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During translation and transcription, DNA exerts its effects in cells through regulatory mechanisms including mRNA stabilization, transcription, and epigenetic changes.1 The pattern of altered gene expression or epigenetic change is of major importance in common malignancies.2, 3 Methylation of the DNA, histone deacetylation, ubiquitination, and phosphorylation are examples of epigenetic change.4 DNA methylation, unlike the other epigenetic changes, does not alter the nucleotide sequence.

Most cytosine-phosphoguanine (CpG) dinucleotides are unevenly distributed throughout the genome and remain in short stretches or clusters (500–2000 bp), called CpG islands.5, 6, 7 These islands are located in the promoter region and are found in half of all human genes.8 In mammals, DNA methylation occurs after replication, when a methyl group (CH3) is added to the 5′ position of cytidyl residues in the dinucleotide sequence CpG9, 10 (Figure 1). Endonucleases, which normally degrade foreign DNA, regulate gene expression by silencing genes when the CpG is methylated.11 CpG islands remain unmethylated in housekeeping genes and methylated or silenced in other genes.12

Figure 1
figure 1

DNA methylation. SAM and other methyl donors bind to DNMTs to methylate the DNA that is tightly wrapped around the histones.

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DNA is methylated by DNA methyltransferases (DNMTs), which transfer the methyl group from S-adenosylmethionine (SAM) to generate patterns of genomic methylation that silence genes13, 14, 15 (Figure 1). The DNMTs known to date are DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMT3L.15, 16 All methyltransferases have homology and different functions. DNMT1 maintains established methylation patterns in hemi-methylated genes by copying methylation patterns from the parent strand to the daughter and is expressed during the S-phase.17 DNMT2, a small protein of 391 amino acids, is known to have weak DNA methyltransferase activity.18 DNMT3a and DNTM3b, referred to as de novo methyltransferases, methylate unmethylated DNA. They initiate normal DNA methylation during embryonic development.19 DNMT3L does not bind to SAM, but increases the binding of DNMT3a to SAM. Since DNMT3L-deficient mice are sterile, DNMT3L is likely to be essential in the methylation process.19

Overexpression of DNMTs can be lethal in animals as well as in human cancers.20 DNMTs are involved in the downregulation of tumor suppressor genes and stimulation of proto-oncogenes.21, 22 Although DNMTs are important in DNA methylation, several findings indicate that DNMTs are not essential for the promotion of carcinogenesis.23, 24, 25

Hypo- and hypermethylation

When gene expression is altered due to DNA methylation, it is usually categorized as due to hypo-methylation or hypermethylation (Figure 2). DNA hypomethylation is associated with gene reactivation and chromosomal instabilities.26, 27 Functional outcomes of hypomethylation include the upregulation or overexpression of transcription of proto-oncogenes, increased recombination and mutation, X-chromosome inactivation, loss of imprinting, reactivation of transposable elements, and demethylation of xenobiotics10, 28, 29 (Figure 3). Activation of proto-oncogenes, reactivation of transposable elements, and loss of imprinting of genes are the results of hypomethylation and all promote cancer.30

Figure 2
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Two types of aberrant methylation, which elucidate certain actions.

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Figure 3
figure 3

Known functional outcomes of DNA hypomethylation.

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When CpG islands are hypermethylated, the activity of the regulatory proteins that promote transcription is restricted due to the tightly packed nucleosomes.8 DNA hypermethylation is involved in gene repression and chromosomal instabilities.31 Results of hypermethylation include suppression of tumor suppressor genes, chromatin condensation, and suppression of DNA repair genes (Figure 4). Tumor suppressor genes contain unmethylated CpG islands in their promoter regions and are methylated in various malignancies.32, 33, 34 Both types of methylation occur simultaneously in various sporadic cancers and affect the function of human tumor suppressor genes and proto-oncogenes.35

Figure 4
figure 4

Known functional outcomes of DNA hypermethylation.

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Detection of DNA methylation

Currently, several methods are used to detect methylated DNA (Table 1).

