethylation prevalence of 6–62% (PTEN), 5–27% (p16), 41–
57% (p14), and 15–57% (RASSF1A), respectively [36], focal hypermethylation of many more
genes has been reported in melanomas. For instance, COL1A2, NPM2, HSPB6, DDIT4L, and
MT1G promoter methylation was increased in eight early passage human melanoma cell
lines/tissues, compared with newborn and adult melanocytes [37]. The validated markers
were with a significant increase in methylation in advanced-stage melanomas. In another
study of 16 melanoma cell lines, an elevated methylation status was reported for the fol-
lowing 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%) [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, com-
pared with 29 benign nevi [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 [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 [41]. In 75% of 16
melanoma cell lines, significant hypomethylation of LINE-1 sequences was found [42].
In a study comprising 46 primary melanomas and a 5-year follow-up period, LINE1
hypomethylation was reported to be accompanied by a shortened relapse-free survival
of the patients and to be significantly associated with metastasis [42]. In a further study,
LINE-1 retroelements were assessed in resected melanoma tissues from 82 patients ranging
in age from 14 to 88 years. LINE-1 methylation was found decreased in melanoma patients
with clinical parameters associated with an adverse prognosis [43].
Int. J. Mol. Sci. 2022, 23, x 5 of 13

Int. J. Mol. Sci. 2022, 23, 1531 5 of 13

5. 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
5. One-Carbon are caused.inItthe
Metabolism hasEtiology
been proposed that either
of Epigenomic active processes,
Aberrations in Melanoma e.g., an
aberrant activity or function of DNMT enzymes, or passive ones, for instance, changes in
While a plethora of DNA methylation alterations occur in melanoma, it remains
epigenetic modifications that regulate targeting of DNA methylation, may be involved
elusive how they are caused. It has been proposed that either active processes, e.g., an
[36].
aberrant activity or function of DNMT enzymes, or passive ones, for instance, changes in
Based on our recent observation in urothelial carcinoma (UC) [44,45], we would like
epigenetic modifications that regulate targeting of DNA methylation, may be involved [36].
to point out a further passive mechanism that may also apply to melanoma. A major risk
Based on our recent observation in urothelial carcinoma (UC) [44,45], we would like
factor for bladder cancer is persistent exposure to the harmful carcinogens of tobacco
to point out a further passive mechanism that may also apply to melanoma. A major
smoking,
risk factorwhich is estimated
for bladder cancer tois account
persistent forexposure
50% of tumors [44] and carcinogens
to the harmful contributes to ofthe high
tobacco
mutational rate in that cancer. There is thus a parallel to the etiology
smoking, which is estimated to account for 50% of tumors [44] and contributes to the high of melanoma, where
exposure torate
mutational another
in thatexogenous carcinogen—harmful
cancer. There is thus a parallel to UVBtheradiation—is the major where
etiology of melanoma, cause.
The previously proposed PrimeEpiHit hypothesis for UC
exposure to another exogenous carcinogen—harmful UVB radiation—is the major cause.[44,45] may, therefore, explain
methylation
The previously alterations
proposedinPrimeEpiHit
CM carcinogenesis.hypothesis According to thismay,
for UC [44,45] modified hypothesis,
therefore, explain
chronic UVB radiation exposure may occasionally also hit
methylation alterations in CM carcinogenesis. According to this modified hypothesis, genes with key functions in
one-carbon-group metabolism. As a result, their transcription
chronic UVB radiation exposure may occasionally also hit genes with key functions in may become impaired, and
subsequently, theirmetabolism.
one-carbon-group epigenetic status As amay be altered.
result, Epigenetic silencing
their transcription may becomebecause of gene
impaired,
disruption has been experimentally demonstrated [46]. Interestingly,
and subsequently, their epigenetic status may be altered. Epigenetic silencing because analyzing a com-
prehensive mortality rate dataset for 30 types of cancer for 52
of gene disruption has been experimentally demonstrated [46]. Interestingly, analyzing provinces in Spain, span-
ning 1978–1992, it has been found that melanoma correlated with
a comprehensive mortality rate dataset for 30 types of cancer for 52 provinces in Spain, bladder and lung can-
cer, suggesting common risk factors and mechanisms [47].
spanning 1978–1992, it has been found that melanoma correlated with bladder and lung Key genes involved in
one-carbon-group
cancer, suggestingmetabolism
common risk of factors
course comprise a very small
and mechanisms [47]. percentage
Key genesof the whole
involved in
genome, but due tometabolism
one-carbon-group the chronic of carcinogen exposure,
course comprise nonetheless,
a very this may occur
small percentage of theatwhole
some
minor frequency,
genome, but due to which appearscarcinogen
the chronic in accordance with the
exposure, low incidence.
nonetheless, this may occur at some
minor Impairment
frequency, of key appears
which genes ofin one-carbon-group
accordance with metabolism causes imbalances in the
the low incidence.
involved methyl of
Impairment group metabolic
key genes pathways. This disturbs
of one-carbon-group metabolism the causes
delicateimbalances
SAM:SAHinratio the
and, consequently,
involved methyl group genome-wide DNA methylation
metabolic pathways. This disturbs alterations,
the delicate including
SAM:SAH LINE-1 hy-
ratio and,
pomethylationgenome-wide
consequently, that contributes DNAtomethylation
genetic instabilities;
alterations,thus, cellular
including transformation
LINE-1 hypomethyla- oc-
curs.that
tion Notably, this process
contributes to genetic could be enhanced
instabilities; by the transformation
thus, cellular well-describedoccurs.
deficiencies
Notably,of
this process could metabolism
one-carbon-group be enhancedassociated
by the well-described
with aging, which deficiencies of one-carbon-group
are likewise characterized
metabolism
by accumulationassociated
of SAH with
andaging,
DNA which are likewise[48].
hypomethylation characterized by accumulation
Age is an important risk factorof
SAH and DNA hypomethylation [48]. Age is an important risk factor
for UC, as well as CM. Figure 1 provides an overview illustration of the key metabolites for UC, as well as CM.
Figure 1 provides
and enzymes an overview
involved in methylillustration
group of and thepolyamine
key metabolites and enzymes
metabolism and theirinvolved in
interac-
methyl
tions. group and polyamine metabolism and their interactions.

