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Epigenetics in cancer: implications for early detection and prevention
Review
Epigenetics and cancer
Epigenetics in cancer: implications for early detection and prevention Mukesh Verma and Sudhir Srivastava
Knowledge of the molecular events that occur during the early stages of cancer has advanced rapidly. The initiation and development of cancer involves several molecular changes, which include epigenetic alterations. Epigenetics is the study of modifications in gene expression that do not involve changes in DNA nucleotide sequences. Modifications in gene expression through methylation of DNA and remodelling of chromatin via histone proteins are believed to be the most important of the epigenetic changes. The study of epigenetics offers great potential for the identification of biomarkers that can be used to detect and diagnose cancer in its earliest stages and to accurately assess individual risk. There has been a recent surge of interest among researchers as variations in the methylation of DNA have been shown to be the most consistent molecular changes in many neoplasms. An important distinction between a genetic and an epigenetic change in cancer is that epigenetic changes can be reversed more easily by use of therapeutic interventions. The discovery of these basic premises should stimulate much future research on epigenetics. Lancet Oncol 2002; 3: 755–63
Epigenetics has an important role in biological research and affects many different areas of study including cancer biology,1,2 viral latency,3–6 activity of mobile elements,7 somatic gene therapy,8–13 cloning and transgenic technologies, genomic imprinting,14,15 and developmental abnormalities.14,15 The definition of epigenetics varies among investigators. One definition describes epigenetics as the study of mitotically heritable changes in gene expression that are not caused by alteration of the DNA sequence.16 Another view is that epigenetics concerns the inheritance of information on the basis of differential gene expression, whereas genetics focuses on the information inherited through gene sequence.17 The mechanism of inheritance for epigenetic modification has yet to be identified. However, regardless of the definition used, the most important difference between an epigenetic mechanism and a genetic mechanism is that epigenetic changes can be reversed by chemical agents. Two major steps in the epigenetic regulation of gene expression are the deacetylation of histones, causing a change in the structure of chromatin, and the methylation of the promoter region of the gene (figures 1 and 2). Methylation is needed for the normal development of cells, and aberrant methylation confers a selective growth advantage that results in cancerous growth.18 The promoter regions of many genes can be methylated in areas called CpG
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Unmethylated CpG
Methylated CpG Promoter
Hypermethylation
Hypomethylation
STOP
START
Silencing of tumour suppressor gene
Activation of proto-oncogene
Figure 1. Epigenetic regulation of gene expression by methylation.
islands; genes under the control of highly methlyated promoters are “silenced”, which means they cannot be transcribed. In this review we discuss the genes that are deregulated by epigenetic mechanisms and could be used as potential biomarkers for cancer progression. We also discuss strategies to prevent these epigenetic events.
Molecular mechanisms involved in epigenetic changes Two major mechanisms in the epigenetic regulation of genes involve changes in the structure of chromatin and methylation of DNA. Chromosomal organisation and histones
The DNA of all eukaryotes is packaged into chromatin, which is made up of histone proteins around which the DNA is coiled. Histones have amino-termial extensions called ‘tails’, which undergo many covalent modifications that are important in both the organisation of chromosomes and the regulation of specific genes. Histone methyltransferases direct site-specific methylation of aminoacid residues such as MV is Program Director and SS is Program Director and Chief of the Cancer Biomarker Research Group at the National Cancer Institute, MD, USA. Correspondence: Dr Mukesh Verma, Cancer Biomarkers Research Group, Division of Cancer Prevention, National Cancer Institute, National Institutes of Health, Executive Plaza North, Room 3144, 6130 Executive Boulevard, MSC 7346, Bethesda, MD 20892-7346, USA. Tel: +1 301 496 3893. Fax: +1 301 402 8990. Email: [email protected]
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Factors: Environmental Hormonal Genetic
Deacetylation inhibitor: Trichostatin Methylation inhibitors: 5-deoxyazacytidine Zebularine
Epigenetics and cancer
Epigenetics regulates: Cell-cycle control DNA damage Apoptosis Invasion X-chromosome inactivation Imprinting Ageing
Hypermethylation APC BRCA1 ER hMLH1 GSTP1 TIMP3 DAPK1 Hypomethylation RAF c-MYC c-FOS c-HA-RAS c-K-RAS
DNA methyl transferases: DNMT1 DNMT3A DNMT3B
Acetylation/deacetylation Histone acetyltransferase Histone deacetylase (Dynamic equilibrium)
Methionine supplement: S-adenosylmethione
Figure 2. Epigenetic targets in cancer detection and risk assessment. The red arrow indicates down-regulation of genes. The green arrow indicates upregulation of genes.
Lys4 and Lys9 in the tail of the histone protein H3. Methylation of Lys4 is important for the maintenance of the structure of euchromatic domains, which are diffuse areas of chromatin where genes are freely accessible and generally active. By contrast, methylation of H3 Lys9 is associated with the initiation and propagation of heterochromatic domains where chromatin is densely packed and the genes are generally inactive.19 The dense, heterochromatic domains are flanked by inverted repeats that act as boundary elements which prevent the tightly packed structure spreading to neighbouring euchromatic regions. Despite the excitement over the mapping of the human genome, many challenges remain in understanding the regulation and transduction of the information contained in genomic DNA.20 At the time of the completion of the Human Genome Project, the expected number of active genes was about 30 000—twice the number of genes coded by a small organism such as Drosophila melanogaster. However, an obvious question is raised by these findings: is DNA alone responsible for generating the full range of information that results in a complex eukaryotic organism such as a human being? Epigenetic changes, at the level of histones, are a crucial feature of a genome-wide mechanism of information storage and retrieval that is only just beginning to be understood. Jenuwein and Allis proposed the concept of a histone code that could substantially extend the information potential of the genetic code.20 There is evidence that histone proteins
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and their associated covalent modifications contribute to a mechanism that can alter the structure of chromatin. Such changes could lead to inherited differences in transcriptional activation (or inactivation) states or to the stable propagation of chromosomes by a specialised centromere higher-order structure. Histone acetylation is a dynamic process that is regulated by two classes of enzymes: the histone acetyltransferases and histone deacetylases. Although many recent studies have focused on promoter-specific acetylation and deacetylation, these mechanisms are part of a broader, more dynamic acetylation mechanism that profoundly affects many nuclear processes. Kruhlak and colleagues observed this broader histone acetylation in cells entering and leaving mitosis.21 In contrast to the hypothesis that acetyltransferases and deacetylases stay bound to mitotic chromosomes to provide an epigenetic imprint for postmitotic activation of the genome, these researchers found that the enzymes are spatially reorganised and displaced from condensing chromosomes when cells undergo division (mitosis). During mitosis, histone acetyltransferases and deacetylases are unable to acetylate or deacetylate chromatin in situ despite remaining catalytically active when isolated from mitotic cells and assayed in vitro. Thus, these enzymes do not stably bind to the genome to function as an epigenetic mechanism of selective postmitotic gene activation. However, evidence does support a role for spatial organisation of these enzymes within the nucleus. Furthermore, their relation to
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euchromatin and heterochromatin postmitotically in the reactivation of the genome also is important for the active organised structure. DNA methylation
In the normal mammalian genome, methylation occurs only at cytosines 5' to guanosines at CpG dinucleotides. Many CpG dinucleotides have been depleted from the eukaryotic genome throughout evolution by spontaneous deamination. The remaining CpGs have a very high frequency of methylation, which facilitates changes in chromatin structure that block the transcription of particular genes, by rendering them inaccessible to cellular transcription machinery. However, throughout the genome short stretches of CpGrich DNA exists. These regions, known as CpG islands, are not highly methylated despite being rich in cytosine and guanine, and are generally located in the promoter regions of “housekeeping” genes which are essential for cell function. The lack of methylation in promoter regions may be a prerequisite for active transcription of the genes under their control. Only two exceptions to this rule have been reported: fully methylated CpG islands in the silenced allele for specific imprinted autosomal genes and multiple silenced genes on the female inactivated X chromosome.22,23 Another emerging concept is that deacetylation of histone proteins is the first step in the recruitment of methyltransferase to the CpG islands, resulting in hypermethylation of the promoter. For cancer prevention strategies to be developed, factors that regulate deacetylation have to be identified. Extensive research is needed in this area, especially because targets for chemoprevention could emerge from these studies. Furthermore, epigenetic regulations occur early in cancer progression, thus providing an opportunity for the development of interventions to prevent further progression. Genetic changes are the defining feature of neoplastic evolution, as suggested by the analysis of chromosomal structure and determination of the chromosomal number.24 On the basis of current knowledge, the role of epigenetics in cancer development is considered to be minor compared with the role of genetic events.25 Recent data, however, have suggested alternative approaches, and research has consequently begun to focus on the contribution of epigenetics to tumorigenesis. Cancer seems to be a process that occurs because of mutations in DNA and epigenetic mechanisms. These processes can be complementary; thus one can predispose the other to induce cancer. Factors affecting epigenetic regulation of genes
Various factors can modify mammalian cells resulting in an epigenetically transformed phenotype, without changing the DNA sequence information of the cell, including radiation, tobacco smoke, stress, hormones (such as oestradiol), base analogues, cadmium, arsenic, nickel, reactive oxygen species, and various other chemicals. Costa and Klein investigated the mechanism of nickel carcinogenesis, focusing primarily on the epigenetic changes associated with the exposure of cells to carcinogenic nickel compounds.26 They studied the