Table 1 Methods of detection for methylated DNA sequences
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Methylated DNA can be detected by converting unmethylated cytosine residues to uracil using sodium bisulfite modification, followed by polymerase chain reaction (PCR) to identify the unmethylated nucleotides.36 Methylation-specific PCR (MSP) detects the methylation of CpG islands, but with higher specificity and sensitivity.37 Quantitative real-time PCR (MethyLight or QM-MSP) is used to detect low levels of methylation that cannot be distinguished using MSP.38, 39

A modification of MSP, termed McrBC-methylation-sensitive-arbitrarily-primed-polymerase-chain-reaction (McrBC-msAP-PCR) requires methylation-specific GTP-dependent restriction endonuclease, McrBC, to detect differentially methylated sites within DNA, where hypermethylation and hypomethylation is observed by DNA fingerprint band intensity.40 The procedure for the methylated CpG island recovery assay (MIRA) requires isolated and sonicated genomic DNA, instead of sodium bisulfite.41 PCR reactions detect CpG island methylation after the DNA is incubated with a matrix containing methyl-CpG binding domain protein-2b (MBD2b) and methyl-CpG binding domain protein 3-like-1, which bind specifically to methylated DNA sequences.41, 42 MIRA can be used to study the methylation status of a wide array of genes in cancers, such as the lung.42 Currently, other array-based methods are being developed to screen the methylation pattern of several genes.39

Differential methylation hybridization (DMH) is an oligonucleotide array-based method, which determines the extent of methylation of CpG islands by comparison with a reference sample.44 Methylation-specific restriction enzymes (MseI) are used to obtain intact CpG islands. The CpG islands are fluorescently labeled and after subsequent PCR, they are hybridized to arrayed oligonucleotides that can discriminate between methylated and unmethylated alleles in regions of interest. Microarray methylation assessment of single samples (MMASS) has been shown to be more sensitive and is an optimized method for detecting the methylated and unmethylated sequences within the entire genome.45 MMASS uses the methylation-specific enzyme, McrBC, instead of MseI. It is able to detect unmethylated sequences more effectively than DMH because McrBC only cleaves methylated sequences and does not require a reference sample.45 Methylation target array (MTA) simultaneously determines whether or not genes and CpG islands in multiple tumors are hypermethylated and can be correlated to clinicopathological features of the patient.46 An advantage of MTA is that a single nylon filter can be used repeatedly to probe for various genes which are indicative of DNA methylation.47

DNA methylation and cancer: overview

Aberrant DNA methylation is one of the many potential causes for the abnormal growth of cancer cells, but it is also known to protect against intestinal cancer.4 Different types of cancers are associated with methylation of tumor suppressor genes and proto-oncogenes, causing alterations in functional gene expression. Cancer-specific DNA methylation patterns have been detected in free-floating DNA released from dead cancer cells.5, 48 A decrease in the expression of tumor suppressor genes correlates with an increase in methylation of DNA in the promoter region.49, 50 Aberrant methylation of tumor suppressor genes in many cancers, resulting in the downregulation of transcriptional activation, has been reported.

In some cancers, both hypermethylation and hypomethylation are observed. Hypomethylation increases progressively with increasing malignancy grade in breast, ovarian, cervical, and brain cancers.51 Breast and colorectal cancers are malignancies commonly caused by regional hypermutability or global hypomethylation.

DNA methylation and breast cancer

Many factors contribute to the pathogenesis of breast cancer, which is one of the most common malignancies among females. These factors include family history, nutrition, age, and epigenetic changes including DNA methylation. Methylation appears to be an early event in the etiology of breast carcinogenesis, resulting in the activation of many oncogenes and silencing of tumor suppressors to promote proliferation of abnormal cells.52, 53 It is debatable, however, whether global hypomethylation or regional hypermethylation occurs first during the development of breast cancer, since the phenomena are independent processes. It is not known whether or not it is possible to inhibit carcinogenesis by inhibiting one of these processes and not the others. Numerous studies have revealed various genes, which are either hypo- or hypermethylated in breast cancer (Table 2).

