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Heritable and reversible mechanisms known as epigenetic alterations do not require direct alterations of DNA sequences, but they can be responsible for modifying gene expression and are related to cancer development.1-6 Although the genetic information provides the sequence for protein synthesis, the epigenetic information provides instructions on how, where, and when the genetic information will be used.7
Several epigenetic mechanisms regulate gene expression: DNA methylation, modifications of histone proteins, and functional noncoding RNA.1 The major form present in mammalian cells is DNA methylation, which is the covalent addition of a methyl group to the carbon-5 position of cytosine predominantly in the CpG dinucleotide.7, 8 This cytosine modification pattern can be transmitted through cell division and may contribute to gene inactivation in cancer.3, 4
DNA methylation is essential for normal embryonic development and has a variety of important functions, such as the regulation of gene expression, control of cell differentiation and development, chromatin modification, mutation accumulation, silencing of endogenous retroviruses, preservation of chromosomal integrity, genomic imprinting control, and X chromosome inactivation.7, 9-13 Initially discovered as a mechanism for the control of development, it plays an important role in many tumor types.14 Genomic methylation patterns are frequently altered in tumor cells, with global hypomethylation accompanying region-specific hypermethylation sites. When hypermethylation occurs within the promoter of a tumor suppressor gene, it can silence expression of the associated gene and provide the cell with a growth advantage in a manner akin to deletions or mutations.7, 10, 15
This review summarizes the main epigenetic mechanisms involved in cancer development, with special focus on DNA methyltransferase enzymes and hypermethylation of tumor suppressor genes.
Methylation Process and Cancer
In mammalian cells, the DNA targets for modification through methylation are cytosine bases adjacent to guanine bases (CpG dinucleotides).16, 17 Sequences of CpG, when found at a high frequency in the genome, are referred to as CpG islands.18 Most of the 29,000 CpG islands found in the human genome are in the promoter regions of almost half the genes and are generally unmethylated in normal cells.3, 10, 19
The modification of cytosine is catalyzed by the enzymes DNA methyltransferases (DNMTs) using S-adenosyl-L-methionine (AdoMet) as the methyl donor (Fig. 1).9, 13, 18 The methyl group of AdoMet is bound to a sulfonium ion that thermodynamically destabilizes the molecule and makes the relatively inert methylthiol of the methionine moiety very reactive toward activated carbon atoms.9 The reaction involves DNMT DNA binding, flipping the target cytosine out of the double helix, and formation of a transient covalent complex with the cytosine residue.17 DNMT adds a cysteine thiolate to the 6-carbon of the substrate cytosine, followed by transfer of the methyl group to the 5-carbon.20 Glycine N-methyltransferase (GNMT) is the main enzyme responsible for the catabolism of excess hepatic AdoMet, and the absence of this enzyme has been associated with a greater methylation process, especially during hepatic tumorigenesis.21
The distribution of methylated and nonmethylated CpG dinucleotides is not random; rather it conforms to a pattern.16 Certain genomic sites, such as pericentromeric regions, imprinted regions, and genes on the inactive X chromosome in females, are hypermethylated, whereas other sites, such as CpG islands, which are often associated with gene promoter regions, are hypomethylated.7, 13, 22
Cancers exhibit at least 2 types of methylation defects: hypomethylation, characterized by a global loss of methylation, and hypermethylation of CpG islands of regulatory regions of tumor suppressor genes.23, 24
Hypomethylation of nonpromoter regions of DNA (known as global hypomethylation) may cause genomic instability and structural changes in chromosomes in cancer, although the relationship between the 2 processes is not clear.10, 25 Two resulting effects of losses of methylation in tumorigenesis have been proposed. First, weakening of transcriptional repression in normally silent regions of the genome could cause the potentially harmful expression of inserted viral genes and of normally silenced genes, such as imprinted genes and genes on the inactive X chromosome. Second, losses of methylation of nuclear structures other than genes could affect the functional stability of chromosomes, such as pericentromeric regions,26 although the loss-of-methylation mechanism in tumors is not known.
Genes affected by hypomethylation includes growth regulatory genes, enzymes, developmentally critical genes, and tissue-specific genes such as germ cell-specific tumor antigen genes.27 The cell-cell adhesion glycoprotein P-cadherin has been found to be overexpressed in breast cancer, whereas its expression is only restricted to the myoepithelial cells in normal breast tissue. Paredes et al stated that this gene promoter methylation could be a putative molecular mechanism responsible for its transcriptional regulation and found a higher percentage of P-cadherin unmethylation in contrast to normal tissue (42% versus 0%). Others genes have been shown to become promoter-hypomethylated during carcinogenesis such as cyclin D2 in gastric cancer28 and MAGE in melanomas.29 It have been demonstrated that cancer cells with hypomethylated oncogenes overexpress its protein, whereas treatment with a methylation inhibitor induces its expression in cell lines not expressing the gene.30 These observations indicate that aberrant hypomethylation in cancer may contribute to oncogene expression.