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distribution of nickel in the cell and compared the genetic and epigenetic changes that occurred. The epigenetic effects included alterations in the concentrations of transcription factors, such as activating transcription factor 1, p53, hypoxia-inducible factor 1␣, and nuclear factor . They also investigated the relation between nickel and calcium metabolism, the effect of reactive oxygen species, and the interactions of nickel with proteins. They concluded that nickel induces various signalling pathways as well as genes that seem to be important for the survival of cancer cells. They also observed that the same genes induced or repressed by nickel are overexpressed or not expressed in nickel-transformed cells, which may represent an important selection process in the mechanism of nickel carcinogenesis. In carcinogenesis research, there is a growing understanding that neoplastic transformation must be viewed as an environmental process.26 Breivik and Gaudernack have developed a model that integrates evolution and carcinogenesis at the molecular level.27,28 This model offers new perspectives, including that of epigenetics, on carcinogenesis and relates the neoplastic process to the basic evolutionary concept of biology. Nutrients can also affect the epigenetic regulation of cancer-associated genes. Altered methionine metabolism is a characteristic trait of malignant cells;29 normal cells can use homocysteine as a direct methionine precursor, whereas transformed cells tend to require methionine in their growth medium. The cycle begins with the conversion of S-adenosylmethionine to S-adenosylhomocysteine by the donation of its methyl group to a methyl acceptor. S-adenosylhomocysteine is then broken down to yield homocysteine and adenosine. Methionine is regenerated from homocysteine by a reaction dependent on folate and cobalamin. The cycle is completed by the conversion of methionine to S-adenosylmethionine. The methionine dependence occurs because of increased transmethylation, not because of decreased methionine biosynthesis. Finally, there is a lower than normal availability of free methionine in transformed cells. Low rations of S-adenosylmethionine to S-adenosylhomocysteine, which were reversible by treatment with S-adenosylmethionine, have been observed in the early stages of liver carcinogenesis.29
Genes that are deregulated by the epigenetic process Several genes have been identified in different cancers that are regulated by the epigenetic process (panel 1). These genes are involved at different stages in the division, differentiation, and proliferation of cells (figure 2). Hypermethylation of tumour suppressor genes
Many tumour supressor genes are inactivated in particular tumour types have highly methylated promoters; under normal conditions, the promoter regions are unmethylated and the gene is transcribed.17,30–36 Genes affected by these modifications include RASSF1, RARbeta, DAPK, p16, p15, MGMT, and GSTP1 in lung cancer; CDKN2A, CALCA, MGMT, and TIMP3p in oesophageal cancer; 14ARF in
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ulcerative colitis; GSTP1 in prostate cancer; and HIC1 and p53 in breast cancer. Whether methylation is the initiating or secondary event in gene silencing has not been established. However, irrespective of its role in the initiation of cancer development, methylation is an important marker for epigenetically mediated loss-of-gene function. Furthermore, these events are of comparable importance to gene mutations for the initiation and propagation of carcinogenesis. Although the functional importance of hypermethylation is apparent, the molecular mechanisms involved are still unclear. The integration of DNA methylation with chromatin organisation and the regulation of histone acetylation and deacetylation may be an important parts of the overall effect.25 However, the involvement of as yet unidentified proteins cannot be ruled out. Proteins that bind preferentially to methylated cytosines in DNA and participate in complexes that contain active histone deacetylases have recently been identified. These proteins, which include methylated DNA-binding protein MECP2 and methyl CpG-binding domain proteins MBD2 and MBD3, provide an important clue to the proposed association of methylation with transcriptionally repressive chromatin.37,38 Furthermore, these proteins contain the histone deacetylases (1 and 2) and associated proteins such as transcriptional corepressors. The existence of such protein complexes suggests that DNA methylation can target chromatin which actively represses gene expression by remodelling and disturbing the dynamic equilibrium of histone acetylase and deacetylase.39 Multiple pathways could be involved throughout the entire process.
Panel 1. Cancer types and genes regulated by epigenetic mechanisms Cancer type
Genes
Breast
p16, BRCA1, GSTP1, DAPK, CDH1, TIMP-3
Brain
p16, p14ARF, MGMT, TIMP-3
Bladder
p16, DAPK, APC
Colon
p16, p14ARF, MGMT, hMLH1, DAPK, TIMP-3, APC
Oesophagus
p16, p14ARF, GSTP1, CDH1, APC
Head and neck
p16, MGMT, DAPK
Kidney
p16, p14ARF, MGMT, GSTP1, TIMP-3, APC
Leukaemia
p15, MGMT, DAPK1, CDH1, p73
Liver
p16, GSTP1, APC
Lymphoma
p16, p15, MGMT, DAPK, p73
Lung
p16, p14ARF, MGMT, GSTP1, DAPK, FHIT, TIMP-3, RARb, RASSF1A
Ovary
p16, BRCA1, DAPK,
Pancreas
p16, MGMT, APC
Stomach
p16, MGMT, APC
Uterus
p16, p14ARF, hMLH1
BRCA1, breast carcinoma 1; GSTP1, glutathione S-transferase P1; DAPK, deathassociated protein kinase; CDH1, E-cadherin; TIMP-3, tissue inhibitor of metalloproteinase 3; MGMT, O6-methylguanine-DNA methyltransferase; APC, adenomatous polyposis coli; FHIT, fragile histidine triad; RAR, retinoic acid receptor beta; RASSF1A, RAS-association-domain family protein 1A.
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Hypomethylation of proto-oncogenes
Several studies have shown low DNA methylation of protooncogenes in cancer cells.40–42 For example, low DNA methylation of Raf, c-Myc, c-Fos, c-H-Ras, and c-K-Ras associated with neoplasia have been reported in rodent liver.42,43 Although some studies have identified hypomethylation of RAS in human cancers,44–46 other studies do not support these findings and some researchers have suggested that DNA methylation is irrelevant to RAS expression.40,47 A significant inverse correlation was found between methylation and the degree of expression of the BCL-2 gene in human B-cell chronic lymphocytic leukaemia.41 Hypomethylation of the third exon of the c-MYC gene has been reported in a various human cancers.48 However, differentiation between cause and effect is difficult, even in the few cases in which associated increased gene expression was documented.29
Epigenetically regulated genes as biomarkers Epigenetic changes have been reported in a several cancer types, but not all of them have been well characterised. Colorectal cancer
Colorectal cancer is an excellent model for studying the genetic basis of cancer—the disease progresses in an orderly manner with very distinct genetic alterations at each stage. Also, epigenetic studies of colorectal tumours have greatly increased our understanding of the associated molecular events in disease progression. Studies of the methylation of DNA in colorectal cancer have provided further information about the pathogenesis of the disease. Progressive methylation of DNA and subsequent silencing of a subset of genes occurs in normal tissues alongside age and time-dependent events which predispose these normal cells to neoplastic transformation. At late stages of disease progression, methylation of DNA is important in a subset of tumours that have epigenetic instability caused by the simultaneous silencing of many genes—this characteristic is called the CpG island methylator phenotype (CIMP).49 CIMP-positive cancers include the majority of tumours with sporadic mismatch-repair deficiency due to hypermethylation of the hMLH1 promoter and also include the majority of tumours with Ki-RAS mutations. In CIMP colorectal tumours, the highly methylated DNA interacts with genetic lesions. CIMP-negative cancers evolve along a more standard genetic-instability pathway, with high rates of p53 mutations and chromosomal changes. Elucidation of the combined epigenetic and genetic events that occur in colorectal cancer has provided a complete molecular understanding of this tumour type and this knowledge could have implications for the diagnosis, prognosis, and treatment of patients with this disease. Multistage carcinogenesis in the colon can be viewed as a series of pathways that are activated (or inactivated) in populations of cells that are selected on the basis of growth (or survival) advantages.49 In most colorectal tumours, the common pathways (which operate simultaneously) are the APC/beta-catenin pathway, which provides a growth advantage via the transcriptional activation of c-MYC and
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other genes; the senescence bypass pathway, which is required for immortalisation of cells possibly through the inactivation of the p53 or the p16 gene;50 and the TGF bypass pathway, which is needed to overcome the inhibition of growth caused by TGF through inactivation of TGF receptor II or activation of Ki-RAS.51 Another unknown pathway could be altered in ageing cells, causing the expansion of the proliferative zone and thus a predisposition to neoplastic transformation. In colon cancer, the specific pathway that is affected is more important than the genes that are altered, which partly explains the large diversity in molecular changes observed in this fairly uniform disease. Furthermore, chromosomal instability, microsatellite instability, and epigenetic instability (CIMP) can be viewed as simply the molecular mechanisms required to generate molecular diversity, but which are also prerequisites for the change from neoplasia to cancerous growth. Further clarification is needed about the underlying causes of chromosomal and epigenetic instability and about whether the type of instability required for tumour growth has clinical implications. Indeed, tumours with microsatellite instability seem to have a more favourable prognosis overall than those without this feature, and the responses to chemotherapy of the two types differ. There are still many unanswered questions, and further studies that integrate patterns of molecular instability, epidemiology, and outcome are needed. For instance, there is evidence that CIMP-negative tumours may have a worse prognosis than CIMP-positive ones because of the high rate of chromosomal changes and p53 mutations, the latter of which is a measure of chemosensitivity in the colon.52 Also, CIMP-positive tumours may be especially sensitive to therapy. This tumour type might also be a possible target for chemoprevention by the inhibition of methylation.53 Prostate adenocarcinoma