Table 2 Genes methylated in breast cancer
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Metastatic breast cancer requires the expression of multiple genes. Regional hypermethylation and global hypomethylation are involved in different stages of breast cancer.76 Global hypomethylation could be a mechanism for late stages of breast cancer while local hypermethylation is plausible for early stages of breast cancer.22, 30 DNA methylation results in altered gene products including cell cycle regulators, steroid receptors, and cell adhesion molecules, which give rise to increased susceptibility to tumor development and decreased detoxification of carcinogens.58 Alterations in the breast cancer susceptibility gene product (BRCA) accounts for half of the inherited breast carcinomas.77 Its methylation is observed in breast and ovarian cancers, but not in colon and liver cancers, or leukemia indicating a tissue-specific process.58 The frequency of methylation of this gene product is 38.5% in sporadic breast cancer.60 Patients with a HER2/Neu-positive tumor indicate a highly aggressive breast cancer that requires special treatment, since it is amplified in 30% of invasive breast carcinomas.55 DNA methylation is prevalent in the highly aggressive HER2/Neu-positive breast cancers; this gene is amplified in 30% of the cancers.55 Increased aberrant methylation of steroid receptor genes and glycoproteins, such as progesterone receptor (PR) and E-cadherin, respectively, are associated with Her2/Neu-positive cancers. Hypermethylation of the GC-rich region and loss of expression in about 80% of invasive lobular carcinomas and lobular carcinoma in situ indicate the importance of the methylation of the CDH1 promoter in the pathogenesis of breast cancer.78 Although many mechanisms, including mutation and loss of heterozygosity (LOH), are attributed to the downregulation of CDH1 in breast cancer, CDH1 promoter methylation is the mostly likely cause.78, 79

The current criteria for detection and prognosis of breast cancer include an abnormal breast biopsy, tumor size, histological grade, estrogen and progesterone receptor status, and presence of the HER2/Neu oncogene.80, 81 Breast cancer can also be diagnosed by detecting the various aberrantly methylated genes. MSP is currently being used to detect the methylation status of various genes in breast biopsy tissues samples.59 Presence of methylated DNA in the nipple duct lavage fluids, needle aspirates of the breast, and molecular staging of sentinel lymph nodes are also used to predict breast cancer development.58 Ductal carcinoma in situ (DCIS), the most frequent breast cancer, can be detected early by observing the methylation of a panel of tumor suppressor or other cancer genes.82, 83 A 60-sample study with ductal lavage fluid from patients with a high risk of developing breast cancer and patients with breast cancer revealed that a nine-gene panel detection system using quantitative methylation-specific polymerase chain reaction (QM-MSP) can detect the rate of cancer cells more effectively than cytological and histological studies alone. Thus, earlier detection of breast cancer formation is possible.84 Aberrant methylation of four genes was detected by QM-MSP in the plasma DNA of patients with breast cancer and tumors were successfully detected in eight of 24 patients with early-stage breast cancer.85 Loss of MGMT (O(6)-methylguanine-DNA methyltransferase) was found to be associated with DNA methylation in a subset of breast cancers.86 Although DNMT3b, a de novo methyl transferase, is overexpressed in 30% of breast cancers, its expression alone was not considered to be a prognostic factor for breast cancer progression.87 Such studies highlight the importance of identifying specific methylated genes for diagnostic purposes, and for monitoring the efficacy of therapeutic modalities.

DNA methylation and colorectal cancer

Colorectal carcinoma is the third most common cancer in developed countries.88 Although age and other demographic and environmental features, including gender, weight, nutritional intake, and alcohol consumption, are prognostic of colorectal cancer, epigenetic alterations are also causal.89 Aberrant methylation is gradually acquired in the early stages of colorectal carcinoma.90 As in breast cancer, both hypomethylation and hypermethylation of genes occur in colorectal cancer91, 92 (Table 3). The genes for p53 and for retinoic acid receptor (RAR) are hypermethylated in colorectal cancer.108 A study with 65 colorectal carcinoma tissues demonstrated hypermethylation of the gene for the cell cycle regulatory protein, cyclin A1, in all cases, and for cadherin-13 in 65% cases.96 Methylation did not correlate significantly with any clinicopathological feature, and changes in methylation appeared in an early phase of colon carcinogenesis.96

Table 3 Genes methylated in colorectal cancer
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Aberrant methylation of some genes, including those for estrogen receptor α (ERα) and myoblast determination 1-protein (MYOD), correlate with aging and the prognosis of colorectal cancer.109 UDP-glucoronosyltransferase (UGT1A1) gene expression is silenced and transcriptional activity is completely repressed in colon cancer cells due to direct methylation of its promoter region.97 Treatment with either the inhibitors of histone deacetylase or demethylating agents restores normal expression of UGT1A1 in hypermethylated cells but has no effect on hypomethylated cells.97

Rhee et al100 reported that DNA methyltransferases, DNMT1 and DNMT3b, are hypermethylated in colorectal cancer. Other studies suggest that methylation depends upon the type of cancer and that colorectal cancer can progress in the absence of DNMT1, as seen in the SW48 colorectal cancer cell line.108, 110 However, methylation can be reinitiated by introducing DNMT1 to the SW48 colon cancer cells lacking DNMT1 and DNMT3b.111 Therefore, DNMT1 is potentially important in the hypermethylation of CpG islands in the promoter region of many genes in human cancer cells.

