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DNA Methylation in Solid Tumors: Functions and Methods of Detection International Journal of Molecular Sciences Review DNA Methylation in Solid Tumors: Functions and Methods of Detection Andrea Martisova , Jitka Holcakova , Nasim Izadi , Ravery Sebuyoya, Roman Hrstka and Martin Bartosik * Masaryk Memorial Cancer Institute, Zluty Kopec 7, 656 53 Brno, Czech Republic; [email protected] (A.M.); [email protected] (J.H.); [email protected] (N.I.); [email protected] (R.S.); [email protected] (R.H.) * Correspondence: [email protected] Abstract: DNA methylation, i.e., addition of methyl group to 50-carbon of cytosine residues in CpG dinucleotides, is an important epigenetic modification regulating gene expression, and thus implied in many cellular processes. Deregulation of DNA methylation is strongly associated with onset of various diseases, including cancer. Here, we review how DNA methylation affects carcinogenesis process and give examples of solid tumors where aberrant DNA methylation is often present. We explain principles of methods developed for DNA methylation analysis at both single gene and whole genome level, based on (i) sodium bisulfite conversion, (ii) methylation-sensitive restriction enzymes, and (iii) interactions of 5-methylcytosine (5mC) with methyl-binding proteins or antibodies against 5mC. In addition to standard methods, we describe recent advances in next generation sequencing technologies applied to DNA methylation analysis, as well as in development of biosensors that represent their cheaper and faster alternatives. Most importantly, we highlight not only advantages, Citation: Martisova, A.; Holcakova, but also disadvantages and challenges of each method. J.; Izadi, N.; Sebuyoya, R.; Hrstka, R.; Bartosik, M. DNA Methylation in Keywords: DNA methylation; epigenetic modification; tumor; tumorigenesis; bisulfite conversion; Solid Tumors: Functions and restriction enzyme; DNA biosensor Methods of Detection. Int. J. Mol. Sci. 2021, 22, 4247. https://doi.org/ 10.3390/ijms22084247 1. Introduction Academic Editors: Maria M. Sasiadek DNA methylation in eukaryotes is an important epigenetic modification that regulates and Pawel Karpinski gene expression. It is vital in embryogenesis, affecting such processes as imprinting, X chromosome inactivation and silencing of repetitive DNA [1]. Its deregulation is associated Received: 8 April 2021 with a range of human diseases, e.g., autoimmune diseases, metabolic disorders, neurolog- Accepted: 16 April 2021 Published: 19 April 2021 ical diseases, and cancer [2]. From a chemical point of view, DNA methylation involves an addition of a methyl group to the 50-carbon of cytosine residues in CpG dinucleotides. Publisher’s Note: MDPI stays neutral CpG dinucleotides are distributed unevenly in mammalian genomes, clustered mainly in with regard to jurisdictional claims in so-called CpG islands (CGIs) that are often found within gene promoters [3]. The main published maps and institutional affil- reason for this is that most 5-methylcytosines (5mCs) that are not clustered in CGIs are iations. in default methylated and undergo over time deamination to thymine [4]. T’s are not recognized by the DNA repair machinery, which results in C to T transition as one of the most frequent mutation in humans. On the contrary, CGI promoters remain mostly unmethylated in the germline. Hence, when spontaneous deamination of C occurs at this site, it leads to the formation of uracil which is effectively removed by uracil-DNA Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. glycosylase and CpG content is retained. CGI methylation in promoters of somatic cells This article is an open access article leads to gene silencing either by direct inhibition of transcriptional factors, or indirectly distributed under the terms and via interaction of 5mCs with methyl-CpG-binding domain (MBD) proteins that repress conditions of the Creative Commons transcription by recruiting enzymes that deacetylate histones [5]. Attribution (CC BY) license (https:// The process of DNA methylation is mediated by different types of DNA methyltrans- creativecommons.org/licenses/by/ ferase enzymes (DNMTs). First, de novo DNA methyltransferases DNMT3a and DNMT3b 4.0/). create a methylation pattern on unmethylated DNA, which is then maintained during Int. J. Mol. Sci. 2021, 22, 4247. https://doi.org/10.3390/ijms22084247 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 4247 2 of 22 subsequent cell division by DNMT1 using hemimethylated strand. These enzymes are essential in embryogenesis and their loss of function is lethal [6]. A reverse process of demethylation may either occur passively by a loss of maintenance during cell division, or, as shown recently, actively via catalytic action of 10–11 translocation (TET) family of proteins that convert 5mC into 5-hydroxymethylcytosine (5hmC) [7]. Further below, we describe an impact of DNA methylation on carcinogenesis process, then illustrate principles of numerous methods that were developed to analyze DNA methylation status of both single genes and whole genome and present the newest advances in development of biosensors that could represent novel, cheaper and faster alternatives to current methods. Importantly, we highlight not only advantages, but also disadvantages and challenges that each method faces. 