The aetiology of prostate cancer, a multifactorial disease, is not completely understood. Environmental, genetic, and epigenetic factors all modulate the expression of genes involved in prostate-cancer development.54,55 One gene that is used as a marker for prostate-cancer detection is GSTP1. GSTP1 is one of a superfamily of enzymes known as the glutathione-S-transferases (GST) that are involved in the detoxification of electrophilic compounds, such as carcinogens, by glutathione conjugation, thus protecting DNA from oxidative damage. Hypermethylation of the GSTP1 promoter is a highly prevalent event in prostate cancer; it is present in potential precursor lesions and is strongly linked to loss of GST expression.54 However, common somatic GSTP1 inactivation prevents any major effects from inherited genotypic variants in the progression of prostate cancer. Jeronimo and colleagues compared patients with prostate cancer (adenocarcinomas and prostatic intraepithelial neoplasia lesions) with a control group consisting of patients with benign prostatic hyperplasia (BPH) and healthy blood donors.55 Tissue samples from the patients with prostate cancer and from patients with BPH were analysed for hypermethylation of GSTP1. No significant effect on
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prostate-cancer risk was detectable for the GSTP1 genotype compared with the control group. Moreover, no association was found between this genotype and tumour or BPH methylation status. Patients with carcinomas that did not show hypermethylation of GSTP1 did not have significant differences in genotypic distribution from the control population. In patients with adenocarcinoma, a strong association between hypermethylation of the GSTP1 promoter and loss of GST expression was observed. However, this trend was not found in prostatic intraepithelial neoplasia or BPH lesions. Although the GSTP1 polymorphism is not associated with altered susceptibility to prostate cancer, somatic promoter hypermethylation is a significant, but not the only, cause of decreased GST function. Ulcerative colitis
Ulcerative colitis is an inflammatory disease of the mucosa and submucosa of the large intestine. The duration and extent to which a patient suffers from ulcerative colitis depend on the development of colorectal carcinoma. The frequency and timing of hypermethylation of the p14ARF gene in ulcerative colitis were determined in patients with ulcerative colitis of different histological stages: adenocarcinoma, dysplasia, non-neoplastic ulcerative colitis mucosae, and ulcerative-colitis-associated carcinogenesis.56 The results indicated that hypermethylation of p14ARF is a relatively common and early event in ulcerative-colitisassociated carcinogenesis. Barrett’s oesophageal cancer
Barrett’s oesophagus is the only known precursor to oesophageal adenocarcinoma. The incidence of oesophageal adenocarcinoma has increased rapidly in more developed countries. Lesions in the cyclin-dependent kinase 4 and 6 inhibitor gene, p16INK4a, occur frequently in oesophageal adenocarcinomas, but their role in neoplastic progression is not well understood. Inactivation of p16 by hypermethylation of the cyclin-dependent kinase (CDKN2A/p16) promoter and loss of heterozygosity of the CDKN2A gene could have a role in the neoplastic progression of Barrett’s oesophagus. Hypermethylation of the CDKN2A promoter in Barrett’s oesophageal adenocarcinomas, premalignant lesions, and normal oesophageal squamous-cell epithelium was studied by the use of methylation-sensitive, singlestrand conformation analysis and sequencing of bisulphitemodified DNA.57 No methylation of the CDKN2A promoter was found in normal oesophageal squamous-cell epithelia. Methylation was detected, however, in 82% of adenocarcinomas and in 30% of premalignant lesions (including in 33% of samples with intestinal metaplasia only). Loss of heterozygosity at the CDKN2A gene locus was found in 68% of adenocarcinomas and in 55% of premalignant lesions. Most samples showed hypermethylation of the CDKN2A promoter, with or without loss of heterozygosity. These results indicate that methylation of the CDKN2A promoter is the predominant mechanism for p16 inactivation. This hypermethylation is a very common event in oesophageal adenocarcinoma and occurs as early as metaplasia.
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frequently methylated. Methylation of these genes correlates with other indices of prognosis and survival. For example, CDH1 methylation-positive status is associated with poor survival.
Transcription
Gastric cancer Relaxed chromatin Transcription factor complex No transcription
v Compact chromatin 5-aza-deoxycytidine
Methylation
Trichostatin A
Methylation binding protein
DNA methyl transferase
Histone deacetylase
Loss of control of the cyclo-oxygenase 2 (COX2) gene, via changes in methylation of the COX2 promoter, may have important consequences in gastric carcinogenesis. Overexpression of COX2 is strongly associated with loss of apoptosis and enhancement of proliferation in the gastrointestinal tract, especially in gastrointestinal Advances in epithelial cells.34 quantification of methylation and high-throughput technology are needed to elucidate further the mechanisms of this cancer Lung cancer
Lung cancer is the most common cause of death from cancer in the USA and kills more than 156 000 people there alone every year. Significant progress has been made during the past 20 years in understanding of the molecular and cellular pathogenesis of lung cancer. Abnormalities in proto-oncogenes, genetic and epigenetic changes in tumour suppressor genes, the role of angiogenesis in the multistage development of the disease, and detection of molecular abnormalities in preinvasive respiratory-tract lesions have been the focus of recent research. Virmani and co-workers developed a panel of markers for aberrant methylation that detects lung cancer at the early stages of development.58 This panel includes the genes: RAR, DAPK, GSTP1, FHIT, RASSF1, MGMT, E-cad, APC, and p16. In particular, the researchers found that RAR and the newly discovered tumour suppressor gene RASSF1 (located in the region 3p21·3) are deleted in many lung and breast cancers and are very methylated in very many small-cell cancers. They also found that APC is methylated in non-small cell cancers.
Figure 3. Inhibition of gene transcription by inhibitors of methyltransferase and histone acetyltransferase.
New approaches including advancements in chip technology, are needed for quantitative high-throughput research and for validation of different markers for riskassessment screening of large populations. Laird and colleagues used a MethylLight assay to analyse a panel of 20 genes from 104 tissue specimens from 51 patients with different stages of Barrett’s oesophagus and associated adenocarcinoma. Histologically identified clinical samples were studied, and epigenomic fingerprints for the different histological stages of oesophageal carcinoma were compared. Oesophageal adenocarcinoma arises through different stages; for example, squamous mucosa, columnar epithelium, Barrett’s oesophagus, dysplasia, and malignancy. The MethylLight study found distinct classes of methylation patterns in the different types of tissue. The frequency of methylation ranged from 15% (CDKN2A) to 60% (MGMT). Altogether, three classes were identified according to the absence (CDKN2A, ESR1, and MYOD1) or presence (CALCA, MGMT, and TIMP3) of methylation in normal oesophageal mucosa and the stomach or the infrequent methylation of normal oesophageal mucosa accompanied by methylation in all normal stomach samples (APC). In other genes, the frequency of hypermethylation was below 5% (ARF, CDH1, CDKN2B, GSTP1, MLH1, PTGS2, and THBS1) or it was completely absent (CTNNB1, RB1, TGFBR2, and TYMS1). Methylation patterns of specific genes at different stages of disease progression can be used as markers, and the degree of methylation reflects the stage of the disease. Bladder cancer
Many genes are methylated during the development of bladder cancer; CDH1, RASSF1A, APC, and CDH13 are
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Lymphoma and leukaemia
Hypermethylation of promoters of several viral and cellular genes is well known to result in silencing of transcription.4,59 For example, transcriptional inactivation of the Epstein-Barr virus (EBV) latency gene Wp due to hypermethylation of its promoter suggests that hypermethylation is one of the mechanisms for regulating genes involved in interaction between infectious agent and host. EBV and a few other viruses are strongly associated with different types of cancer including lymphoma and leukaemia.60 Takacs and colleagues analysed the methylation patterns of CpG dinucleotides in latent membrane protein 1 (LMP1) regulatory sequences, a bidirectional promoter region of latent EBV genomes, by use of bisulphite-induced modification of DNA followed by automated fluorescence genomic sequencing.3 Transcripts
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for two latent membrane proteins, LMP1 (a transforming protein) and LMP2B, are initiated in this region in opposite directions. The results showed that B-cell lines and a clone expressing LMP1 carried EBV genomes with unmethylated or hypomethylated LMP1 regulatory sequences, whereas highly methylated CpG dinucleotides were present at each position or at discrete sites within hypermethylated regions in LMP1-negative cells. Comparison of high-resolution methylation maps suggests that CpG methylation directly interferes with the binding of nuclear factors LBF2, LBF3, LBF5, LBF6, LBF7, and AML1/LBF1. The maps also show that methylation of CpGs within an E-box sequence (where activators as well as repressors can bind) is not the major mechanism in silencing the LMP1 promoter. Factors that recruit methyltransferase to the CpG islands differ from regular transcription factors. Hypermethylation of LMP1 regulatory sequences or regions within these sequences in LMP1-negative cells suggest a role for an indirect mechanism in silencing the LMP1 promoter, via methylcytosine-binding proteins.60 Kaposi’s sarcoma
Kaposi’s sarcoma-associated herpesvirus (KSHV) is strongly linked to Kaposi’s sarcoma, primary effusion lymphomas, and a subset of multicentric Castleman’s disease. The mechanism by which this virus establishes latency and reactivation is not understood completely.61,62 The KSHV lytic transactivator (Lyta), mainly encoded by the ORF50 gene, is a lytic switch for viral reactivation from latency. It is essential for keeping the virus alive and for maintaining the latency. Chen and colleagues showed that the Lyta promoter region was heavily methylated in latently infected cells.62 The researchers then reversed the methylation of Lyta promoter by treating primary-effusion lymphoma-delivered cell lines with tetradecanoylphorbol acetate, promoting the lytic phase of KSHV. This experiment shows that demethylation of the Lyta promoter region is essential for the expression of Lyta. In samples from a latently infected KSHV carrier, this region remained methylated, which suggests a relation among a demethylation in the Lyta promoter, the reactivation of KSHV, and the development of KSHVassociated diseases. Epigenetic regulation has been observed in other cancer-associated viruses such as hepatitis B virus and papillomavirus. Studies of the mechanisms of interactions between host and pathogen could provide molecular targets for intervention.