Figure 1.
Figure 1. “One-carbon
“One-carbonmetabolism”
metabolism” [49,50] including
[49,50] thethe
including polyamine metabolic
polyamine pathway
metabolic [51]. [51].
pathway Me-
tabolites: 5-MTHF = 5-methyl tetrahydrofolate, 5,10-MTHF = 5,10-methylene tetrahydrofolate, CYS
Metabolites: 5-MTHF = 5-methyl tetrahydrofolate, 5,10-MTHF = 5,10-methylene tetrahydrofolate,
Int. J. Mol. Sci. 2022, 23, 1531 6 of 13

CYS = cystathionine, dcSAM = decarboxylated S-adenosyl-L-methionine, HCY = homocysteine, MET

= methionine, MTA = methylthioadenosine, SAM = S-adenosyl methionine, SAH = S-adenosyl homo-
cysteine, THF = tetrahydrofolate. Enzymes: AHCY = adenosyl homocysteinase, AMD1 = adenosyl
methionine decarboxylase, ARG1 = arginase 1, BHMT = betaine homocysteine S-methyltransferase,
CBS = cystathionine ß-synthase, COMT = catechol-O methyltransferase, DNMT = DNA methyltrans-
ferase, GAMT = guanidinoacetate N-methyltransferase, GNMT = glycine methyltransferase, MAT =
methionine adenosyl transferase, MTR = methionine synthase, MTHFR = methylene tetrahydrofolate
reductase, MTRR = methionine synthase reductase, ODC1 = ornithine decarboxylase 1, SHMT =
serine hydroxymethyl transferase, SMOX = spermine oxidase, SMS = spermine synthase, SRM =
spermidine synthase Co-factors: B6 = vitamin B6, B12 = vitamin B12, UHRF1 = ubiquitin-like with
PHD and ring-finger domains 1.