2. Role of DNA Methylation in Tumorigenesis Given the critical role of regulatory pathways in which DNA methylation plays a part (especially at transcriptional level), aberrant DNA methylation has been generally associated with a range of human diseases, including cancer [8]. Many cancerous cells are characterized by abnormal methylation patterns compared to their physiological counter- parts. It is well known that hypermethylation (i.e., higher rate of methylation) of promoter regions could lead to transcriptional silencing of certain tumor suppressor genes and also contributes to control of many regulatory proteins and enzymes. In contrast, hypomethy- lation (i.e., lower rate of methylation) during very early stages of cancer development is associated with promotion of genomic instability and cell transformation [9]. 2.1. DNA Hypermethylation Methylation status of CGIs is highly controlled and protected from DNMTs by several mechanisms including active demethylation, active transcription, replication timing and local chromatin structure. Problems with disruption of any of the mentioned mechanisms would grant DNMTs access to CGIs, in turn resulting in inactivation of many cellular pathways. The following examples show that DNA hypermethylation of known tumor suppressors is indeed involved in solid tumor carcinogenesis. Cell cycle regulation genes, e.g., p16INK4a, p15INK4a, or retinoblastoma (Rb) commonly undergo DNA methylation-mediated silencing in a variety of cancers [10]. For instance, overexpression of tumor suppressor p16INK4a has been involved in cellular senescence, aging and cell cycle progression [11]. This protein blocks cell cycle progression by inhibit- ing cyclin-dependent kinases 4 and 6, which mediate phosphorylation of retinoblastoma protein (pRB) in G1 phase, consequently blocking the cell cycle progression. Increased silencing of p16INK4 due to DNA methylation leads to an increased level of pRB phospho- rylation, and hence in unlimited cell proliferation [11]. Hypermethylated p16INK4a gene promoters were found in various solid tumors, such as hepatocellular carcinoma [12], lung [13], pancreatic, breast, cervical or bladder carcinomas, as well as melanomas and gliomas [14]. Moreover, in non-small cell lung carcinomas (NSCLC), loss of expression of p16INK4a is associated with poor survival [15]. Various critical genes related to DNA repair processes are also hypermethylated in tumor tissues. For instance, hypermethylation pattern of BRCA1, which is involved in DNA repair of double-stranded breaks and is essential for preserving genome integrity, was found in ovarian and breast carcinomas [16]. Silencing of genes involved in cell adhesion may lead to tumor aggressiveness and tumor progression [9], as was shown for CD97, CTNNA1, DLC1, and HAPLN2 genes in which hypermethylation was associated with poorer survival of patients with ovarian cancer [17]. Furthermore, genes linked with cancer cell survival having proapoptotic functions were inactivated by hypermethylation, as in the case of XAF1 (XIAP-associated factor 1) gene that is frequently hypermethylated in human urogenital cancers and contributes to the malignant progression of tumors [18], or CASP8 (caspase 8) gene that was reported to be hypermethylated after glioblastoma multiforme relapse [19]. Int. J. Mol. Sci. 2021, 22, 4247 3 of 22 Interestingly, there is also a strong association between DNA methylation and short noncoding microRNAs. It was shown that microRNA biogenesis depends on DNA methy- lation and that methylated microRNAs are significantly enriched within cancerous pheno- types compared to unmethylated microRNAs [20]. 2.2. DNA Hypomethylation Overall gene methylation can also decrease as a result of global or gene wide hy- pomethylation effect. This phenomenon is accompanied by increased DNA damage, chromatin decondensation, and chromosome instability [21]. Repetitive sequences like long and short interspersed nuclear elements, and classical satellites, are normally heavily methylated [21,22]. The cells with abnormal histology due to aging or cancer, however, of- ten show a noticeable loss of DNA methylation (hypomethylation) of these regions [21]. For instance, decrease in DNA methylation of human pericentromeric repeat
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