mice have shown that inhibition of DNA methylation can suppress tumour initiation, which suggests that this approach might be useful as a preventive strategy. Cytosine analogues are being used to treat preneoplastic syndromes in clinical trials. Trials of antisense oligodeoxynucleotides against methyltransferase have shown a decrease in carcinogenesis. Although these studies have yielded promising, preliminary clinical information, a relation between clinical efficacy and target-gene demethylation has not yet been found. However, further studies in this area should clarify this situation. The reactivation of epigenetically silenced genes has therapeutic benefits.66–68 Indeed, azacytidine and its derivatives have been used in the clinic with beneficial results.69 The mechanisms underlying these effects, however, have not been fully elucidated. The doses of 5-azacytidine currently used are toxic, possibly due to effects not directly related to demethylation. The recent observation that low doses of azacytidine can facilitate reactivation of hypermethylated genes with inhibitors of histone deacetylase has created much interest. Trials of a clinically available histone deacetylase inhibitor, phenylbutyrate, combined with low doses of azacytidine are in the planning stages. These trials will include haemopoietic and solid tumours, and the researchers will monitor gene-reactivation events. Whether such an approach will specifically reactivate genes for better control of the neoplastic process or whether other genes will be affected, resulting in toxic effects in normal cells is not known. The initial results might justify further development of this therapeutic concept and further increase interest in clinical aspects of epigenetics in cancer.70–73 Mintz and Debinski have suggested immune-based therapies for Xchromosome-linked malignant glioma in which epigenetic and genetic regulation have a significant role.74
Summary Defininition of how hypermethylation of promoter regions participate in gene silencing and how loss of methylation alters chromosome structure is difficult. Nevertheless, evidence of abnormal methylation of DNA in cancer cells has potential importance in the clinical setting. One possible therapeutic approach is the use of CpG hypermethylation events as tumour biomarkers. The changes in methylation in promoter regions potentially provide a positive signal for cancer cells that can be detected by conventional techniques. On the basis of recent research, use of multiple markers for predicting one tumour type
Therapeutic potential of epigenetic changes Epigenetic changes are potentially good therapeutic targets because of their reversibility (figure 3). Markers for aberrant methylation may also be very useful for monitoring of the onset and progression of cancer. Treatment with inhibitors of DNA methylation can restore the activities of dormant genes such as CDKN2A and decrease the growth rate of cancer cells in a heritable manner. Similarly, the capacity of cells to repair DNA can be restored by activation of MLH1. Partial reversing the cancer phenotype by the use of methylation inhibitors should, therefore, be possible.63–65 Experiments with Min
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Search strategy and selection criteria Data for this review were identified by searches of Current Contents, the National Library of Medicine database, EndNotes, Pubmed with the search terms: “acetyl transferase”, “biomarker”, “cancer”, “cancer prevention”, “chromatin”, “chromatin modelling”, “chromosomal organisation”, “deacetylase”, “DNA damage”, “DNA repair”, “early detection of cancer”, “epigenome”, “epigenetics”, “histone”, “methylation”, “microarray”, “oncogene”, “risk assessment”, “tumour suppressor gene”, “virus”. Only papers published in English between 1983 and 2002 were selected.
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seems reasonable. However, the findings must be validated by trials with large sample sizes and clinical evidence to correlate epigenetic changes with clinical outcome. Since almost every tumour type seems to have many independent promoter-hypermethylation events, reasonably sized marker panels might be constructed for each tumour type to provide indices for early detection and monitoring of cancer risk. Early detection of cancer will allow therapeutic interventions to be applied to inhibit further development of cancer. Such information could also be used in the development of strategies to prevent cancer. Conflict of interest
None declared. References
1 Baylin SB. Tying it all together: epigenetics, genetics, cell cycle, and cancer. Science 1997; 277: 1948–49. 2 Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002; 3: 415–28. 3 Takacs M, Salamon D, Myohanen S, et al. Epigenetics of latent Epstein-Barr virus genomes: high resolution methylation analysis of the bidirectional promoter region of latent membrane protein 1 and 2B genes. Biol Chem 2001; 382: 699–705. 4 Tierney RJ, Kirby HE, Nagra JK, et al. Methylation of transcription factor binding sites in the Epstein-Barr virus latent cycle promoter Wp coincides with promoter down-regulation during virus-induced B-cell transformation. J Virol 2000; 74: 10468–79. 5 Robertson KD. The role of DNA methylation in modulating Epstein-Barr virus gene expression. Curr Top Microbiol Immunol 2000; 249: 21–34. 6 Tao Q, Swinnen LJ, Yang J, et al. Methylation status of the Epstein-Barr virus major latent promoter C in iatrogenic B cell lymphoproliferative disease: application of PCR-based analysis. Am J Pathol 1999; 155: 619–25. 7 Hagan CR, Rudin CM. Mobile genetic element activation and genotoxic cancer therapy: potential clinical implications. Am J Pharmacogenomics 2002; 2: 25–35. 8 Nelson WG, De Marzo AM, Deweese TL, et al. Preneoplastic prostate lesions: an opportunity for prostate cancer prevention. Ann N Y Acad Sci 2001; 952: 135–44. 9 Rideout WM III Eggan K, Jaenisch R. Nuclear cloning and epigenetic reprogramming of the genome. Science 2001; 293: 1093–98. 10 Nelson WG, De Marzo AM, DeWeese TL. The molecular pathogenesis of prostate cancer: implications for prostate cancer prevention. Urology 2001; 57 (Suppl 1): 39–45. 11 Howell CY, Bestor TH, Ding F, et al. Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene. Cell 2001; 104: 829–38. 12 El-Osta A, Wolffe AP. DNA methylation and histone deacetylation in the control of gene expression: basic biochemistry to human development and disease. Gene Expr 2000; 9: 63–75. 13 Zuccotti M, Garagna S, Redi CA. Nuclear transfer, genome reprogramming and novel opportunities in cell therapy. J Endocrinol Invest 2000; 23: 623–29. 14 Feinberg AP. Cancer epigenetics takes center stage. Proc Natl Acad Sci USA 2001; 98: 392–4. 15 Feinberg AP. DNA methylation, genomic imprinting and cancer. Curr Top Microbiol Immunol 2000; 249: 87–99. 16 Lengauer C, Issa JP. The role of epigenetics in cancer: DNA methylation, imprinting and the epigenetics of cancer—an American Association for Cancer Research special conference. Las Croabas, Puerto Rico, 12–16 December, 1997. Mol Med Today 1998; 4: 102–03. 17 Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet 1999; 21: 163–67. 18 Hu L, Troyanovsky B, Zhang X, et al. Differences in the immunogenicity of latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus genomes derived from LMP1-positive and negative nasopharyngeal carcinoma. Cancer Res 2000; 60: 5589–93. 19 Rice JC, Allis CD. Histone methylation versus histone acetylation: new insights into epigenetic regulation. Curr Opin Cell Biol 2001; 13: 263–73.
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20 Jenuwein T, Allis CD. Translating the histone code. Science 2001; 293: 1074–80. 21 Kruhlak MJ, Hendzel MJ, Fischle W, et al. Regulation of global acetylation in mitosis through loss of histone acetyltransferases and deacetylases from chromatin. J Biol Chem 2001; 276: 38307–19. 22 Surani MA. Genetics: immaculate misconception. Nature 2002; 416: 491–93. 23 Surani MA. Imprinting and the initiation of gene silencing in the germ line. Cell 1998; 93: 309–12. 24 Kinzler KW, Vogelstein B. Landscaping the cancer terrain. Science 1998; 280: 1036–37. 25 Baylin SB, Herman JG. DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 2000; 16:168–74. 26 Costa M, Klein CB. Nickel carcinogenesis, mutation, epigenetics, or selection. Environ Health Perspect 1999; 107: A438–39. 27 Breivik J, Gaudernack G. Genomic instability, DNA methylation, and natural selection in colorectal carcinogenesis. Semin Cancer Biol 1999; 9: 245–54. 28 Breivik J, Gaudernack G. Carcinogenesis and natural selection: a new perspective to the genetics and epigenetics of colorectal cancer. Adv Cancer Res 1999; 76: 187–212. 29 Laird PW, Jaenisch R. The role of DNA methylation in cancer genetic and epigenetics. Annu Rev Genet 1996; 30: 441–64. 30 Esteller M, Catasus L, Matias-Guiu X, et al. hMLH1 promoter hypermethylation is an early event in human endometrial tumorigenesis. Am J Pathol 1999; 155: 1767–72. 31 Esteller M, Hamilton SR, Burger PC, et al. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 1999; 59: 793–97. 32 Esteller M, Sanchez-Cespedes M, Rosell R, et al. Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer Res 1999; 59: 67–70. 33 Tycko B. Epigenetic gene silencing in cancer. J Clin Invest 2000; 105: 401–07. 34 Akhtar M, Cheng Y, Magno RM, et al. Promoter methylation regulates helicobacter pylori-stimulated cyclooxygenase-2 expression in gastric epithelial cells. Cancer Res 2001; 61: 2399–403. 35 Ahluwalia A, Yan P, Hurteau JA, et al. DNA methylation and ovarian cancer I: analysis of CpG island hypermethylation in human ovarian cancer using differential methylation hybridization. Gynecol Oncol 2001; 82: 261–68. 36 Ahluwalia A, Hurteau JA, Bigsby RM, Nephew KP. DNA methylation in ovarian cancer. II: expression of DNA methyltransferases in ovarian cancer cell lines and normal ovarian epithelial cells. Gynecol Oncol 2001; 82: 299–304. 