The PrimeEpiHit hypothesis assumes that, e.g., imbalances in polyamine biosynthesis

cause deficiencies in methyl group levels in proliferating cancer cells. Specifically, aber-
rantly increased methylation of the ODC1 gene in proliferating cells leads to trapping
of SAM as decarboxylated S-adenosylmethionine (dcSAM), which inhibits methylation
reactions and impedes the supply of methyl groups for DNA methylation [52]. In support
of this hypothesis, we demonstrated that the 50 -regulatory region of ODC1 is hyperme-
thylated in most analyzed early UC (pTa/pT1) specimens and that its promoter activity
can be efficiently repressed by DNA methylation [45]. Moreover, ODC1 gene repression
in uroepithelial cells by RNA interference in vitro induced LINE-1 demethylation, LINE-1
transcriptional activation, and DNA double-strand breaks [45].
Table 1 presents an overview of expression data for key genes in one-carbon-group
metabolism and DNA methyltransferases in melanoma based on the results of the compre-
hensive TCGA study [53]. While these results require confirmation in independent studies,
ODC1 is downregulated in melanoma, compared with controls as well. In contrast, ODC1
expression is upregulated in many other cancer types, e.g., prostate, colon, and esophageal
carcinoma show an elevated expression [53].

Table 1. TCGA data for skin cutaneous melanoma (SKCM). Fold change in comparison with
healthy controls [53]. The samples consisted of 67 (20%) primary cutaneous melanomas (all originat-
ing from non-glabrous skin) and 266 (80%) metastases [54].

Gene Fold Change Gene Fold Change

FOLR2 0.38 DNMT3a 0.919
GNMT 0.471 AHCYL1 0.988
BHMT2 0.48 MTHFR 0.99
MAT2B 0.489 DNMT1 1.01
ODC1 0.533 BHMT 1.08
SHMT1 0.559 MAT2A 1.11
MGMT 0.612 AHCYL2 1.13
TCN2 0.654 SMS 1.34
AMD1 0.706 AHCY 1.52
UHRF1 0.877 SMOX 1.72
DNMT3b 0.893 ARG1 2.11
CBS 0.904 FOLR3 2.3

Thus, ODC1 DNA methylation status, the potential impairment of its activity, and its
potential role for cancer initiation and propagation should be investigated in melanoma.
In addition, an extended examination of further epigenetic disturbances of key factors
of the one-carbon-group metabolism, e.g., FOLRs, GNMT, MGMT, etc., influencing the
epigenome may reveal new potential targets for diagnosis and prognosis of CM.
Int. J. Mol. Sci. 2022, 23, 1531 7 of 13

