**2. Asbestos-induced molecular alterations**

As previously mentioned, asbestos exposure is a primary cause of the development of pleural mesothelioma. Molecular analyses show that asbestos-related carcinogenesis is caused by chronic inflammation that both promote the release of oxygen free radicals that alter intracellular components, and DNA mutation and its consequent transformation. Asbestos fibers also contain iron ions and can induce hemolysis by sequestering iron from hemoglobin. This is particularly important since free iron disproportionately releases H2O2, which consequently releases hydroxyl radicals (OH) that oxidize DNA, and release nucleic acids, proteins, and lipids. This process is exacerbated by the release of cytokines including tumor necrosis factor-alpha from macrophages and high mobility of box group 1 (HMGB1) proteins from necrotic cells, leading to an amplification of the inflammatory response and an increase in cells that are driven to oxidative damage. Damaged oxidized DNA, if not properly repaired, is highly mutagenic and can lead to genomic instability. A multitude of oxidative DNA lesions includes oxidation of DNA bases, baseless sites, single-strand breaking, double-strand breaking, and interchain breaking, all of which require different pathways for proper repair. These last two chain-breaking types are particularly toxic, since they cause replication collapse, as well as allow chromosome rearrangements, chromosome gains, losses, or fragmentation [2].

There are other mechanisms involved in how asbestos fibers cause MPM (**Figure 1**). Four proposed models related to asbestos fibers induce genetic and cellular damage to cells, in addition to the previously mentioned chronic inflammation. The different molecular models involved in asbestos exposure are explained below:

I.Reactive oxygen species generated by asbestos fibers with their surface exposed leads to DNA damage and cell membrane rupture. Macrophages that *Epigenomics in Malignant Pleural Mesothelioma DOI: http://dx.doi.org/10.5772/intechopen.105408*

#### **Figure 1.**

*Possible oncogenic mechanisms induced by asbestos. Abbreviations: HMGB1 = high-mobility group box 1. ROS = reactive oxygen species. TGF-B = transforming growth factor beta. VEGF = vascular endothelial growth factor.*

engulf asbestos fibers but cannot digest them also produce abundant reactive oxygen species.


(GAMB1) is released from mesothelial cells, which are exposed to asbestos and then undergo necrotic cell death, promoting an inflammatory response. Thus, aberrantly activated signaling between mesothelial cells, inflammatory cells, fibroblasts, and other stromal cells can create a set of mesothelial cells, which harbor aneuploidy and DNA damage, potentially developing cancer cells and together all these phenomena form a tumoral microenvironment that supports and nurtures them [6–8].

#### **2.1 DNA methylation**

Methylated DNA studied through immunoprecipitation grounded on next-generation sequencing makes it possible to analyze the DNA methylome, which constitutes a useful and efficient tool in the approach of cancer epigenomics [5, 9, 10].

An important and widely described phenomenon in the development of MPM is the epigenetic dysregulation that promotes changes in gene expression [11]. DNA methylation modifications play an important role in the malignant transformation of mesothelioma. Survival in MPM has been attributed to promoter methylation and silencing of genes such as SFRP4, SFRP5, FHIT, and SLCA20.

The methylated CpG islands have been shown to affect different process, such as uncontrolled cell proliferation & differentiation and dysregulations in apoptosis, in the oncogenic process of MPM. It is important to mention that asbestos fibers have been related with increased prevalence of aberrant promoter methylation by controlling the APC and RASSF1 genes, directly affecting the cell cycle [1–4].

Epigenetic modifications require active maintenance and are potentially reversible, characteristics that make them targets for therapeutic strategies. Multiple DNA methyltransferases and histone deacetylases (HDACs) participate on the regulation of some tumor suppressor genes by gene silencing and chromatin compaction. Therefore, changes in these two enzymes promote disturbances in gene expression and allow deflections in cell proliferation, differentiation, and apoptosis. When HDACs are inhibited, there is a massive production of superoxide radicals and the caspase system is activated, leading to cell death. Additionally, hyperacetylation of non-histone proteins takes place, promoting angiogenesis and tumor cells motility and invasion [12].

DNA modifications are not the only mechanisms involved in tumorogenesis. Epigenetic changes also play an important role in oncogenesis through changes in DNA-associated proteins, modifying their expression. In this regard, the most important changes are DNA methylation and histone deacetylation. These changes lead to important modifications in DNA activity and expression. As a result of this process, some proteins involved in tumorogenesis can be induced and modulated, for example, epidermal growth receptor factor, tumor necrosis factor-alpha protein fusion peptide, transforming growth factor-beta and others. As mentioned above, these changes are induced by epigenetic mechanisms that are potentially reversible [12, 13].

