CpG Island Methylation in Cancer
CpG Island Methylation in Cancer
By Guillermo Garcia-Manero, MD, and Jean-Pierre Issa, MD
The field of dna methylation recently has generated considerable interest thanks to several conceptual and technical advances. First, the discovery that epigenetic gene silencing through DNA methylation plays a role in the development of several disease entities, including cancer, has triggered clinical interest in this phenomenon. Second, the development of new technologies to analyze the methylation state of multiple genes in a relatively short time, and to detect DNA methylation abnormalities in serum, has raised the hope of rapidly finding clinical applications for this research. Third, the cloning of several DNA methyltransferases, enzymes with the capacity of methylating DNA, and the discovery of mutations in one of these in a rare, hereditary syndrome has increased interest in the role of the methylation machinery in disease. Finally, the possibility of reversing DNA methylation changes pharmacologically has opened new areas of research in the treatment and prevention of cancer. Here, we will summarize our present understanding of DNA methylation and its clinical and biological implications.
DNA Methylation
CpG methylation is the addition of a methyl group to the fifth position of the cytosine ring in a cytosine guanine dinucleotide (CpG). CpG dinucleotides (CpGs) have a peculiar distribution in the human genome. Overall, CpG sites are present at a lower frequency than expected in vertebrate DNA.1 CpG sites are found in two distinct areas: Most CpG sites are located in peri-centromeric satellite DNA and within repeat elements (Alus and LINEs) present in non-coding intergenic areas. Most of these are methylated in normal tissues. This methylation appears to play a role in chromosomal stability, and has also been proposed to serve as a defense mechanism against the recombinant properties of these repeat elements.2 CpG sites can also be found in close proximity to the 5´end of many genes, involving both promoter regions and 5´ untranslated regions. In these areas, a near normal representation of CpGs can be found, and for that reason they are known as CpG islands. It is estimated that there are close to 45,000 of these islands in the human genome.3 Most of these CpG islands are not methylated in normal tissues, regardless of the expression status of the associated gene.1
DNA Methylation, Gene Silencing, and Disease
The association between DNA methylation and epigenetic gene silencing recently has raised considerable interest in this phenomenon. Epigenetic silencing refers to stable, non-genetic inactivation of gene expression that is clonally inherited and thought to be irreversible under most "normal" situations.4 A role for DNA methylation in silencing was revealed by the observed inverse relationship between the transcriptional activity of a particular promoter and its methylation status.5 CpG island methylation-related silencing is important for imprinting and X-chromosome inactivation in women.6,7 Other than this physiologic role, aberrant CpG island methylation has been implicated in certain disease processes, such as the Fragile X syndrome, aging, and in cancer.8-11 DNA methylation has been implicated in oncogenesis by silencing the expression of genes crucial for cell function, such as tumor suppressor genes. This phenomenon represents a molecular alternative gene mutation and/or deletion for the inactivation of critical genes. DNA methylation primarily appears to induce silencing by attracting a protein complex that includes methylated DNA binding proteins and histone deacetylases, ultimately resulting in the formation of a closed chromatin structure.12 Mutations in the methylated-DNA binding protein MECP2 have recently been implicated in Rett syndrome, a hereditary progressive neurodevelopmental disorder.13
Regulation of DNA Methylation
The regulation of CpG island methylation is relatively poorly understood, and little is known about the mechanisms that lead to aberrant methylation in cancer. Several enzymes have been implicated in the methylation of CpGs. They are known as DNA methyltransferases. DNMT1 was the first enzyme cloned with the capacity to add methyl groups to cytosines.14 It is now thought that DNMT1 is primarily a maintenance enzyme, responsible for reproducing patterns of DNA methylation after replication. It does so by using hemimethylated DNA as a template after DNA replication, and it may have little affect on the generation of de novo methylation patterns. Recently, two novel DNA methyltransferases have been cloned, DNMT3a and DNMT3b.15 These two enzymes have been shown to have de novo methylation activity, and their lack of expression is lethal in a knock out mouse model.16 Of interest, the ICF syndrome, a rare autosomal recessive disorder that consists of immunodeficiency, centromeric chromosomal instability, and facial abnormalities, has recently been shown to be caused by loss of function mutations in the DNMT3b gene.13,16 Hypomethylation, primarily of centromeric DNA, is a central characteristic of this disease and likely plays a role in the generation of the phenotype.17 The expression of DNMT3a and DNMT3b has not been correlated with CpG island methylation in cancer, but it is not known whether activating mutations of these genes are present in human neoplasms.18
