**3. DNA methylation**

 The first epigenetic modification to be identified was DNA methylation [28], which is based on stable and heritable changes in gene structure without a change in DNA sequence [12]. Methylation refers to the addition of a methyl group to the fifth carbon in cytosine forming 5-methylcytosine (5-mC), which is mediated by DNMTs [29]. Generally, methylation occurs in intergenic regions and repetitive sequences such as satellite repeats, and long and short interspersed nuclear elements, while CpG islands of gene promoters often are unmethylated [5, 7, 30]. Interestingly, the global effects of epigenetic alterations in gene regulatory sequences from over 100 cancer cell lines have been identified by the ENCODE project [31]. Normally, DNA

methylation is critical for maintaining pluripotency, X chromosome inactivation, and genome imprinting [12]. Aberrant DNA methylation is one of the hallmarks of cancer [32].

 Methylation of DNA is catalyzed by the enzymes DNMT3A and DNMT3B and is then maintained by the major DNA methyltransferase DNMT1, which is also assisted by DNMT3A and DNMT3B [9, 12]. A recent study found that DNMT1, DNMT3A, and DNMT3B are themselves differentially methylated in PDAC [33]. Besides, a very recent finding suggests that the interactions between TP53 and H3K4, MLL3 and MOZ genes play a major role in chromatin regulation [34]. The methylation of tumor suppressor genes is the best-characterized epigenetic event in several malignancies, including PDAC [11]. In fact, several genes such as APC, BRCA1, P16INK4a, P15INK4b, RARβ, and p73 are frequently methylated [10]. Recent studies have revealed that apparent DNA methylation occurred in critical signaling pathways in PDAC such as TGFβ, WNT, integrin, cell adhesion, and axon guidance signaling pathways [35]. Likewise, TGFβ induces epithelial-mesenchymal transition (EMT) by enhancing hypermethylation of CpG islands in the VAV1 gene promoter [36] (see **Figure 2**). Furthermore, the WNT signaling pathway is a target of hypermethylation in PDAC. This has been found for WNT ligands WNT5A, WNT7A, and WNT9A, or the cell surface receptor FZD9, or the cytoplasmic transducer APC2, the nuclear factors SOX1, SOX7, SOX14, and SOX17, and the pathway inhibitors FRZB, SFRP1, SFRP2, KREMEN2, NKD2, and WIF1. Strikingly, the tumor suppressor candidate HIC1 is hypermethylated, which is acting as a transcriptional repressor for abnormal survival circuits of the transcription factors involved in the WNT signaling pathway [33, 37].

Furthermore, several studies demonstrated that promoter DNA hypermethylation is associated with the transcriptional repression of multiple microRNAs (miRNAs). This results into upregulation of oncogenic target genes of the microR-NAs, such as observed for the downregulation of miR-181b, which promotes the

#### **Figure 2.**

*Schematic diagram of signaling pathways in PDAC, which are deregulated by DNA methylation. Critical tumor suppressors and transcription factors are silenced, whereas oncogenes are activated. This deregulation promotes EMT and metastasis.* 



#### **Table 1.**

*A series of methylated genes in PDAC.* 

 expression of BCL 2 [38]. Moreover, downregulation of the miR-29 family was associated with the overexpression of the DNA methyltransferases DNMT3A and DNM3B [39]. The noncoding RNAs and antisense RNA sequences are strongly involved in the respective DNA hypermethylation process, which silences important genes such as polycomb group (PcGs), which in turn may expose these regions to DNA methylation changes [40]. More examples are listed in **Table 1**, all of which are related to PDAC.