6. Epigenetic Diagnosis Based on Differential Chromatin Organization and DNA

Methylation in Melanoma
Sensitive and specific biomarkers are urgently needed for early diagnosis and classifi-
cation, prognosis, and risk assessment to optimize individualized therapeutic choices in
melanoma. If apparently complete remission is achieved by therapy, modern and mean-
ingful biomarkers are needed to support the decision to stop treatment at the earliest and
the right point, to concomitantly minimize the risk of recurrence and avoid the adverse
events associated with unnecessary prolonged therapeutic treatment. As partly summa-
rized above, distinct melanoma phenotypes could be driven by distinct aberrations of their
epigenome. It is believed that epigenetic alterations exist in cancer, within the chromatin or-
ganization, and within the methylome, which comprises all methylated CpG dinucleotides,
consistent within a distinct cancer cell population or even few that may be largely consistent
between various cancer entities, opening new perspectives for pan-cancer diagnosis.
Focusing first on cancer-specific differential chromatin organization, for taking advan-
tage of it in cancer diagnosis and therapy, we suggest screening after sites of chromatin
with distinct differential organized structures for various melanoma stages, e.g., the first
mesenchymal stage, which arises after the epithelial–mesenchymal transition. Once we
detect such promising chromatin areas with, e.g., a more relaxed chromatin organization in
a pathogenic melanoma cell population, we would be able to target it either for diagnosis
or even for therapy. For diagnosis, e.g., taking advantage of the limited effectiveness of
micrococcus nuclease (MN) on closed chromatin, it would be possible to pretreat melanoma
and reference DNA and provide cancer-cell-originated DNA templates of MN-affected
integrity for assaying them by sensitive PCR assays, as previously demonstrated [55]. For
therapy, those cancer-cell-specific, differential organized chromatin areas could be targeted
by CRISPR/Cas in vivo, taking advantage of its selective preference feature. Evolved
as an adaptive immunity system that protects bacteria and archaea against phages and
plasmids [56] by directing sequence-specific Cas9 endonuclease-mediated double-strand
DNA cleavage (DSB) to the intruder’s DNA, and hence destroying its genetic informa-
tion [57], this system has evolutionarily never been encountered as a substrate DNA, which
is organized in complex, high-order, structured chromatin and, hence, has a high pref-
erence for binding to more easily accessible chromatin regions [58–60]. Among others,
interesting concrete targets suggested here would be the genome-wide-distributed LINE-1
retrotransposons, which are densely methylated at their CpG dense promoter regions,
tightly packaged into inactive chromatin, and poorly accessible for transcription in healthy
differentiated somatic cells [61]. On the other hand, LINE-1 hypomethylation and activa-
tion is a common phenomenon in many different cancers [62,63], interestingly associated
with cancer progression, becoming more pronounced in high-stage and high-grade can-
cer [64,65], and it is established that activation prevents chromatin compaction [66]. These
new perspectives concerning the mentioned epigenetic alterations and LINE-1 should be
considered and studied for diagnosis and therapy in melanoma.
A further basic approach we pursued is to find within a certain distinct cancer cell
population of interest, differentially methylated CpG dinucleotides forming a distinct,
consistent, and characteristic methylation signature for this cell population. In our former
investigations on prostate cancer and urothelial cancer specimens from patients, we were
able to discover such unique DNA methylation signatures. The proposed strategy [45] is
to screen using DNA methylation array technology after such differentially methylated
CpG regions, and once detected, to validate and precisely dissect the cancer cell-specific
DNA methylation pattern by bisulfite genomic sequencing [67]. Afterward, based on
this information, we can define the outmost most suitable and robust MSPCR primers to
sensitively detect this methylation signature. In Figure 2A,B, we show an example of how
clear this differential methylation occurs between cancer and reference samples when the
analysis is based on only a few CpG dinucleotides covered by the array probes, instead
of surveying whole CpG islands, due to their implied functional relevance. In all cancer
samples, we uncovered many consistently hypomethylated (blue) and hypermethylated
Int. J. Mol. Sci. 2022, 23, x 8 of 13

Int. J. Mol. Sci. 2022, 23, 1531 surveying whole CpG islands, due to their implied functional relevance. In all cancer 8 of 13
samples, we uncovered many consistently hypomethylated (blue) and hypermethylated
(red) CpG-rich probes associated with the named genes. To illustrate this, we decided to
choose
(red) independent
CpG-rich probesdata, recently
associated published
with the namedby others
genes. [68]. With respect
To illustrate to decided
this, we the role toof
epigenetic aberrations in melanoma, the authors reported in their study
choose independent data, recently published by others [68]. With respect to the role of that a hierar-
chical clustering
epigenetic analysis
aberrations provides stratification
in melanoma, into two in
the authors reported DNA methylation
their study that aepigenotypes
hierarchical
with high-analysis
clustering and low-methylation subgroups.
provides stratification Interestingly,
into the prognosis
two DNA methylation was significantly
epigenotypes with
worseand
high- in high-methylation cases [68]. Interestingly,
low-methylation subgroups. Our own bioinformatic
the prognosis analysis of these publicly
was significantly worse
available
in data (GEO
high-methylation database
cases [68]. Our own under the analysis
bioinformatic accession number
of these publiclyGSE140171
available
(GSM4155680-GSM4155734)) independently confirmed and extended
data (GEO database under the accession number GSE140171 (GSM4155680-GSM4155734)) this methylo-
me-based stratification to more subgroups, as shown in Figure 2D.
independently confirmed and extended this methylome-based stratification to more sub-This subgrouping
remainsas
groups, toshown
be validated and2D.
in Figure correlated with the clinical
This subgrouping outcome,
remains to estimate
to be validated anditscorrelated
potential
for prognosis.
with the clinical outcome, to estimate its potential for prognosis.

