The role of DNA methylation in human pancreatic neuroendocrine tumours

Abstract

Pancreatic neuroendocrine tumours (PNETs) are the second most common pancreatic tumour. However, relatively little is known about their tumourigenic drivers, other than mutations involving the multiple endocrine neoplasia 1 (MEN1), ATRX chromatin remodeler, and death domain-associated protein genes, which are found in ~40% of sporadic PNETs. PNETs have a low mutational burden, thereby suggesting that other factors likely contribute to their development, including epigenetic regulators. One such epigenetic process, DNA methylation, silences gene transcription via 5'methylcytosine (5mC), and this is usually facilitated by DNA methyltransferase enzymes at CpG-rich areas around gene promoters. However, 5'hydroxymethylcytosine, which is the first epigenetic mark during cytosine demethylation, and opposes the function of 5mC, is associated with gene transcription, although the significance of this remains unknown, as it is indistinguishable from 5mC when conventional bisulfite conversion techniques are solely used. Advances in array-based technologies have facilitated the investigation of PNET methylomes and enabled PNETs to be clustered by methylome signatures, which has assisted in prognosis and discovery of new aberrantly regulated genes contributing to tumourigenesis. This review will discuss the biology of DNA methylation, its role in PNET development, and impact on prognostication and discovery of epigenome-targeted therapies.

Keywords: epigenetics; hydroxymethylation; menin; methylation; multiple endocrine neoplasia type 1; pancreatic neuroendocrine tumours.

Figure 1

Relationship between histone and DNA methylation with chromatin state. In chromosomes, DNA is usually tightly wrapped and packaged around histone proteins when not being actively transcribed. DNA methylation is catalysed by DNMT enzymes which ensure that cytosines at CpG sites remain methylated and this prevents transcriptional machinery from binding to these sections of DNA. Histone and DNA methylation work together to either allow or prevent DNA transcription. Thus, sections of DNA are ‘marked’ for transcription with both histone and DNA modifications to determine which parts of DNA are unwound from histone proteins to enable transcriptional machinery to access DNA. TET enzymes ensure that DNA remains unmethylated, thereby allowing transcription factors to bind to DNA, whereas the methylation mark H3K27 tri-methylation (H3K27me3), catalysed by EZH2, is associated with heterochromatin and keeps DNA wound tightly around histone proteins. Menin catalyses the addition of a methyl group by MLL1/2 (KMT2A/B) to form the active histone methylation mark H3K4 tri-methylation (H3K4me3), which unwinds DNA.

Figure 2

The dynamic DNA methylome cycle. In the dynamic DNA methylome, 5’methylcytosine (5mC) undergoes consecutive oxidative steps to form 5’hydroxymethylcytosine (5hmC), 5’formylcytosine (5fC) and 5’carboxylcytosine (5caC) and then back to an unmodified cytosine (C), which can re-enter the cycle following re-methylation by DNA Methyltransferase (DNMT) enzymes to 5mC. The DNA methylome is linked with the tricarboxylic acid (TCA) cycle, which is also known as the Krebs or citric acid cycle. The TCA cycle provides alpha-ketoglutarate which is required for active demethylation by ten-eleven-translocase (TET) and by histone lysine demethylase (KDM) enzymes including KDM5B which demethylates H3K4me3, H3K4 dimethylation (H3K4me2), and H3K4 mono-methylation (H3K4me1). Loss of menin leads to increased DNMT1 and subsequent DNA methylation, as well as a loss of the active histone mark H3K4me3, which also protects against DNA methylation.

Figure 3

DNA methylation in normal (panel A) and cancer (panel B) states. (A) A typical strand of DNA with a CpG island I (CGI) in normal tissue. CGIs are flanked on either side by shores (within 2 kB of the CGI), shelves (within 4 kB), and the open sea (>4kB). CpG sites occur more frequently in CpG islands when compared to the rest of the genome and are usually hypomethylated (blue circles), whilst 5’hydroxymethylcytosine (5hmC) marks (green circles) tend to be present at the shores of CGI and protect against DNA methyltransferases (DNMTs), and subsequent 5’methylcytosine (5mC) marks (red circles) are found less frequently outside of CGIs. H3K4 tri-methylation (H3K4me3) marks are associated with regions of DNA hypomethylation and H3K4 mono-methylation (H3K4me1) marks are associated with regions enriched in 5hmC. CpG sites in the open sea (i.e. >4 kB away from a CGI) tend to be methylated. (B) In cancer, aberrant DNA methylation occurs with a loss of 5hmC marks (green circles) that results in an inability to protect against DNMTs, which leads to the usually hypomethylated cytosines (blue circles) in CGI becoming methylated (red circles) by DNMTs that in turn leads to transcriptional silencing. Scattered CpGs outside the CGI (shelves and open sea) become progressively hypomethylated in malignancy.