37 Ng HH, Zhang Y, Hendrich B, et al. MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat Genet 1999; 23: 58–61. 38 Wade PA, Gegonne A, Jones PL, et al. Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation. Nat Genet 1999; 23: 62–66. 39 Nan BC, Shao DM, Chen HL, et al. Alteration of Nacetylglucosaminyltransferases in pancreatic carcinoma. Glycoconj J 1998; 15: 1033–37. 40 Barbieri R, Mischiati C, Piva R, et al. DNA methylation of the Haras-1 oncogene in neoplastic cells. Anticancer Res 1989; 9: 1787–91. 41 Hanada M, Delia D, Aiello A, et al. Bcl-2 gene hypomethylation and high-level expression in B-cell chronic lymphocytic leukemia. Blood 1993; 82: 1820–28. 42 Ray JS, Harbison ML, McClain RM, Goodman JI. Alterations in the methylation status and expression of the raf oncogene in phenobarbital-induced and spontaneous B6C3F1 mouse live tumors. Mol Carcinog 1994; 9: 155–66. 43 Rao PM, Antony A, Rajalakshmi S, Sarma DS. Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver. Carcinogenesis 1989; 10: 933–37. 44 Vachtenheim J, Horakova I, Novotna H. Hypomethylation of CCGG sites in the 3’ region of H-ras protooncogene is frequent and is associated with H-ras allele loss in non-small cell lung cancer. Cancer Res 1994; 54: 1145–48. 45 Feinberg AP, Vogelstein B. Hypomethylation of ras oncogenes in primary human cancers. Biochem Biophys Res Commun 1983; 111: 47–54. 46 Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of
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47 48 49 50 51 52
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some human cancers from their normal counterparts. Nature 1983; 301: 89–92. Chandler LA, DeClerck YA, Bogenmann E, Jones PA. Patterns of DNA methylation and gene expression in human tumor cell lines. Cancer Res 1986; 46: 2944–49. Stephenson J, Akdag R, Ozbek N, Mufti GJ. Methylation status within exon 3 of the c-myc gene as a prognostic marker in myeloma and leukaemia. Leuk Res 1993; 17: 291–93. Issa JP. The epigenetics of colorectal cancer. Ann N Y Acad Sci 2000; 910: 140–53; 153–55. Yeager TR, DeVries S, Jarrard DF, et al. Overcoming cellular senescence in human cancer pathogenesis. Genes Dev 1998; 12: 163–74. Markowitz S, Wang J, Myeroff L, et al. Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science 1995; 268: 1336–38. Boland CR, Thibodeau SN, Hamilton SR, et al. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 1998; 58: 5248–57. Zheng M, Wang H, Zhang H, et al. The influence of the p53 gene on the in vitro chemosensitivity of colorectal cancer cells. J Cancer Res Clin Oncol 1999; 125: 357–60. Jeronimo C, Varzim G, Henrique R, et al. I105V polymorphism and promoter methylation of the GSTP1 gene in prostate adenocarcinoma. Cancer Epidemiol Biomarkers Prev 2002; 11: 445–50. Jeronimo C, Usadel H, Henrique R, et al. Quantitation of GSTP1 methylation in non-neoplastic prostatic tissue and organ-confined prostate adenocarcinoma. J Natl Cancer Inst 2001; 93: 1747–52. Sato F, Harpaz N, Shibata D, et al. Hypermethylation of the p14 (ARF) gene in ulcerative colitis-associated colorectal carcinogenesis. Cancer Res 2002; 62: 1148–51. Eads CA, Lord RV, Wickramasinghe K, et al. Epigenetic patterns in the progression of esophageal adenocarcinoma. Cancer Res 2001; 61: 3410–18. Virmani AK, Tsou JA, Siegmund KD, et al. Hierarchical clustering of lung cancer cell lines using DNA methylation markers. Cancer Epidemiol Biomarkers Prev 2002; 11: 291–97. Salamon D, Takacs M, Ujvari D, et al. Protein-DNA binding and CpG methylation at nucleotide resolution of latency-associated promoters Qp, Cp, and LMP1p of Epstein-Barr virus. J Virol 2001; 75: 2584–96. Verma M, Lambert PF, Srivastava SK. Meeting highlights: National Cancer Institute workshop on molecular signatures of infectious agents. Dis Markers 2001; 17: 191–201.
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61 Laman H, Boshoff C. Is KSHV lytic growth induced by a methylation-sensitive switch? Trends Microbiol 2001; 9: 464–66. 62 Chen J, Ueda K, Sakakibara S, et al. Activation of latent Kaposi’s sarcoma-associated herpesvirus by demethylation of the promoter of the lytic transactivator. Proc Natl Acad Sci USA 2001; 98: 4119–24. 63 Worm J, Guldberg P. DNA methylation: an epigenetic pathway to cancer and a promising target for anticancer therapy. J Oral Pathol Med 2002; 31: 443–49. 64 Christman JK. 5-Azacytidine and 5-aza-2’-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 2002; 21: 5483–95. 65 Brown R, Strathdee G. Epigenomics and epigenetic therapy of cancer. Trends Mol Med 2002; 8 (Suppl 4): S43–48. 66 Sirchia SM, Ren M, Pili R, et al. Endogenous reactivation of the RARbeta2 tumor suppressor gene epigenetically silenced in breast cancer. Cancer Res 2002; 62: 2455–61. 67 Lin X, Asgari K, Putzi MJ, et al. Reversal of GSTP1 CpG island hypermethylation and reactivation of pi-class glutathione S-transferase (GSTP1) expression in human prostate cancer cells by treatment with procainamide. Cancer Res 2001; 61: 8611–16. 68 Suh SI, Pyun HY, Cho JW, et al. 5-Aza-2’-deoxycytidine leads to down-regulation of aberrant p16INK4a RNA transcripts and restores the functional retinoblastoma protein pathway in hepatocellular carcinoma cell lines. Cancer Lett 2000; 160: 81–88. 69 Momparler RL, Cote S, Eliopoulos N. Pharmacological approach for optimization of the dose schedule of 5-Aza-2’-deoxycytidine (decitabine) for the therapy of leukemia. Leukemia 1997; 11 (Suppl 1): S1–6. 70 Piekarz RL, Robey R, Sandor V, et al. Inhibitor of histone deacetylation, depsipeptide (FR901228), in the treatment of peripheral and cutaneous T-cell lymphoma: a case report. Blood 2001; 98: 2865–68. 71 Bovenzi V, Momparler RL. Antineoplastic action of 5-aza-2’deoxycytidine and histone deacetylase inhibitor and their effect on the expression of retinoic acid receptor beta and estrogen receptor alpha genes in breast carcinoma cells. Cancer Chemother Pharmacol 2001; 48: 71–76. 72 Kikuchi T, Toyota M, Itoh F, et al. Inactivation of p57KIP2 by regional promoter hypermethylation and histone deacetylation in human tumors. Oncogene 2002; 21: 2741–49. 73 Kikuchi T, Itoh F, Toyota M, et al. Aberrant methylation and histone deacetylation of cyclooxygenase 2 in gastric cancer. Int J Cancer 2002; 97: 272–77. 74 Mintz A, Debinski W. Cancer genetics/epigenetics and the X chromosome: possible new links for malignant glioma pathogenesis and immune-based therapies. Crit Rev Oncog 2000; 11: 77–95.
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Epigenetics and cancer
Epigenetics in cancer: implications for early detection and prevention Mukesh Verma and Sudhir Srivastava
Knowledge of the molecular events that occur during the early stages of cancer has advanced rapidly. The initiation and development of cancer involves several molecular changes, which include epigenetic alterations. Epigenetics is the study of modifications in gene expression that do not involve changes in DNA nucleotide sequences. Modifications in gene expression through methylation of DNA and remodelling of chromatin via histone proteins are believed to be the most important of the epigenetic changes. The study of epigenetics offers great potential for the identification of biomarkers that can be used to detect and diagnose cancer in its earliest stages and to accurately assess individual risk. There has been a recent surge of interest among researchers as variations in the methylation of DNA have been shown to be the most consistent molecular changes in many neoplasms. An important distinction between a genetic and an epigenetic change in cancer is that epigenetic changes can be reversed more easily by use of therapeutic interventions. The discovery of these basic premises should stimulate much future research on epigenetics. Lancet Oncol 2002; 3: 755–63
Epigenetics has an important role in biological research and affects many different areas of study including cancer biology,1,2 viral latency,3–6 activity of mobile elements,7 somatic gene therapy,8–13 cloning and transgenic technologies, genomic imprinting,14,15 and developmental abnormalities.14,15 The definition of epigenetics varies among investigators. One definition describes epigenetics as the study of mitotically heritable changes in gene expression that are not caused by alteration of the DNA sequence.16 Another view is that epigenetics concerns the inheritance of information on the basis of differential gene expression, whereas genetics focuses on the information inherited through gene sequence.17 The mechanism of inheritance for epigenetic modification has yet to be identified. However, regardless of the definition used, the most important difference between an epigenetic mechanism and a genetic mechanism is that epigenetic changes can be reversed by chemical agents. Two major steps in the epigenetic regulation of gene expression are the deacetylation of histones, causing a change in the structure of chromatin, and the methylation of the promoter region of the gene (figures 1 and 2). Methylation is needed for the normal development of cells, and aberrant methylation confers a selective growth advantage that results in cancerous growth.18 The promoter regions of many genes can be methylated in areas called CpG
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Unmethylated CpG
Methylated CpG Promoter
Hypermethylation
Hypomethylation
STOP
START
Silencing of tumour suppressor gene
Activation of proto-oncogene
Figure 1. Epigenetic regulation of gene expression by methylation.
islands; genes under the control of highly methlyated promoters are “silenced”, which means they cannot be transcribed. In this review we discuss the genes that are deregulated by epigenetic mechanisms and could be used as potential biomarkers for cancer progression. We also discuss strategies to prevent these epigenetic events.