Colorectal tumors are often identified by the level of their microsatellite instability (MSI), which is a defect in the ability of repairing mistakes during DNA replication. MSI, stratified as MSI high, MSI low, and MSI stable, is commonly correlated with the degree of the colorectal cancer methylator phenotype (CIMP) when diagnosing colorectal cancer at the molecular level.112 CIMP is distinguished as CIMP(+) or CIMP(−), although the existence of CIMP is still controversial. Supporters suggest an association with microsatellite instability (MSI) and proximal location of the colonic tumor.113 A study with 106 primary colorectal tumors, however, did not support the existence of CIMP in human colorectal cancer and, consequently, it was regarded as a statistical artifact.114 Colorectal cancer cannot be identified on the basis of tumor methylation status and CIMP alone.

Hereditary non-polyposis colorectal cancer (HNPCC, Lynch syndrome) accounts for 2–4% of all colorectal cancers and aberrant methylation of the mismatch repair genes, human mutL homolog 1 (hMLH1) or hMLH2, are the basis for the cancer.115 The combination of MSI-H and CIMP(−) is commonly observed in HNPCC.112 In a study with 97 colorectal adenoma cases, hMLH1 methylation was more frequently observed in overweight or obese patients.93 High-level MSI sporadic colon cancer and HNPCC share histological features, proximal tumor location, and presence of tumor-infiltrating lymphocytes. They differ, however, in having widespread promoter hypermethylation of specific genes such as hMLH1 and BRAF.112 Fewer methylated genes are found in HNPCC than in high-level MSI colorectal tumors.116

Inhibitors of DNA methylation and demethylators

DNA methylation is a reversible process in which genes can be demethylated and restored to their original expression and function. DNA methylation inhibitors have been investigated as anticancer agents, since they block the activity of DNMTs and thus activate tumor suppressor genes54 (Figure 5). The antisense oligonucleotide to human DNMT1, MG98, acts as a DNA methylation inhibitor and downregulates the activity of DNMT1. It has had promising results in clinical trials in treating cancers of the head and neck.117 Recently, two novel inhibitors, NSC303530 and NSC401077, were shown to inhibit DNMTs in vitro and in vivo by blocking the active site of DNMT1.118 These inhibitors have also been proposed as potential antitumor drugs.118

Figure 5
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DNA methylation inhibitors. Successful inhibition of DNA methylation inhibitors (DNMTis) can prevent the suppression of tumor suppressor genes and continuous activation of proto-oncogenes.

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Demethylation of aberrantly silenced genes can restore gene expression and function. Azacitidine (5-aza-C) and decitabine (5-aza-2′-deoxycytidine or 5-aza-2′-CdR) are two potent DNA demethylating agents. 5-aza-C was developed as a cancer chemotherapeutic agent and was thought to inhibit the enzymes that methylate the cytosine residues in DNA.10, 119 However, it is now considered that it demethylates methylated DNA and acts as an anti-tumor agent in many cancer cells.120 Azacitidine is more toxic than its analog, decitabine.22

Decitabine has a short in vivo half-life and is able to reactivate previously silenced genes.121, 122 In clinical investigations in treating leukemia, decitabine was found to be most effective in an intensive dose with a short treatment time.123 Decitabine is a potent cytotoxic agent and shows in vitro antitumor activity against breast cancer cells.124 Both azacitidine and decitabine are effective in treating leukemia, which is characterized by hypermethylation.35, 125

Zebularine, another potent demethylator, was found to be effective against cancer cells in many studies. In earlier studies, it was found to have toxic effects in cancer cell lines.73 It has been shown to remove 25–60% of the methyl groups from methylated genes in a panel of seven human tumor cell lines.94 The most promising features of the drug include its stability, low toxicity to normal cells, and that it can be taken orally.126

Administration of decitabine can reactivate silenced tumor suppressor genes and the histone deacetylase inhibitor, LAQ824 (LAQ), can activate genes related to cell cycle arrest.123, 127 These agents can synergistically produce greater antineoplastic effects on MDA-MB-231 breast cancer cells, thereby ensuring application of these agents for future clinical trials.127 Lower doses of azacitidine and trichostatin A (TSA) are required to re-express ER in MDA-MB-231 (ER−) cells, when the drugs are used in combination than alone.58 Thus, the combination of drugs, which directly affects DNA methylation with drugs, causing other epigenetic changes, has considerable potential in increasing therapeutic affects.