(A) (B)

(C) (D)

Figure 2.2. Bioinformatics analysis of DNA methylation

Figure methylation microarray data from from female
female and
and male
male
melanoma samples and primary human melanocytes (PMC) [68]. We
melanoma samples and primary human melanocytes (PMC) [68]. We identified many short CpG- identified many short
CpG-rich
rich DNA DNA fragments
fragments (60nt),(60nt),
which which are consistently
are consistently differentially
differentially methylated
methylated in all
in almost almost all
cancer
cancer samples (27 female and 27 male) by direct comparison with the control, in this case, PMC,
samples (27 female and 27 male) by direct comparison with the control, in this case, PMC, both
both unmethylated (A) and methylated (B). This approach reveals differential DNA methylation
unmethylated (A) and methylated (B). This approach reveals differential DNA methylation based on
based on every single array-probe (blue squares, hypomethylated; red squares, hypermethylated)
every singleofarray-probe
consisting (blue squares,
2–3 differentially hypomethylated;
methylated CpG dinucleotidesred squares, hypermethylated)
and provides a new valuableconsisting
level of
of 2–3 differentially
resolution methylated CpG
of DNA methylation dinucleotides
analyses in additionandto provides a new
the classical CpG valuable
island level of resolution
analysis. Based on
of
allDNA methylation
400.000 CpG probes analyses in addition
analyzed for everytosingle
the classical
sample,CpG we island analysis.
were able BasedPrincipal
to perform on all 400.000
com-
ponent
CpG analysis
probes (PCA)
analyzed for(C) andsingle
every subgroup classification
sample, we were able(D)toand name Principal
perform the relevant genes. At
component global
analysis
DNA (C)
(PCA) methylomics
and subgrouplevel, the female(D)
classification (F)and
andname
malethe(M) melanoma
relevant genes.samples
At globalmix with
DNA each other
methylomics
(Figure 2C).
level, the female (F) and male (M) melanoma samples mix with each other (Figure 2C).

Furthermore,
Furthermore,we werecently
recentlypresented
presenteda new
a newmethodological
methodological approach
approachto separate cell-
to separate
free DNADNA
cell-free fromfrom
cellular DNADNA
cellular and unreservedly
and unreservedlyapplyapply
bisulfite genomic
bisulfite sequencing
genomic and
sequencing
MSPCR
and MSPCR on this
onpure
this cell-free DNA. It
pure cell-free is established
DNA. that all tumors
It is established that all shed
tumorstheir cell-free
shed their
DNAs that bear their unique DNA methylation patterns into the bloodstream
cell-free DNAs that bear their unique DNA methylation patterns into the bloodstream [67], hence
providing
[67], henceanproviding
easily accessible
an easilyand exciting and
accessible Achilles’ heelAchilles’
exciting of cancerheel
for effective
of cancerdiagnosis.
for effec-
For instance, PTEN promoter methylation was reported in cell-free DNA from 62% of the
melanoma serum samples examined by pyrosequencing, indicating a good correlation with
the same epigenetic alteration found in paired melanoma tissues [69]. Furthermore, LINE-
Int. J. Mol. Sci. 2022, 23, 1531 9 of 13

1 hypomethylation, detected in both tissues and plasma-circulating DNA of melanoma