Molecular mechanisms involved in epigenetic changes Two major mechanisms in the epigenetic regulation of genes involve changes in the structure of chromatin and methylation of DNA. Chromosomal organisation and histones
The DNA of all eukaryotes is packaged into chromatin, which is made up of histone proteins around which the DNA is coiled. Histones have amino-termial extensions called ‘tails’, which undergo many covalent modifications that are important in both the organisation of chromosomes and the regulation of specific genes. Histone methyltransferases direct site-specific methylation of aminoacid residues such as MV is Program Director and SS is Program Director and Chief of the Cancer Biomarker Research Group at the National Cancer Institute, MD, USA. Correspondence: Dr Mukesh Verma, Cancer Biomarkers Research Group, Division of Cancer Prevention, National Cancer Institute, National Institutes of Health, Executive Plaza North, Room 3144, 6130 Executive Boulevard, MSC 7346, Bethesda, MD 20892-7346, USA. Tel: +1 301 496 3893. Fax: +1 301 402 8990. Email: [email protected]
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Factors: Environmental Hormonal Genetic
Deacetylation inhibitor: Trichostatin Methylation inhibitors: 5-deoxyazacytidine Zebularine
Epigenetics and cancer
Epigenetics regulates: Cell-cycle control DNA damage Apoptosis Invasion X-chromosome inactivation Imprinting Ageing
Hypermethylation APC BRCA1 ER hMLH1 GSTP1 TIMP3 DAPK1 Hypomethylation RAF c-MYC c-FOS c-HA-RAS c-K-RAS
DNA methyl transferases: DNMT1 DNMT3A DNMT3B
Acetylation/deacetylation Histone acetyltransferase Histone deacetylase (Dynamic equilibrium)
Methionine supplement: S-adenosylmethione
Figure 2. Epigenetic targets in cancer detection and risk assessment. The red arrow indicates down-regulation of genes. The green arrow indicates upregulation of genes.
Lys4 and Lys9 in the tail of the histone protein H3. Methylation of Lys4 is important for the maintenance of the structure of euchromatic domains, which are diffuse areas of chromatin where genes are freely accessible and generally active. By contrast, methylation of H3 Lys9 is associated with the initiation and propagation of heterochromatic domains where chromatin is densely packed and the genes are generally inactive.19 The dense, heterochromatic domains are flanked by inverted repeats that act as boundary elements which prevent the tightly packed structure spreading to neighbouring euchromatic regions. Despite the excitement over the mapping of the human genome, many challenges remain in understanding the regulation and transduction of the information contained in genomic DNA.20 At the time of the completion of the Human Genome Project, the expected number of active genes was about 30 000—twice the number of genes coded by a small organism such as Drosophila melanogaster. However, an obvious question is raised by these findings: is DNA alone responsible for generating the full range of information that results in a complex eukaryotic organism such as a human being? Epigenetic changes, at the level of histones, are a crucial feature of a genome-wide mechanism of information storage and retrieval that is only just beginning to be understood. Jenuwein and Allis proposed the concept of a histone code that could substantially extend the information potential of the genetic code.20 There is evidence that histone proteins
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and their associated covalent modifications contribute to a mechanism that can alter the structure of chromatin. Such changes could lead to inherited differences in transcriptional activation (or inactivation) states or to the stable propagation of chromosomes by a specialised centromere higher-order structure. Histone acetylation is a dynamic process that is regulated by two classes of enzymes: the histone acetyltransferases and histone deacetylases. Although many recent studies have focused on promoter-specific acetylation and deacetylation, these mechanisms are part of a broader, more dynamic acetylation mechanism that profoundly affects many nuclear processes. Kruhlak and colleagues observed this broader histone acetylation in cells entering and leaving mitosis.21 In contrast to the hypothesis that acetyltransferases and deacetylases stay bound to mitotic chromosomes to provide an epigenetic imprint for postmitotic activation of the genome, these researchers found that the enzymes are spatially reorganised and displaced from condensing chromosomes when cells undergo division (mitosis). During mitosis, histone acetyltransferases and deacetylases are unable to acetylate or deacetylate chromatin in situ despite remaining catalytically active when isolated from mitotic cells and assayed in vitro. Thus, these enzymes do not stably bind to the genome to function as an epigenetic mechanism of selective postmitotic gene activation. However, evidence does support a role for spatial organisation of these enzymes within the nucleus. Furthermore, their relation to
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euchromatin and heterochromatin postmitotically in the reactivation of the genome also is important for the active organised structure. DNA methylation
In the normal mammalian genome, methylation occurs only at cytosines 5' to guanosines at CpG dinucleotides. Many CpG dinucleotides have been depleted from the eukaryotic genome throughout evolution by spontaneous deamination. The remaining CpGs have a very high frequency of methylation, which facilitates changes in chromatin structure that block the transcription of particular genes, by rendering them inaccessible to cellular transcription machinery. However, throughout the genome short stretches of CpGrich DNA exists. These regions, known as CpG islands, are not highly methylated despite being rich in cytosine and guanine, and are generally located in the promoter regions of “housekeeping” genes which are essential for cell function. The lack of methylation in promoter regions may be a prerequisite for active transcription of the genes under their control. Only two exceptions to this rule have been reported: fully methylated CpG islands in the silenced allele for specific imprinted autosomal genes and multiple silenced genes on the female inactivated X chromosome.22,23 Another emerging concept is that deacetylation of histone proteins is the first step in the recruitment of methyltransferase to the CpG islands, resulting in hypermethylation of the promoter. For cancer prevention strategies to be developed, factors that regulate deacetylation have to be identified. Extensive research is needed in this area, especially because targets for chemoprevention could emerge from these studies. Furthermore, epigenetic regulations occur early in cancer progression, thus providing an opportunity for the development of interventions to prevent further progression. Genetic changes are the defining feature of neoplastic evolution, as suggested by the analysis of chromosomal structure and determination of the chromosomal number.24 On the basis of current knowledge, the role of epigenetics in cancer development is considered to be minor compared with the role of genetic events.25 Recent data, however, have suggested alternative approaches, and research has consequently begun to focus on the contribution of epigenetics to tumorigenesis. Cancer seems to be a process that occurs because of mutations in DNA and epigenetic mechanisms. These processes can be complementary; thus one can predispose the other to induce cancer. Factors affecting epigenetic regulation of genes
Various factors can modify mammalian cells resulting in an epigenetically transformed phenotype, without changing the DNA sequence information of the cell, including radiation, tobacco smoke, stress, hormones (such as oestradiol), base analogues, cadmium, arsenic, nickel, reactive oxygen species, and various other chemicals. Costa and Klein investigated the mechanism of nickel carcinogenesis, focusing primarily on the epigenetic changes associated with the exposure of cells to carcinogenic nickel compounds.26 They studied the
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distribution of nickel in the cell and compared the genetic and epigenetic changes that occurred. The epigenetic effects included alterations in the concentrations of transcription factors, such as activating transcription factor 1, p53, hypoxia-inducible factor 1␣, and nuclear factor . They also investigated the relation between nickel and calcium metabolism, the effect of reactive oxygen species, and the interactions of nickel with proteins. They concluded that nickel induces various signalling pathways as well as genes that seem to be important for the survival of cancer cells. They also observed that the same genes induced or repressed by nickel are overexpressed or not expressed in nickel-transformed cells, which may represent an important selection process in the mechanism of nickel carcinogenesis. In carcinogenesis research, there is a growing understanding that neoplastic transformation must be viewed as an environmental process.26 Breivik and Gaudernack have developed a model that integrates evolution and carcinogenesis at the molecular level.27,28 This model offers new perspectives, including that of epigenetics, on carcinogenesis and relates the neoplastic process to the basic evolutionary concept of biology. Nutrients can also affect the epigenetic regulation of cancer-associated genes. Altered methionine metabolism is a characteristic trait of malignant cells;29 normal cells can use homocysteine as a direct methionine precursor, whereas transformed cells tend to require methionine in their growth medium. The cycle begins with the conversion of S-adenosylmethionine to S-adenosylhomocysteine by the donation of its methyl group to a methyl acceptor. S-adenosylhomocysteine is then broken down to yield homocysteine and adenosine. Methionine is regenerated from homocysteine by a reaction dependent on folate and cobalamin. The cycle is completed by the conversion of methionine to S-adenosylmethionine. The methionine dependence occurs because of increased transmethylation, not because of decreased methionine biosynthesis. Finally, there is a lower than normal availability of free methionine in transformed cells. Low rations of S-adenosylmethionine to S-adenosylhomocysteine, which were reversible by treatment with S-adenosylmethionine, have been observed in the early stages of liver carcinogenesis.29
Genes that are deregulated by the epigenetic process Several genes have been identified in different cancers that are regulated by the epigenetic process (panel 1). These genes are involved at different stages in the division, differentiation, and proliferation of cells (figure 2). Hypermethylation of tumour suppressor genes
Many tumour supressor genes are inactivated in particular tumour types have highly methylated promoters; under normal conditions, the promoter regions are unmethylated and the gene is transcribed.17,30–36 Genes affected by these modifications include RASSF1, RARbeta, DAPK, p16, p15, MGMT, and GSTP1 in lung cancer; CDKN2A, CALCA, MGMT, and TIMP3p in oesophageal cancer; 14ARF in
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ulcerative colitis; GSTP1 in prostate cancer; and HIC1 and p53 in breast cancer. Whether methylation is the initiating or secondary event in gene silencing has not been established. However, irrespective of its role in the initiation of cancer development, methylation is an important marker for epigenetically mediated loss-of-gene function. Furthermore, these events are of comparable importance to gene mutations for the initiation and propagation of carcinogenesis. Although the functional importance of hypermethylation is apparent, the molecular mechanisms involved are still unclear. The integration of DNA methylation with chromatin organisation and the regulation of histone acetylation and deacetylation may be an important parts of the overall effect.25 However, the involvement of as yet unidentified proteins cannot be ruled out. Proteins that bind preferentially to methylated cytosines in DNA and participate in complexes that contain active histone deacetylases have recently been identified. These proteins, which include methylated DNA-binding protein MECP2 and methyl CpG-binding domain proteins MBD2 and MBD3, provide an important clue to the proposed association of methylation with transcriptionally repressive chromatin.37,38 Furthermore, these proteins contain the histone deacetylases (1 and 2) and associated proteins such as transcriptional corepressors. The existence of such protein complexes suggests that DNA methylation can target chromatin which actively represses gene expression by remodelling and disturbing the dynamic equilibrium of histone acetylase and deacetylase.39 Multiple pathways could be involved throughout the entire process.