Hypermethylation of CpG islands in gene promoter regions may be involved in carcinogenesis as a result of 3 possible mechanisms: cytosine methylation facilitates gene mutation as 5-methylcytosine is deaminated to thymine,8, 31 aberrant DNA methylation may be associated with allelic loss,7, 32 and tumor suppressor genes may be inactivated by DNA hypermethylation.3, 33
Cytosine methylation can increase mutation rates because of the spontaneous hydrolytic deamination of methylated cytosine, which causes C→T transition mutation.8, 10, 18 This phenomenon was used to explain the high incidence of CpG to TpG transition mutations observed in the p53 tumor suppressor gene.34 The epigenetic silencing of the DNA repair enzyme O6-methylguanine DNA methyltransferase (O6-MGMT) is another example of how abnormal methylation may lead to increased rates of mutation.35 The O6-MGMT protein removes carcinogen-induced O6-methylguanine adducts from DNA, which produce G→A transition mutations if left unrepaired.10 Tumors with silenced O6-MGMT alleles, such as p5335 and K-ras,36 seem to be predisposed to mutation in key genes.
The hypermethylation of gene promoter regions blocks its transcription, contributing to inactivation of tumor suppressor genes in cancer. Many tumor suppressor genes in cancer have been found to have promoter hypermethylation (Table 1). Cell cycle-regulating genes such as p15 and p16 have been shown to be affected by CpG island hypermethylation. The inactivation of these 2 genes results in Rb phosphorylation, entry into the S phase, and subsequently in cell proliferation.38 DAP-kinase expression, a positive mediator of apoptosis, is frequently lost in metastatic lung tumors. In addition, restoration of its protein to the physiologic levels suppressed their ability to form metastases.43, 57 This observation indicates that tumor suppressor gene promoter hypermethylation contributes to cancer development.
Table 1. Most Studied Hypermethylated Promoters of Genes Implicated in Carcinogenesis
According to the Knudson 2-hit hypothesis, promoter methylation may occur on a gene acting on the wild-type allele, while the other is mutated, contributing to the biallelic inactivation of tumor suppressor genes, either as a primary or a second hit in both familial and sporadic forms of cancer.26, 58 In this case, genetic and epigenetic changes can collaborate to prevent the expression of a functional gene product in cancer cells.59 Otherwise, hypermethylation of both alleles may also be present in some cases (Fig. 2).26
It is important to note that only methylation within or around the promoter region is associated with gene silencing. Dense methylation within the body of a gene, even within CpG islands, does not hinder transcription.26, 60
It has been recognized that aberrant hypermethylation events can occur early in tumorigenesis, predisposing cells to malignant transformation.3, 7, 14 Renal tumors were demonstrated to have their average number of methylated CpG islands increase significantly and progressively from precancerous conditions to invasive tumors,12 and thus, precursor lesions of oral,42 liver,61 and uterine cervix62 cancers have been the focus of studies. Moreover, methylation of genes such as APC in the formation of intestinal polyps, H19 in preneoplastic kidney parenchyma of Wilms tumor patients, and RB1 in familial cases of unilateral retinoblastoma is almost certainly implicated in the earliest stages of tumorigenesis.3
Mechanisms of Silencing
In higher eukaryotes, DNA methylation and histone modifications appear to be the main events responsible for the formation of transcriptionally active or inactive chromatin.63
DNA methylation inhibits transcription by interfering with its initiation.3 Because 5-methylcytosine is in the major groove of the DNA helix,9 it is possible that this modified cytosine interferes directly with the binding of transcription factors.22, 64 Many factors are known to bind CpG-containing sequences, and some of these fail to bind when the CpG is methylated.22 However, it is unlikely to be a widespread mechanism for transcriptional silencing because most transcription factors do not have CpG dinucleotides within their DNA binding sites.7 Another possible mechanism is that specific transcriptional repressors may recognize methyl-CpG and turn off transcription.64
Four proteins with a methyl-CpG-binding domain (MeCP2, MBD1, MBD2, and MBD3) recognize methylated DNA and are implicated in transcriptional repression. These proteins also have an affinity for histone-modifying enzymes that cause chromatin condensation and gene silencing.65 MeCP2 contains both a methyl-CpG-binding domain and a transcriptional repression domain that can be tethered to another protein called Sin3A, which interacts with histone deacetylase, a member of another transcriptional repression system.66