Cancer Prevention, Dietary Intake, and DNA Methylation

Nutrition influences susceptibility to cancer. Approximately 35–50% of all cancers have a dietary component in their etiology.128 Some food constituents can promote the onset of cancer. Deficiency of fiber, folic acid, methionine/choline, zinc, selenium, and chemicals found only in fruits and vegetables all can also cause cancer.129 Excess intake of alcohol, animal fats, and salt promote cancer.130 It was predicted that the incidence of cancer in vegetarian subjects would be lower than in those on a meat diet. Even though the vegetarian diet has a lower intake of vitamin B and lower content of methionine, both of which are essential for eventual methylation, this does not cause cancer.131

The role of folic acid in cancer is still controversial. The methyl groups of 5-methylenetetrahydrofolate is the precursor of the methyl group of methionine and thereby of SAM.132 Low intake of folate combined with high alcohol intake can result in global hypomethylation and cause colorectal cancer. An increased risk of breast cancer occurs if folic acid is not metabolized correctly and the resultant supply of methyl groups to DNA in pre-menopausal women is insufficient.113, 132

Low intake of folate is associated with an increased risk of colorectal cancer.113 In vitro studies have indicated that colon cancer cell lines, when deprived of folic acid, have decreased viability.132 Uracil is misincorporated into DNA as a result of folate deficiency. However, once a cancerous lesion is present, folate intake enhances tumor growth.133 Methionine is taken as a nutritional supplement in adulthood to correct some genetically based epigenetic defects.29 However, excess intake of methionine can also impair DNA methylation. Nevertheless, folate deficiency, leading to aberrant methylation of DNA, is not the sole cause of colon carcinogenesis.

Caffeic acid and chlorogenic acid, two catechol containing coffee polyphenols, inhibit DNA methylation.134 They increase the formation of S-adenosyl-L-homocysteine (SAH), an inhibitor of DNA methylation. Partial inhibition of methylation in the promoter region of the retinoic acid receptor beta (RAR-β) gene by both caffeic acid and chlorgenic acid was demonstrated in breast cancer cell lines MCF-7 and MDA-MB-231.135

DNA Methylation and Aging

The risk of cancer increases with age. Only 10% of children have a chance of getting cancer, whereas adults have a 35% chance, because methylation of CpG islands in non-malignant tissues increases but the total number of methylated cysteine residues decreases with age.81, 136, 137 Individual genes are progressively methylated during aging due to chromosomal instability.138 Genes that change methylation status with age are tissue specific. The c-myc gene is hypomethylated in the spleen and the c-fox is hypermethylated in the liver but not in the spleen.28

The normal colonic mucosa of older females has higher methylation levels, making these cells more susceptible to differentiate into malignant cells.103, 139 Hypermethylation does not always result in malignancy. For example, hypermethylation of the estrogen receptor gene was observed in both normal and cancer colon tissues, suggesting that the relationship between hypermethylation and age in cancer might not be simple, and requires a more careful analysis.8

Concluding remarks

DNA methylation is important in gene regulation and expression. It is imperative to learn more about the regulation of how this simple and basic process becomes aberrant. The complete biological mechanisms that initiate and maintain methylation of DNA need to be fully explained. Both hypomethylation and hypermethylation of proto-oncogenes and/or tumor suppressor genes occurs in various cancers. Many known genes are aberrantly methylated in breast and colorectal cancers.

In studies with established inhibitors of DNA methylation and demethylation, some genes were shown to be able to resume normal function. Zebularine was shown to be effective, without many toxic affects, in clinical trials. Despite constant efforts, the most effective and least toxic drugs are yet to be discovered. Indeed, even more questions arise from the plethora of recent advances in our understanding of the underlying mechanisms of methylation in cancer and other malignancies.