Detection of DNA Methylation
From the above information, it is clear that the analysis of the methylation state of specific genes in diverse disease entities can potentially provide useful clinical information, similar to the analysis of gene mutations or other abnormalities in these same disorders. For example, methylation of specific genes may provide prognostic information and could potentially be used to screen for the presence of cancer.19 Therefore, the development of rapid and sensitive techniques to study DNA methylation is important for such translational research. Over the past few years, the study of DNA methylation has been greatly facilitated by the realization that bisulfite treatment of DNA results in the conversion of unmethylated cytosines to uracil, leaving most methylated cytosines intact.20 Several bisulfite-PCR based techniques have now been developed that allow quantitative or very sensitive detection of gene-specific methylation.21,22 In addition, several techniques were developed to clone genes based on aberrant CpG island methylation.23,24 These developments are providing a more complete picture of the DNA methylation changes in cancer and their potential clinical implications.
The CpG Island Methylator Phenotype: A New Classification of Disease?
The methylation analysis of multiple genes in individual patient samples has revealed considerable variation in the degree of methylation, suggesting that the process is not random, but likely results from specific defects in methylation control. Thus, an extensive analysis of colon cancer has revealed a subset of cases that evolves with a high degree of methylation and silencing—a phenotype termed CpG Island Methylator Phenotype.25 This concept has now been verified in other tumor types, including gastric and hepatocellular cancers and hematopoietic malignancies.26 The importance of this phenomenon is that the clinical and biological characteristics of the different methylation groups appear to be substantially different,27 and this may have clinical implications such as different prognosis and different responses to specific therapeutic interventions.
Methylation Inhibitors for the Treatment and Prevention of Cancer
The data previously discussed lend strong support to the notion that aberrant CpG island methylation participates in the molecular pathogenesis of different neoplasms. An obvious correlate to this concept is whether methylation inhibition can be used in the prevention and treatment of these diseases. In fact, cytosine analogues that significantly inhibit DNA methylation were described several years ago, and have been in clinical development primarily in hematopoietic malignancies.28,29 In addition, pharmacologic and/or genetic reductions in methylation have demonstrated chemopreventive properties in animal models of lung and colon cancer.30,31 Important issues that remain to be clarified are the optimal doses and schedules of administration of methylation inhibitors to target demethylation (rather than cytotoxicity that occurs at high doses) and the selection of patients who might best benefit from these agents.
Future Implications
Although the concept of DNA methylation is not new, its study in human disease is in its infancy. New molecular techniques are allowing the rapid analysis of multiple genes in multiple samples and will certainly clarify the importance of this epigenetic phenomenon in disease. The data emerging on methylator phenotypes may add to the molecular classification of neoplasia, with potential implications for treatment and prognosis. The recent cloning of new DNA methyltransferases will help clarify the regulatory mechanisms of DNA methylation. Finally, the development of specific methylation inhibitors holds promise of a novel approach to cancer prevention and treatment. (Dr. Garcia-Manero is an Assistant Professor of Medicine, Department of Leukemia, University of Texas M.D. Anderson Cancer Center and Division of Oncology, University of Texas-Houston Health Science Center; and Dr. Issa is an Associate Professor of Medicine, Department of Leukemia, University of Texas M.D. Anderson Cancer Center, Houston, Texas.)
Acknowledgements
Research in the authors’ laboratories is supported by grants from the Leukemia and Lymphoma Society of America, The American Cancer Society, and the National Institutes of Health. Dr. Garcia-Manero is the recipient of an Award from the CapCure Foundation.
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