**Abstract**

Pancreatic cancer is the fourth leading cause of cancer deaths, with a low 5-year survival rate of about 7% due to its highly invasive nature. Pancreatic ductal adenocarcinoma (PDAC) comprises more than 90% of all pancreatic cancer cases. At the time of detection, around 80% of cases harbor metastases due to the lack of early diagnosis. For decades, scientists have primarily focused on dissecting the origin of pancreatic cancer through genetic alterations and their contribution to diagnosis. Recently, PDAC research has turned into epigenetics to revolutionize our understanding about the silencing of critical regulatory genes. Epigenetic events can be divided mechanistically into various components, including DNA methylation, histone posttranslational modification, nucleosome remodeling, and regulation of transcription or translation by microRNA. The identified epigenetic processes in PDAC contribute to its specific epigenotype and are correlated phenotypic features. Strikingly, some of them have been suggested to have potential as cancer biomarkers, for disease monitoring, prognosis, and risk validation. As epigenetic aberrations are reversible, their correction will become as a promising therapeutic target.

**Keywords:** PDAC, epigenetics, DNA methylation, histone modification, microRNA, 3' UTR

#### **1. Introduction**

Pancreatic ductal adenocarcinoma (PDAC) comprises more than 90% of all pancreatic cancer cases. It is highly aggressive, extremely lethal and shows resistance to chemotherapy [1–3]. At diagnosis, around 80% of PDAC cases have already metastasized, thus rendering the current therapeutic options practically ineffective. In line with this, potentially curative surgical resection is limited to a very small portion of patients [4].

On the other hand, cancer metastasis is associated not only with simple gene/ protein expression models but also with the existence of epigenetic mechanisms [5], which complicates this process through DNA methylation, histone modifications, and microRNA regulation (see **Figure 1**). Recent studies uncovered the regulatory mechanisms of each process and their key role in EMT and cancer metastasis [6].

#### **2. PDAC from genetics to epigenetics**

Historically, the development of PDAC was attributed to DNA mutations, which are classified into three main types: oncogenes (KRAS, BRAF, AKT2, MYB, and AIBI),

#### **Figure 1.**

*Schematic diagram of epigenetic mechanisms influencing gene expression. DNA methylation is an epigenetic mechanism through which cytosine residues within CpG regions are covalently modified (left). In addition, the modification of histones has two consequences on genes, which pending on the type of modification and the target residues can either activate or repress the target gene (middle). The epigenetic mechanism is also influenced by microRNAs (miRNAs). These are small noncoding RNAs, which have a proximal length of 22 nucleotides. Functionally, the miRNAs influence gene expression through base pairing with 3′ UTRs of messenger RNAs (right).* 

 tumor suppressor genes (p16, CDKN2A, p53, p21, BRCA2, and SMAD4), and genome maintenance and repair genes (MLH, MSH2, and BRCA2) [1, 2]. Several studies explained the complexity of genetic aberrations and their regulatory signaling pathways [3]. Although a large variety of signal transduction pathways have already been studied in PDAC, much less is known about the cross talk between epigenetic mechanism and signaling pathways typical for PDAC [1]. Strikingly, there are also particular cases where signaling pathways are altered, which directly affect important components of the epigenetic machinery. Therefore, a clear understanding of the epigenetic mechanisms and their implication in PDAC development will open new avenues of therapy. This approach will exploit the intricate process through which cells induce changes at transcription level [4–6].

Epigenetic mechanisms are defined in a way that they can both silence or activate genes without alteration to the DNA sequence itself. Mechanistically, epigenetic changes represent DNA hypermethylation or hypomethylation, histonebased mechanisms that include posttranslational modifications and nucleosome remodeling, as well as aberrant expression of microRNAs [5, 7]. These modifications affect chromatin structure and promoter accessibility, which contribute to genetic alterations [8].

In PDAC, the famous mutant gene KRAS and its downstream signaling cascade are an example for the low therapeutic effect, which is accomplished by current therapies against this gene and its downstream effectors. Interestingly, recent studies demonstrate that dysregulation of epigenetic regulators is essential for PDAC progression as well as for that of many other tumors [9]. Genomic deletions, mutations, and rearrangements frequently target genes encoding components of the chromatin remodeling complex (SWI/SNF), which have been identified in 10–15% of PDAC patients [10].

In general, the discovery of the involvement of the epigenetic effect in cancer added a new concept of gene therapy and informative markers for the diagnosis and prognosis for many malignancies [11]. Whole genomic sequencing studies have revealed driver mutations in epigenetic regulators in various cancer types such as IDH1/2, DNMT3A, KDM6A, DNMT3B, SMARCB1, and CREBBP/EP300 [12]. In PDAC, the sequencing experiments showed more pathogenic mutations in genes encoding multiple components of the SWI/SNF complexes, including ARID1A, ARID1B, PBRM1, SMARCA2, and SMARCA4 [13]. Additionally, recent studies found mutations in important epigenetic regulators like histone methyltransferase enzymes MLL1, MLL2, and MLL3 and histone demethylase KDM6A [13, 14]. Specifically, KDM6A has been found mutated in 5–10% of PDAC patients [15].

Several studies on familial PDAC have shown an association between DNA repair genes' dysfunction and those genes that are responsible for this inheritance (BRCA2, BRCA1, CDKN2A/p16, STK11/LKB1) [13, 16]. Strikingly, the altered DNA repair system is a hallmark of cancer, which causes genome instability and DNA damage [17]. Each cell contains a specific enzymatic system termed the DNA mismatch repair (MMR), which is responsible for detecting and correcting DNA replication errors [18, 19]. Loss or silencing of any protein in this system leads to the accumulation of gene mutations. In this regard, studies reported that MMR inactivation was caused by the epigenetic silencing of the hMLH1 and hMLH2 genes [20, 21]. The hMLH1 protein is one component of a family of seven members of MMR proteins that work coordinately to regulate DNA replication error in humans [20]. In this context, hypermethylation of the hMLH1 promoter has been shown to be an early detection marker of esophageal cancer and also a prognostic marker in colorectal and pancreatic cancers. On the other hand, this mechanism cannot be generalized, as methylation of the hMLH2 gene results less clear into gene inactivation, because the respective promoter is a weak target for CpG island methylation [16, 17, 20–22].

Another example of an affected DNA repair gene is the O6 -methylguanine-DNA transferase (MGMT), which is most intensively regulated by CpG promoter methylation [23]. MGMT is responsible for removing alkyl groups from O6 in guanine and thus prevents mismatch errors during DNA replication. The silencing of the MGMT activity in human colorectal adenomas has been linked to K-ras GC → AT transition mutations [24, 25]. Interestingly, the epigenetic silencing of MGMT has two main effects in human cancer. First, it reveals a new mutator pathway that causes the accumulation of G-to-A transition mutations. Second, there is a strong and significant positive association between MGMT promoter hypermethylation and enhanced tumor sensitivity to alkylating drugs. These findings highlight the significance of MGMT promoter hypermethylation in translational cancer research [17, 26, 27].