In recent years, inhibiting tyrosinase-like receptors (RTKs) has been used as a therapeutic target because MPM cells have been shown to express high levels of receptors that can bind to key molecules, such as epidermal growth receptor factor (EGFR) and platelet-derived growth factor (PDGF), fibroblast growth receptor factor (FGFR-1y3), transforming growth factor-beta (TGF-B), insulin-like growth factor (IGF-1R), and tumor necrosis factor-alpha protein fusion peptide (NGRhTNF-alpha). All these molecules undergo through epigenetic changes and play a dead serious role in tumor invasion and angiogenesis [12, 13].

*Epigenomics in Malignant Pleural Mesothelioma DOI: http://dx.doi.org/10.5772/intechopen.105408*

Numerous genes have been shown to be epigenetically downregulated, as the DNA methylation of transcriptional promoters. These changes deregulate several signaling pathways, including the WNT pathway, in which several negative regulators are hypermethylated and silenced [14, 15]. The global epigenetic profile determined by high-throughput analysis differs between MPM and normal pleura, showing that MPM has aberrant methylation in the CpG islands, as has been mentioned [16, 17]. These data support the hypothesis that a specific DNA methylation pathway is induced during mesothelial carcinogenesis.

Kim et al. [1] carried out a study in a patient with MPM, 122 differently regulated genes were found, 118 genes were down-regulated and four were up-regulated by hypomethylation. Therefore, MPM cells may be epigenetically regulated, and DNA methylation plays a main role in intratumorally heterogeneity, characteristic that boost MPM more aggressiveness.

#### **2.2 Factors associated with methylation**

There are sundry important factors that have been related with DNA methylation of gene loci in MPM such as age-related changes, ethnicity, histological subtype, and asbestos exposure. These factors could explain discrepancies between DNA methylation frequencies in published studies, as well as the experimental method used to detect it. In patients diagnosed with MPM, an increased DNA methylation associated with increased age has been reported. Some studies have shown that methylation status of the IGFBP2 (insulin growth factor binding protein) locus and GDF10 (bone morphogenetic protein) locus is significantly higher in MPM in Japanese patients compared with US patients [18, 19].

There are some concrete characteristics that are related to specific genes, for example; RASSF1 suppressor gene has been reported to have a significantly higher frequency of aberrant methylation in epithelioid MPM than in the sarcomatoid subtype [20, 21]. Methylation of MT2A gene, is shown to differ between these two histological subtypes. Epithelioid and sarcomatoid mesotheliomas also have different methylation changes at 87 CpG islands [22, 23]. MT1A and MT2A gene loci associated with DNA methylation have also been described in MPM.

CpG island methylation in the CCND2, CDKN2A, CDKN2B, HPPBP1, and RASSF1 genes has been studied in correlation with asbestos exposure. The RASSF1 DNA methylation locus is related with a higher number of asbestos bodies in the lung. There are different methylation profiles in MPM according with its exposure to asbestos and a positive association between asbestos fiber load and CDKN2A, CDKN2B, RASSF1 methylation status, and MT1A at another 100 loci.

#### **2.3 Methylation and diagnosis through DNA**

Some differences have been described in DNA methylation for sundry genes between MPM, lung adenocarcinoma, and in non-malignant lung tissues. That's why, at these days, DNA methylation is an important tool in the diagnosis of MPM [20, 24]. Thus, the DNA methylation profile has potential helpfulness in the diagnostic of MPM and reject of other differential diagnoses. It has been demonstrated by high-throughput analyses for methylation, spanning several thousand CpG islands. It was recently suggested that DNA methylation at three specific loci: TMEM30B, KAZALD1, and MAPK13, could be useful in the differential diagnosis of MPM. In the near future, MPM diagnosis may be based on the methylation profile, but by now,

further studies in larger populations are necessary before using a limited number of hypermethylated loci [19–21].

Other studies have shown alterations in the methylation status of individual genes, such as those HIC1, PYCARD, LZTS1, and SLC6A20. All of these genes have been associated with a good or bad prognosis [22, 23]. Besides, patients with MPM and a low frequency of DNA methylation had longer survival [22–24].

In view of the aberrant epigenetic events observed in MPM and the clinical value of histone deacetylase inhibitors (HDACis), the latter is currently being studied as a potential diagnostic method. However, insufficient data is yet available on the regulation of histone modifications, despite their crucial role in maintaining chromatin stability. These data are needed to support clinical trials based on HDACis [6, 7, 25, 26].

#### **2.4 Epigenetic regulation in mesothelioma gene expression**

Each nucleosome is made up of 147 base pairs (bp) of DNA wrapped twice around a histone octamer. Epigenetic regulation of gene expression occurs in the context of chromatin, the basic unit of the nucleosome. Lysine-rich histone tails extend from the nucleosome and provide sites for covalent and reversible binding, promoting processes such as acetylation, methylation, ubiquitination, phosphorylation and SUMOylation, which produce the activation or inhibition of gene expression [8, 27].