Histones, nuclear proteins that interact with DNA to form nucleosomes, are also, in addition to being responsible for packing DNA within chromosomes, essential for transcription regulation.11, 67 Histone modifications such as acetylation and methylation may be read by the DNA methylation machinery, leading to either methylation of or failure to methylate a particular CpG dinucleotide.22 Histone acetylation occurs at sites where transcription takes place, resulting in chromatin decondensation (euchromatin) to permit binding of transcription factors to DNA.18, 66 Acetylation is controlled by histone acetylases and histone deacetylases (HDACs).68 Deacetylation of these proteins (in particular H3 and H4) by HDAC leads to a tighter nucleosomal packing and the formation of a compacted chromatin environment (heterochromatin) that inhibits transcription.3, 11 Moreover, histone methylation is linked to euchromatic and heterochromatic states.68, 69 Methylation of lysine 9 in the core histone H3 is associated with silenced genes,70 whereas methylation of lysine 4 in histone H3 is a feature of active genes.22
DNA methylation and histone modifications are intricately connected with each other.8, 25 Thus, the methylation level is connected to the broad organization of chromatin, with unmethylated DNA usually being part of euchromatin, whereas heavily methylated DNA is part of heterochromatin.9 HDAC may play an important role, in cooperation with DNA methyltransferases, in maintaining tumor suppressor gene silencing.33 DNA methylation recruits methyl-CpG-binding proteins and their associated corepressors and HDACs, resulting in tighter packaging of DNA and reduced access of transcription factors to their binding sites (Fig. 3).11, 71, 72
Indeed, DNA methylation appears to be the dominant mechanism of silencing genes.26 Drugs that inhibit HDAC can increase the expression of genes with unmethylated promoters, but they cannot induce the reexpression of hypermethylated genes in cancer cells. However, if some demethylation is first effected by low doses of demethylating drugs, histone deacetylase inhibitors act synergistically in reexpressing the silent gene.73, 74 Thus, the use of a combination of inhibitors of DNMTs and histone deacetylases is an attractive therapeutic strategy.26
DNA Methyltransferases (DNMTs)
Three DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) are responsible for adding methyl groups to CpG dinucleotides.7, 14
DNMT1 is often referred as the maintenance methyltransferase because it is believed to be the primary enzyme responsible for copying methylation patterns after DNA replication.7, 23, 61, 75 Its predominant splicing isoform in somatic cells in humans comprises 1616 amino acids, with a large N-terminal regulatory domain and a smaller C-terminal catalytic domain.9 It is the most abundant DNMT targeted to replication foci and, under experimental conditions, has up to a 50-fold preference for hemimethylated DNA substrate.15, 76
This enzyme can maintain CpG methylation after DNA replication by methylating the daughter DNA strand, using the methylation pattern of the parental strand as a template (Fig. 4A).9, 16, 77 Its inactivation produces global demethylation, which is consistent with the finding that DNMT1 is required for maintenance methylation.77 The structural and mechanistic basis for the specificity of the enzyme for CpG sites as well as its preference for hemimethylated DNA is still unknown.9
Three sequences in the N-terminal increase the precision of maintenance methylation and give the enzyme direct access to the nuclear replication site: the proliferating cell nuclear antigen (PCNA) binding domain,78 the replication foci targeting sequence79 and the polybromo homology domain.17, 80 PCNA is required for DNA replication, and the DNMT1-PCNA interaction may allow the newly synthesized daughter strands to be rapidly remethylated before being packaged into chromatin.7, 78 This tight association of the DNMT1 with the replication machinery allows DNMT1 to bind newly replicated and still naked DNA.17
Otherwise, some genes may make this interaction difficult with replicating foci. The cell-cycle regulator p21 can disrupt the DNMT-PCNA interaction, suggesting that p21 may negatively regulate methylation by blocking access of DNMT to PCNA,78 particularly during DNA damage, when p21 protein is induced.81 It was also demonstrated that p21 may inhibit DNMT1 gene expression.81 The retinoblastoma gene product Rb, another cell-cycle regulator protein, can bind to DNMT1 and inhibit its methyltransferase activity during DNA replication in the cell cycle.82 Loss of functional Rb may grant DNMT1 free access to the genome, which could allow for aberrant de novo methylation of CpG.7 These observations point to a complicated network of connections between DNMT1 and several cellular proteins involved in gene regulation and epigenetic signaling during cell replication.63
Although DNMT1 is the major DNMT in humans, 2 other enzymes, DNMT3a and DNMT3b, have also been shown to possess DNMT activity.12 They catalyze DNA methylation at CpG dinucleotides in unmethylated genomic sequences.33