patients, seems to be a hallmark of the metastatic capacity of primary melanomas, and
LINE-1 hypomethylation may predict the overall survival in stage III CM patients [70].
MSPCR has been improved by us and enables, for the first time, relative quantification
of DNA methylation in samples with identical and unequal genetic settings [71]. This is of
importance for cancer samples, which often yield genetic aberrations, including melanoma,
characterized by a higher number of chromosomal structural aberrations [72], and bladder
carcinoma, characterized by early (pTa, pT1) chromosomal changes and imbalances [73].
A conventional normalization of MSPCR by using a housekeeping gene such as GAPDH
or the ACTB gene may result in false results, especially if tumor samples from different
patients must be compared, because, e.g., the copy number of the DNA segment containing
the housekeeping gene chosen for normalization may vary in cancer DNA. The reliable
measurement of DNA methylation by using MSPCR is based on DNA amplification, and
it is extremely hindered by a variable number of template segments. In contrast, by
using the idiolocal normalization of real-time, methylation-specific PCR (IDLN–MSP) [71],
the normalization loci are chosen adjacent to the targeted loci. With this approach, it is
guaranteed that in real-time MSPCR, the number of normalization loci is always as high as
the number of targeted loci, reducing false-negative results in a significant manner, and this
contributes to a significant improvement of DNA methylation measurements by MSPCR in
tumor samples of different patient origin [71].

7. Conclusions
Surveying the known literature on melanoma genetics and epigenetics, one may easily
infer that epigenetic restructuring is a major driver of melanoma development. Epigenetic
changes are the only known alterations in cancer that hold the potential to be largely
consistent in all relevant cells of a certain tumor stage. This is of high relevance for therapy
and diagnosis. Additionally, we believe that, on that basis, even pan-cancer biomarkers
will be defined in near future. For sure, it is yet not clear to which extent melanoma
cell dedifferentiation resort to epigenetic programs of stem cells. Defining this fact may
help us to better understand the nature of this deadly cancer and more successfully infer
from our knowledge in stem cell research to target its vulnerability. Certainly, global,
comprehensive epigenetic reorganization occurs. On the other hand, specific functionally
relevant epigenetic reconfiguration of tumor suppressor genes and oncogenes also occurs.
Focusing on the invasive and metastasizing melanoma cell population, it appears likely
that one will be able to uncover specific chromatin and DNA methylation changes that
exhibit consistent occurrence in all relevant cancer cells of the same stage. Thus, these
may be preferred targets for therapy and diagnosis. Tumor-specific, epigenetically relaxed
repetitive elements and other euchromatic regions may present new targets for CRISPR/Cas
targeting to induce DSBs, taking advantage of the already disturbed DSB repair mechanisms.
On the other hand, to widen the resolution of CpG differential methylation analyses on
single, differentially methylated CpG sites holds the potential to reveal new consistently
occurring differentially methylated sites for exploration in diagnosis. Here, one focus may
be on cfDNA exhibiting melanoma cell-specific differentially methylated signatures relying
on single, meaningful CpG-rich gene promoter regions, e.g., RASSF1A, LINE-1, or ODC1.

8. Material and Methods

The aim of this research was to provide a concise overview of epigenetic alterations in
melanoma and provide evidence for their functional relevance in pathogenicity and/or
possible suitability for their exploitation in diagnosis. We selected relevant publications on
incidence and etiology of melanoma, epigenomics alterations of melanoma progression,
including histone modifications and one-carbon metabolism, and epigenetic diagnosis
based on differential analysis of epigenomics data. To perform our computational analysis,
we downloaded publicly available DNA methylation data from female and male melanoma
samples and primary human melanocytes (PMCs) from the Gene Expression Omnibus
Int. J. Mol. Sci. 2022, 23, 1531 10 of 13

(GEO) database, under the accession number GSE140171, deposited by Yamamoto et al., as
described in [68]. We analyzed the data by in-house-developed bioinformatics methods as
described in [74,75].

Author Contributions: Conceptualization, S.S.; writing—original draft preparation, S.S.; writing—

review and editing, W.A.S., L.E., M.L.B., M.H., M.J.A.-B. and D.G.; figures, P.O., L.E., M.J.A.-B. and
D.G.; bioinformatics analyses, M.J.A.-B. and D.G. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was supported by Heinrich-Heine University, Düsseldorf, Germany. Heinrich-
Heine University/University Hospital has supported L.E. and S.S., M.J.A.-B. was supported by a
grant of Ministry of Economy and Competitiveness, Spain, MINECO No. PID2020-119715GB-I00
co-funded by the European Regional Development Fund (ERDF/ESF, Investing in your future), and
D.G. and M.J.A.-B. by a European Union (H2020-FETOPEN, Project 899417) grant.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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