DNA methylations represent the most important mechanism regulating major changes in gene expression during normal cell cycle and tissue differentiation, as well as long-term repression of imprinted alleles, germ cell-restricted genes, repetitive DNA, and sequences. Endogenous retrovirals [27–29]. Normal somatic cells have three major DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. All these enzymes mediate the transfer of a methyl group from S-adenosyl-methionine to the 5′ position of cytosine in the context of CpG. CpG dinucleotide groups are found in the promoters of approximately 60% of genes. Furthermore, most of these islands are unmethylated, allowing for a relaxed structure (euchromatin) and active transcription [30]. Some other CpG dinucleotides and CpG islands, which are often hypermethylated in normal cells, are scattered throughout the genome [31]. Although there is considerable overlap, DNMT1 preferentially binds hypermethylated DNA and works primarily as a housekeeping methyltransferase, restoring DNA methylation patterns during the process of DNA repair or replication. On the other hand, DNMT3A and 3B mediate de novo DNA methylation after recognition of unmethylated or hypermethylated DNA [30, 31].

It is important to recapitulate that methylation-sensitive transcription factor binding is inhibited by DNA methylation, and these changes promote the recruitment of the CpG methyl-binding domain (MBD) and relevant proteins such as UHRF1, syn3a-containing repressor complexes, NCoRs and histone deacetylases (HDACs), resulting in silent transcriptional heterochromatin output [32–34].

During the process of malignant transformation, the aberrant orientation and overexpression of some factors involved in DNA methylation promote the epigenetic silencing of genes related to differentiation, many of which are tumor suppressors. On the other hand, tumor suppressor genes can be inactivated by DNA methylation through transitional mutations resulting from deamination of 5-methylcytosine (5-MC) or adduct formation with environmental carcinogens such as benzopyrene [35].

DNA demethylation occurs passively during DNA replication [36, 37]. In addition, DNA can be actively demethylated by oxidation of 5-MC to 5-hydroxymethylcytosine, a ten-eleven translocation (TET) enzyme-mediated reaction [20].

#### *Epigenomics in Malignant Pleural Mesothelioma DOI: http://dx.doi.org/10.5772/intechopen.105408*

The total amount of methylated CpGs, during malignant transformation, is up to 50%, excluding CpG promoter islands. The genome-wide DNA demethylation is importantly related to a deficient DNA repair process [38–41]. Besides, it can promote unrepression of imprinted alleles, endogenous retroviruses, and transposable elements, inducing genomic instability [42, 43]. On the other hand, the mechanisms that mediate this phenomenon, such as decreased expression of methyltransferase 1 [44–46] glycosylase-mediated cleavage of 5-MC and aberrant expression/orientation of TET proteins, have not been fully elucidated [38].

The most widely characterized histone modifications in normal cells and malignant cells have been the acetylation-deacetylation and the methylation-demethylation [1, 2, 43, 47]. Histone acetylation is mediated by a variety of histone acetyltransferases (HAT), increasing the net negative charge leading to DNA repulsion, chromatin relaxation, and gene expression. Some non-histone proteins, including Hsp90, SP1, p53, and HDAC1, are targets for HAT and HDAC. In the other hand, histone deacetylation is regulated by HDAC [48].

Histone lysine methylation is mediated by a variety of histone methyltransferases (KMTs), lysine mediating monomethylation/dimethylation/trimethylation of specific residues, whereas histone demethylation is mediated by histone demethylases [47, 49, 50]. Histone modifications are highly dynamic in response to environmental signals [51, 52]. Unlike histone acetylation, histone lysine methylation does not modify the charge of core histones. Furthermore, histone lysine methylation can promote or inhibit gene expression.

ATP-dependent chromatin remodeling complexes have emerged, in recent years, as critical mediators of the epigenetic regulation of gene expression in normal and malignant cells [53, 54]. To date, four gene families have been described including switch/non-fermentable sucrose (SWI/SNF), SWI mimetic (ISWI), DNA-binding helicase chromodomain (CHD), and INO80, named for their ability to regulate inositol-responsive gene expression. All these complexes have multiple subunits with diverse isoforms and exhibit pleiotropic functions including regulation of gene expression, maintenance of chromatin structure, replication of pericentromeric heterochromatin, repression of ribosomal RNA, and repair of cell damage. DNA [55]. There are several mechanisms by which different families remodel chromatin. For example, the SWI/SNF complexes expose DNA by disassembling the nucleosome, while members of the ISWI, INO80, and CDH families reposition (slide) the nucleosomes and extend the intervening DNA, promoting access to transcriptional factors. These complexes also have an important role in maintaining chromatin structure and genome stability, through mechanisms that reassemble the nucleosome [53, 55].

Studies in transcriptome analysis have revealed that almost 90% of the genome is transcribed as non-coding RNAs (IncRNAs), which are critical mediators of chromatin structure and gene expression in normal cells and malignant transformation [56–58]. Besides, lncRNAs participate in the recruitment of DNMTs and histone methyltransferases to chromatin [59], adding another layer of epigenetic regulation in normal cells which is altered in malignant tumors.