Panel 1. Cancer types and genes regulated by epigenetic mechanisms Cancer type
Genes
Breast
p16, BRCA1, GSTP1, DAPK, CDH1, TIMP-3
Brain
p16, p14ARF, MGMT, TIMP-3
Bladder
p16, DAPK, APC
Colon
p16, p14ARF, MGMT, hMLH1, DAPK, TIMP-3, APC
Oesophagus
p16, p14ARF, GSTP1, CDH1, APC
Head and neck
p16, MGMT, DAPK
Kidney
p16, p14ARF, MGMT, GSTP1, TIMP-3, APC
Leukaemia
p15, MGMT, DAPK1, CDH1, p73
Liver
p16, GSTP1, APC
Lymphoma
p16, p15, MGMT, DAPK, p73
Lung
p16, p14ARF, MGMT, GSTP1, DAPK, FHIT, TIMP-3, RARb, RASSF1A
Ovary
p16, BRCA1, DAPK,
Pancreas
p16, MGMT, APC
Stomach
p16, MGMT, APC
Uterus
p16, p14ARF, hMLH1
BRCA1, breast carcinoma 1; GSTP1, glutathione S-transferase P1; DAPK, deathassociated protein kinase; CDH1, E-cadherin; TIMP-3, tissue inhibitor of metalloproteinase 3; MGMT, O6-methylguanine-DNA methyltransferase; APC, adenomatous polyposis coli; FHIT, fragile histidine triad; RAR, retinoic acid receptor beta; RASSF1A, RAS-association-domain family protein 1A.
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Hypomethylation of proto-oncogenes
Several studies have shown low DNA methylation of protooncogenes in cancer cells.40–42 For example, low DNA methylation of Raf, c-Myc, c-Fos, c-H-Ras, and c-K-Ras associated with neoplasia have been reported in rodent liver.42,43 Although some studies have identified hypomethylation of RAS in human cancers,44–46 other studies do not support these findings and some researchers have suggested that DNA methylation is irrelevant to RAS expression.40,47 A significant inverse correlation was found between methylation and the degree of expression of the BCL-2 gene in human B-cell chronic lymphocytic leukaemia.41 Hypomethylation of the third exon of the c-MYC gene has been reported in a various human cancers.48 However, differentiation between cause and effect is difficult, even in the few cases in which associated increased gene expression was documented.29
Epigenetically regulated genes as biomarkers Epigenetic changes have been reported in a several cancer types, but not all of them have been well characterised. Colorectal cancer
Colorectal cancer is an excellent model for studying the genetic basis of cancer—the disease progresses in an orderly manner with very distinct genetic alterations at each stage. Also, epigenetic studies of colorectal tumours have greatly increased our understanding of the associated molecular events in disease progression. Studies of the methylation of DNA in colorectal cancer have provided further information about the pathogenesis of the disease. Progressive methylation of DNA and subsequent silencing of a subset of genes occurs in normal tissues alongside age and time-dependent events which predispose these normal cells to neoplastic transformation. At late stages of disease progression, methylation of DNA is important in a subset of tumours that have epigenetic instability caused by the simultaneous silencing of many genes—this characteristic is called the CpG island methylator phenotype (CIMP).49 CIMP-positive cancers include the majority of tumours with sporadic mismatch-repair deficiency due to hypermethylation of the hMLH1 promoter and also include the majority of tumours with Ki-RAS mutations. In CIMP colorectal tumours, the highly methylated DNA interacts with genetic lesions. CIMP-negative cancers evolve along a more standard genetic-instability pathway, with high rates of p53 mutations and chromosomal changes. Elucidation of the combined epigenetic and genetic events that occur in colorectal cancer has provided a complete molecular understanding of this tumour type and this knowledge could have implications for the diagnosis, prognosis, and treatment of patients with this disease. Multistage carcinogenesis in the colon can be viewed as a series of pathways that are activated (or inactivated) in populations of cells that are selected on the basis of growth (or survival) advantages.49 In most colorectal tumours, the common pathways (which operate simultaneously) are the APC/beta-catenin pathway, which provides a growth advantage via the transcriptional activation of c-MYC and
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other genes; the senescence bypass pathway, which is required for immortalisation of cells possibly through the inactivation of the p53 or the p16 gene;50 and the TGF bypass pathway, which is needed to overcome the inhibition of growth caused by TGF through inactivation of TGF receptor II or activation of Ki-RAS.51 Another unknown pathway could be altered in ageing cells, causing the expansion of the proliferative zone and thus a predisposition to neoplastic transformation. In colon cancer, the specific pathway that is affected is more important than the genes that are altered, which partly explains the large diversity in molecular changes observed in this fairly uniform disease. Furthermore, chromosomal instability, microsatellite instability, and epigenetic instability (CIMP) can be viewed as simply the molecular mechanisms required to generate molecular diversity, but which are also prerequisites for the change from neoplasia to cancerous growth. Further clarification is needed about the underlying causes of chromosomal and epigenetic instability and about whether the type of instability required for tumour growth has clinical implications. Indeed, tumours with microsatellite instability seem to have a more favourable prognosis overall than those without this feature, and the responses to chemotherapy of the two types differ. There are still many unanswered questions, and further studies that integrate patterns of molecular instability, epidemiology, and outcome are needed. For instance, there is evidence that CIMP-negative tumours may have a worse prognosis than CIMP-positive ones because of the high rate of chromosomal changes and p53 mutations, the latter of which is a measure of chemosensitivity in the colon.52 Also, CIMP-positive tumours may be especially sensitive to therapy. This tumour type might also be a possible target for chemoprevention by the inhibition of methylation.53 Prostate adenocarcinoma
The aetiology of prostate cancer, a multifactorial disease, is not completely understood. Environmental, genetic, and epigenetic factors all modulate the expression of genes involved in prostate-cancer development.54,55 One gene that is used as a marker for prostate-cancer detection is GSTP1. GSTP1 is one of a superfamily of enzymes known as the glutathione-S-transferases (GST) that are involved in the detoxification of electrophilic compounds, such as carcinogens, by glutathione conjugation, thus protecting DNA from oxidative damage. Hypermethylation of the GSTP1 promoter is a highly prevalent event in prostate cancer; it is present in potential precursor lesions and is strongly linked to loss of GST expression.54 However, common somatic GSTP1 inactivation prevents any major effects from inherited genotypic variants in the progression of prostate cancer. Jeronimo and colleagues compared patients with prostate cancer (adenocarcinomas and prostatic intraepithelial neoplasia lesions) with a control group consisting of patients with benign prostatic hyperplasia (BPH) and healthy blood donors.55 Tissue samples from the patients with prostate cancer and from patients with BPH were analysed for hypermethylation of GSTP1. No significant effect on
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prostate-cancer risk was detectable for the GSTP1 genotype compared with the control group. Moreover, no association was found between this genotype and tumour or BPH methylation status. Patients with carcinomas that did not show hypermethylation of GSTP1 did not have significant differences in genotypic distribution from the control population. In patients with adenocarcinoma, a strong association between hypermethylation of the GSTP1 promoter and loss of GST expression was observed. However, this trend was not found in prostatic intraepithelial neoplasia or BPH lesions. Although the GSTP1 polymorphism is not associated with altered susceptibility to prostate cancer, somatic promoter hypermethylation is a significant, but not the only, cause of decreased GST function. Ulcerative colitis
Ulcerative colitis is an inflammatory disease of the mucosa and submucosa of the large intestine. The duration and extent to which a patient suffers from ulcerative colitis depend on the development of colorectal carcinoma. The frequency and timing of hypermethylation of the p14ARF gene in ulcerative colitis were determined in patients with ulcerative colitis of different histological stages: adenocarcinoma, dysplasia, non-neoplastic ulcerative colitis mucosae, and ulcerative-colitis-associated carcinogenesis.56 The results indicated that hypermethylation of p14ARF is a relatively common and early event in ulcerative-colitisassociated carcinogenesis. Barrett’s oesophageal cancer
Barrett’s oesophagus is the only known precursor to oesophageal adenocarcinoma. The incidence of oesophageal adenocarcinoma has increased rapidly in more developed countries. Lesions in the cyclin-dependent kinase 4 and 6 inhibitor gene, p16INK4a, occur frequently in oesophageal adenocarcinomas, but their role in neoplastic progression is not well understood. Inactivation of p16 by hypermethylation of the cyclin-dependent kinase (CDKN2A/p16) promoter and loss of heterozygosity of the CDKN2A gene could have a role in the neoplastic progression of Barrett’s oesophagus. Hypermethylation of the CDKN2A promoter in Barrett’s oesophageal adenocarcinomas, premalignant lesions, and normal oesophageal squamous-cell epithelium was studied by the use of methylation-sensitive, singlestrand conformation analysis and sequencing of bisulphitemodified DNA.57 No methylation of the CDKN2A promoter was found in normal oesophageal squamous-cell epithelia. Methylation was detected, however, in 82% of adenocarcinomas and in 30% of premalignant lesions (including in 33% of samples with intestinal metaplasia only). Loss of heterozygosity at the CDKN2A gene locus was found in 68% of adenocarcinomas and in 55% of premalignant lesions. Most samples showed hypermethylation of the CDKN2A promoter, with or without loss of heterozygosity. These results indicate that methylation of the CDKN2A promoter is the predominant mechanism for p16 inactivation. This hypermethylation is a very common event in oesophageal adenocarcinoma and occurs as early as metaplasia.
