Large scale de-methylation of the genome was one of the first identified epigenetic phenomena in 1983 implicated in tumourigenesis (Gama-Sosa et al., 1983). Since then, it has been recognised as a common feature in all tumours (Feinberg et al., 2006). DNA hypomethylation promotes tumour development through a number of methods including: 1) Activation of oncogenes and genes associated with metastasis and tumour aggression, 2) Activation of repetitive sequences, 3) Chromosomal instability, 4) Aneuploidy and recombination and 5) Biallelic transcription of imprinted genes (Herceg and Vaissiere, 2011).
Silencing of parasitic sequences X-chromosome inactivation Correct organisation of chromatin in active and
inactive states Tissue specific DNA methylation Genetic imprinting Persistence of 5mC residues Global genomic hypomethylation Hypermethylation of CpG islands of tumour suppressor genes Mutations Chromosome instability Aneuploidy Activation of transposons Up-regulation of gene expression Gene inactivation N o r m a l c e l l C a n c e r c e l l
33
DNA methylation levels are reduced 20%_60% in cancer cells compared to normal cells. This mainly reflects the DNA hypomethylation detected at coding regions and introns of genes and the repetitive regions (Esteller and Herman, 2002). Considering that transcription of repetitive regions is primarily suppressed through their heavy methylation and the fact that approximately half of the human genome is comprised of repetitive regions, including mobile transposons and satellites that are dispersed throughout the entire genome, it is not surprising that most of the observed DNA hypomethylation in cancers corresponds to these regions (Ehrlich, 2009; Herceg and Vaissiere, 2011). Thus hypomethylation of transposable elements and pericentromeric repeats results in their activation, followed by instability and rearrangement of the chromosomes and aneuploidy (Esteller and Herman, 2002; Strathdee and Brown, 2002; Ehrlich, 2009).
Other than hypomethylation of the whole genome, hypomethylation of specific genes such as oncogenes, genes associated with metastasis, and drug resistance genes have been detected in mammalian tumours (Ehrlich, 2009). For example the BCL-2 oncogene and the gene encoding urokinase-type plasminogen activator (uPA) are hypomethylated and over- transcribed in human B-cell chronic lymphocytic leukaemia and in breast cancer with increased aggressiveness, respectively (Laird and Jaenisch, 1996; Ehrlich, 2009).
Another consequence of DNA hypomethylation in tumours is loss of imprinting (LOI). LOI results in biallelic expression of the previously imprinted genes, hence increasing the expression levels of these genes by two-folds (Laird and Jaenisch, 1996). As most of the imprinted genes are related to biological functions such as regulation of cell growth, cell signalling, cell cycle and transport; their over-expression has major consequences in normal development and function of a cell and has been implicated in many human disorders, such as cancers (Feinberg et al., 2006). For example LOI of IGF2 gene is correlated with five-fold increase in the risk of colorectal cancer and is commonly linked with Wilms’ tumour (Herceg and Vaissiere, 2011; Laird and Jaenisch, 1996).
The mechanism of DNA hypomethylation has been largely unexplored. This is due to the nature of hypomethylated regions and their high repetitiveness. However, recent emergence of whole genome methylation profiling techniques and discovery of single copy-hypomethylated genes in tumours, known as germ cell-specific genes or cancer-germline (CG) genes, has led to recognition of DNA hypomethylation in tumours as a selective event rather than a non-
34
selective, random procedure (De Smet and Loriot, 2010; De Smet et al., 2004). Thus regions between 1kb to several megabases undergo hypomethylation in cancers while other regions escape these waves of DNA hypomethylation. This leads to formation of a mosaic DNA methylation pattern (De Smet and Loriot, 2010). As a result, DNA hypomethylation patterns are tumour-type specific. In fact, activation levels, types of CG genes and some repetitive sequences have shown tumour specificity (De Smet and Loriot, 2010; Akers et al., 2010). For example the NBL2, a DNA repeat in the acrocentric chromosomes, is hypomethylated in neuroblastomas and hepatocellular carcinoma and is hypermethylated in ovarian cancer and Wilms’ tumours while showing intermediate methylation levels in normal tissues (De Smet and Loriot, 2010).
So far about 50 CG genes have been identified in humans (De Smet and Loriot, 2010). As these genes are hypermethylated in normal somatic cells and hypomethylated in germ cells, their expression is restricted to germ cells. Although the functions of all these genes are not entirely clear, it is thought that they express tumour-specific antigens and may be involved in providing them with stem cell-like properties such as immortality (De Smet and Loriot, 2010; Akers et al., 2010; Loriot et al., 2006). In addition, there has been some evidence that CG antigens are hallmarks of normal stem cells. This supports the stem cell origin of tumours, as tumour cells also express these antigens (this model is explained in section 1.3.5.3). However, the role of CG genes in stem cells and provision of the tumours with stem cell-like properties is inconclusive and requires further investigation (Akers et al., 2010).
Several models for mechanisms of DNA hypomethylation in tumours have been proposed. One model that seems most likely is the demethylation/remethylation model. This model is based on the same principals observed in reprogramming events during embryogenesis. The entire genome of a tumour cell undergoes non-selective, active demethylation. This is followed by a selective de novo methylation of the genome. This re-methylation only takes place at regions that are not protected by transcription factors. Therefore, the selectiveness of DNA hypomethylation is based on a protection mechanism against re-methylation (Figure 1.12) (De Smet and Loriot, 2010). For example, the melanoma antigen-A1 (MAGE-A1) CG gene is methylated and in-active in somatic cells while it is hypomethylated and active in tumours. Therefore, MAGE-A1 becomes de-methylated and it is protected against re- methylation by de novo methyltransferases in tumour cells. This protection against DNA
35
methylation is achieved by binding of E-twenty six (ETS)-TF to the regulatory regions of this gene, thus inhibiting the access of de novo methyltransferases to these regions. It has been shown that removal of ETS transcription factor results in methylation of MAGE-A1 in tumour cells (De Smet et al., 2004; De Smet and Loriot, 2010).
Figure 1.12. Demethylation/remethylation model for site specific DNA hypomethylation in tumours. Methylation is removed non-selectively from the entire genome. Methylation is restored by de novo methyltransferases at the regions that are not protected from re- methylation by transcription factors. This leads to selective hypomethylation. This model is one of the proposed models for DNA hypomethylation in tumours. However, based on current evidence, it appears that it is also the most likely model. Red circles: methylated cytosine, green oval: transcription factor, blue oval: active demethylase (the nature of these enzymes is not known), gray oval; maintenance methyltransferase, yellow oval: de novo methyltransferase. This image was modified from De Smet and Loriot, 2010.
36