Because DNMT3a and DNMT3b cannot differentiate between unmethylated and hemimethylated CpG sites, they obviously cannot copy a specific pattern of methylation or contribute to the maintenance of methylation pattern.9 Because they show no preference for hemimethylated DNA, both enzymes appear to function as de novo methyltransferases77 and show a disperse distribution throughout the nucleus not associated with replication sites, even during S-phase (Fig. 4B).17 This finding suggests that these DNMTs utilize a different mechanism for accessing the densely packed chromatin and for interacting with their target sites that may involve auxiliary factors such as chromatin remodeling complexes.17
DNMT3a and DNMT3b are highly expressed in early embryonic cells, the stage in which most programmed de novo methylation events occur, are downregulated after differentiation and in adult somatic tissues, and are overexpressed in tumor cells.3, 22, 77, 83, 84 DNMT3b has been shown to play a crucial role in incorporating de novo hypermethylation of promoter CpG islands, a possible mechanism for tumor suppressor gene inactivation within human cancer cells.8, 18
Another member of the DNMT3 family is DNMT3L, a regulatory factor for de novo methylation without methylation capacities. Its amino acid sequence is very similar to that of DNMT3a and DNMT3b but lacks the residues required for DNA methyltransferase activity in the C-terminal domain.8
DNMT and Cancer
Although the DNMT1 and DNMT3 families have been considered maintenance and de novo methyltransferases, respectively, it is likely that all 3 DNMTs possess both functions in vivo, particularly during carcinogenesis.7
Excessive amounts of DNMT1, which cannot target replication foci, may participate in the de novo methylation of CpG islands that are not methylated in normal cells,85 supporting the idea that DNMT can contribute to tumor progression through CpG island methylation-mediated gene inactivation.86 Thus, its increased expression may play an important role in the malignant progression of cancer, leading to aberrant methylation in many important tumor suppressor genes.23
Increased expression of DNMT protein may be an early and significant event in urothelial,85 hepatic,38, 61, 87-89 gastric,90 pancreatic,91 lung,23, 51, 52 breast,92 and uterine cervix62 carcinogenesis (Table 2).
Table 2. Summary of DNA Methyltransferase Studies in Human Cancer Tissues and Cell Lines
NA, not available; IHC, immunohistochemistry; qRT-PCR, real-time PCR; NBH, Northern blot hybridization; ↑, increased expression; ↓, decreased expression.
In contrast to genetic alterations, epigenetic changes in cancer are potentially reversible, which has spurred the development of pharmacologic inhibitors of DNA methylation and histone deacetylation.96 Indeed, the reactivation of epigenetically silenced genes in cancer may have a profound antitumor effect, thereby being a rational target for therapy and prevention.26
DNA methylation inhibitors such as 5-aza-2′-deoxycytidine (decitabine) can be utilized to reverse the effects of methylation, including the reduction of mutations at methylated CpG sites, reactivation of genes suppressed by hypermethylation, and restoration of cell growth control.97, 98 Treatment of cultured cells with this drug has been shown to cause cell growth inhibition, G2/M arrest, and cell apoptosis.83 The disadvantage of this demethylating agent is its myelosuppressive effect, particularly when used at high doses.10
The combination of DNMTs and HDAC inhibitors may have an advantage in the treatment of cancer.26 The use of a histone deacetylase inhibitor such as trichostatin A and phenylbutyrate in combination with 5-aza-2′-deoxycytidine has resulted in a strong synergistic growth inhibition in both cell lines and tumor.99, 100
Aberrant methylation of genes controlling cell proliferation, metastasis, apoptosis, and drug susceptibility has been identified in multiple cancer types. The individual determination of the cases showing hypermethylation of these tumor suppressor genes by molecular techniques such as methylation-specific polymerase chain reaction may contribute to determining the best treatment strategy for each case. So, the addition of DNA methylation inhibitors to the current therapy shows great possibility for improving cancer therapy.
We have described the main epigenetic mechanisms for regulating gene transcription, mainly of tumor suppressor genes. Among these are DNA methylation and histone modification such as deacetylation, intricately related to chromatin configuration. This transcriptional control already exists in normal cells and is essential for adequate development. However, the aberrant epigenetic regulation of gene expression plays an important role in cancer development, as do the genetic alterations, making it an important topic for molecular oncology research.
Because it is a potentially reversible change, the epigenetic event represents new opportunities for the clinical management of cancer through the development of strategies to reverse gene silencing. Further, the associated molecular changes (such as DNMT/HDAC overexpression and gene promoter hypermethylation) may serve as markers for risk assessment, diagnosis and prognosis of cancer.