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frequently methylated. Methylation of these genes correlates with other indices of prognosis and survival. For example, CDH1 methylation-positive status is associated with poor survival.
Transcription
Gastric cancer Relaxed chromatin Transcription factor complex No transcription
v Compact chromatin 5-aza-deoxycytidine
Methylation
Trichostatin A
Methylation binding protein
DNA methyl transferase
Histone deacetylase
Loss of control of the cyclo-oxygenase 2 (COX2) gene, via changes in methylation of the COX2 promoter, may have important consequences in gastric carcinogenesis. Overexpression of COX2 is strongly associated with loss of apoptosis and enhancement of proliferation in the gastrointestinal tract, especially in gastrointestinal Advances in epithelial cells.34 quantification of methylation and high-throughput technology are needed to elucidate further the mechanisms of this cancer Lung cancer
Lung cancer is the most common cause of death from cancer in the USA and kills more than 156 000 people there alone every year. Significant progress has been made during the past 20 years in understanding of the molecular and cellular pathogenesis of lung cancer. Abnormalities in proto-oncogenes, genetic and epigenetic changes in tumour suppressor genes, the role of angiogenesis in the multistage development of the disease, and detection of molecular abnormalities in preinvasive respiratory-tract lesions have been the focus of recent research. Virmani and co-workers developed a panel of markers for aberrant methylation that detects lung cancer at the early stages of development.58 This panel includes the genes: RAR, DAPK, GSTP1, FHIT, RASSF1, MGMT, E-cad, APC, and p16. In particular, the researchers found that RAR and the newly discovered tumour suppressor gene RASSF1 (located in the region 3p21·3) are deleted in many lung and breast cancers and are very methylated in very many small-cell cancers. They also found that APC is methylated in non-small cell cancers.
Figure 3. Inhibition of gene transcription by inhibitors of methyltransferase and histone acetyltransferase.
New approaches including advancements in chip technology, are needed for quantitative high-throughput research and for validation of different markers for riskassessment screening of large populations. Laird and colleagues used a MethylLight assay to analyse a panel of 20 genes from 104 tissue specimens from 51 patients with different stages of Barrett’s oesophagus and associated adenocarcinoma. Histologically identified clinical samples were studied, and epigenomic fingerprints for the different histological stages of oesophageal carcinoma were compared. Oesophageal adenocarcinoma arises through different stages; for example, squamous mucosa, columnar epithelium, Barrett’s oesophagus, dysplasia, and malignancy. The MethylLight study found distinct classes of methylation patterns in the different types of tissue. The frequency of methylation ranged from 15% (CDKN2A) to 60% (MGMT). Altogether, three classes were identified according to the absence (CDKN2A, ESR1, and MYOD1) or presence (CALCA, MGMT, and TIMP3) of methylation in normal oesophageal mucosa and the stomach or the infrequent methylation of normal oesophageal mucosa accompanied by methylation in all normal stomach samples (APC). In other genes, the frequency of hypermethylation was below 5% (ARF, CDH1, CDKN2B, GSTP1, MLH1, PTGS2, and THBS1) or it was completely absent (CTNNB1, RB1, TGFBR2, and TYMS1). Methylation patterns of specific genes at different stages of disease progression can be used as markers, and the degree of methylation reflects the stage of the disease. Bladder cancer
Many genes are methylated during the development of bladder cancer; CDH1, RASSF1A, APC, and CDH13 are
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Hypermethylation of promoters of several viral and cellular genes is well known to result in silencing of transcription.4,59 For example, transcriptional inactivation of the Epstein-Barr virus (EBV) latency gene Wp due to hypermethylation of its promoter suggests that hypermethylation is one of the mechanisms for regulating genes involved in interaction between infectious agent and host. EBV and a few other viruses are strongly associated with different types of cancer including lymphoma and leukaemia.60 Takacs and colleagues analysed the methylation patterns of CpG dinucleotides in latent membrane protein 1 (LMP1) regulatory sequences, a bidirectional promoter region of latent EBV genomes, by use of bisulphite-induced modification of DNA followed by automated fluorescence genomic sequencing.3 Transcripts
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for two latent membrane proteins, LMP1 (a transforming protein) and LMP2B, are initiated in this region in opposite directions. The results showed that B-cell lines and a clone expressing LMP1 carried EBV genomes with unmethylated or hypomethylated LMP1 regulatory sequences, whereas highly methylated CpG dinucleotides were present at each position or at discrete sites within hypermethylated regions in LMP1-negative cells. Comparison of high-resolution methylation maps suggests that CpG methylation directly interferes with the binding of nuclear factors LBF2, LBF3, LBF5, LBF6, LBF7, and AML1/LBF1. The maps also show that methylation of CpGs within an E-box sequence (where activators as well as repressors can bind) is not the major mechanism in silencing the LMP1 promoter. Factors that recruit methyltransferase to the CpG islands differ from regular transcription factors. Hypermethylation of LMP1 regulatory sequences or regions within these sequences in LMP1-negative cells suggest a role for an indirect mechanism in silencing the LMP1 promoter, via methylcytosine-binding proteins.60 Kaposi’s sarcoma
Kaposi’s sarcoma-associated herpesvirus (KSHV) is strongly linked to Kaposi’s sarcoma, primary effusion lymphomas, and a subset of multicentric Castleman’s disease. The mechanism by which this virus establishes latency and reactivation is not understood completely.61,62 The KSHV lytic transactivator (Lyta), mainly encoded by the ORF50 gene, is a lytic switch for viral reactivation from latency. It is essential for keeping the virus alive and for maintaining the latency. Chen and colleagues showed that the Lyta promoter region was heavily methylated in latently infected cells.62 The researchers then reversed the methylation of Lyta promoter by treating primary-effusion lymphoma-delivered cell lines with tetradecanoylphorbol acetate, promoting the lytic phase of KSHV. This experiment shows that demethylation of the Lyta promoter region is essential for the expression of Lyta. In samples from a latently infected KSHV carrier, this region remained methylated, which suggests a relation among a demethylation in the Lyta promoter, the reactivation of KSHV, and the development of KSHVassociated diseases. Epigenetic regulation has been observed in other cancer-associated viruses such as hepatitis B virus and papillomavirus. Studies of the mechanisms of interactions between host and pathogen could provide molecular targets for intervention.
mice have shown that inhibition of DNA methylation can suppress tumour initiation, which suggests that this approach might be useful as a preventive strategy. Cytosine analogues are being used to treat preneoplastic syndromes in clinical trials. Trials of antisense oligodeoxynucleotides against methyltransferase have shown a decrease in carcinogenesis. Although these studies have yielded promising, preliminary clinical information, a relation between clinical efficacy and target-gene demethylation has not yet been found. However, further studies in this area should clarify this situation. The reactivation of epigenetically silenced genes has therapeutic benefits.66–68 Indeed, azacytidine and its derivatives have been used in the clinic with beneficial results.69 The mechanisms underlying these effects, however, have not been fully elucidated. The doses of 5-azacytidine currently used are toxic, possibly due to effects not directly related to demethylation. The recent observation that low doses of azacytidine can facilitate reactivation of hypermethylated genes with inhibitors of histone deacetylase has created much interest. Trials of a clinically available histone deacetylase inhibitor, phenylbutyrate, combined with low doses of azacytidine are in the planning stages. These trials will include haemopoietic and solid tumours, and the researchers will monitor gene-reactivation events. Whether such an approach will specifically reactivate genes for better control of the neoplastic process or whether other genes will be affected, resulting in toxic effects in normal cells is not known. The initial results might justify further development of this therapeutic concept and further increase interest in clinical aspects of epigenetics in cancer.70–73 Mintz and Debinski have suggested immune-based therapies for Xchromosome-linked malignant glioma in which epigenetic and genetic regulation have a significant role.74
Summary Defininition of how hypermethylation of promoter regions participate in gene silencing and how loss of methylation alters chromosome structure is difficult. Nevertheless, evidence of abnormal methylation of DNA in cancer cells has potential importance in the clinical setting. One possible therapeutic approach is the use of CpG hypermethylation events as tumour biomarkers. The changes in methylation in promoter regions potentially provide a positive signal for cancer cells that can be detected by conventional techniques. On the basis of recent research, use of multiple markers for predicting one tumour type
Therapeutic potential of epigenetic changes Epigenetic changes are potentially good therapeutic targets because of their reversibility (figure 3). Markers for aberrant methylation may also be very useful for monitoring of the onset and progression of cancer. Treatment with inhibitors of DNA methylation can restore the activities of dormant genes such as CDKN2A and decrease the growth rate of cancer cells in a heritable manner. Similarly, the capacity of cells to repair DNA can be restored by activation of MLH1. Partial reversing the cancer phenotype by the use of methylation inhibitors should, therefore, be possible.63–65 Experiments with Min
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Search strategy and selection criteria Data for this review were identified by searches of Current Contents, the National Library of Medicine database, EndNotes, Pubmed with the search terms: “acetyl transferase”, “biomarker”, “cancer”, “cancer prevention”, “chromatin”, “chromatin modelling”, “chromosomal organisation”, “deacetylase”, “DNA damage”, “DNA repair”, “early detection of cancer”, “epigenome”, “epigenetics”, “histone”, “methylation”, “microarray”, “oncogene”, “risk assessment”, “tumour suppressor gene”, “virus”. Only papers published in English between 1983 and 2002 were selected.
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seems reasonable. However, the findings must be validated by trials with large sample sizes and clinical evidence to correlate epigenetic changes with clinical outcome. Since almost every tumour type seems to have many independent promoter-hypermethylation events, reasonably sized marker panels might be constructed for each tumour type to provide indices for early detection and monitoring of cancer risk. Early detection of cancer will allow therapeutic interventions to be applied to inhibit further development of cancer. Such information could also be used in the development of strategies to prevent cancer. Conflict of interest
None declared. References
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