Key words: CpG Island, DNA Methylation, Epigenetics, Microarray, Ovarian Cancer

Abstract

Hypermethylation of CpG islands, an epigenetic event that is not accompanied by changes in DNA sequence, represents an alternative mechanism to deletions or mutations to inactivate tumor suppressor genes. Recent evidence supports the notion that CpG island hypermethylation, by silencing key cancer-related genes, plays a major causal role in cancer. However, a long-standing issue in the field is the sequence of molecular events leading to epigenetic gene silencing. A new model has been proposed that chromatin remodeling, as a result of histone deacetylation and methylation, is the primary event in abrogating transcriptional initiation; subsequently, CpG island hypermethylation establishes a permanent state of gene silencing. Accumulating evidence indicates that CpG island hypermethylation is an early event in cancer development and, in some cases, may precede the neoplastic process. Because of their heritable nature, hypermethylated CpG islands leave “molecular footprints” in evolving cancer cells and can be used as molecular markers to reconstruct epigenetic progression during tumorigenesis. Furthermore, hypermethylated CpG islands are proving to be useful for molecular classification of different cancer types.

INTRODUCTION

Epigenetics can be defined as a heritable change in gene expression that is not accompanied by changes in DNA sequence. Methylation of cytosine residues at CpG dinucleotides is the major epigenetic modification in mammalian genomes and known to have profound effects on gene expression. This epigenetic event occurs globally in the normal genome, and 70-80% of all CpG dinucleotides are heavily methylated in human cells (1). However, ~1-kb stretches of GC-rich DNA, called CpG islands, seem to be protected from the modification (2). About 60% of human genes are associated with unique CpG islands (2), and recently it has been estimated that the human genome contains about 29,000 CpG islands (3, 4). These normally unmethylated CpG islands may become methylated in cancer cells, and the event is associated with loss of expression of flanking genes (5). It has been estimated that aberrant methylation is initiated at ~1.4% of 45,000 CpG islands in the human genome, and may continue to accumulate in as many as 10% of the islands during tumor development (6). Thus, abnormal de novo methylation of CpG islands in human cancer cells represents one of the most prevalent molecular markers yet identified, and the list of methylated genes identified in various cancer types continues to grow. Further, classifying CpG islands on the basis of epigenetic status in cancer has practical applications (7, 8), and distinct methylation profiles for various cancers are beginning to emerge (6, 9-15). Nonetheless, the importance of epigenetic changes to cancer remains to be elucidated, and why cancer cells fail to maintain the CpG islands in an unmethylated state is currently unresolved.

In this review, we discuss how methylation imbalances occur in cancer cells and potential mechanisms responsible for CpG island methylation. We also address the issues of whether CpG island methylation is a cause or consequence of gene silencing (i.e., the chicken and the egg question) and whether this abnormal event precedes neoplasm and progressively accumulates in multiple gene loci during the development of cancer. Our observations on the relevance of CpG island methylation profiles to a specific disease, ovarian cancer, will be discussed.

The interplay of genetic and epigenetic alterations in gene silencing

Molecular mechanisms of CpG island hypermethylation – the cause or the consequence of gene silencing?

While CpG island hypermethylation is a common occurrence in cancer, the mechanisms that determine aberrant methylation in cancer are beginning to be revealed. Loss of cell cycle control resulting in unrestrained cell proliferation is a hallmark trait of cancer cells, and increased cell proliferation might be required for epigenetic changes in cancer cells (28). Failure of CpG islands to become remethylated in nondividing cells (28) suggests that de novo methylation of CpG islands occurs only in cells that are dividing. However, CpG island methylation in non-dividing cells has been reported (29), and different mechanisms underlying CpG island methylation may exist in different cancers. Several key enzymes that regulate DNA methylation, such as the DNA methyltransferases (DNMTs), are likely candidates to assess for alterations that would contribute to abnormal de novo methylation and lead to cancer development. Overexpression of all the DNMTs at the mRNA level has been shown for several cancers (30-37), and increased DNMT1 expression in normal cells can cause aberrant de novo methylation of CpG islands (38) and promoter cellular transformation (39, 40). The mRNA levels of the DNMTs are differentially regulated during the cell cycle (41), and improper DNMT expression during the cell cycle may contribute to methylation alterations typically seen in cancer cells. Furthermore, alterations in DNMT function will result in gains or losses (42-44) in DNA methylation, as well as cause shifts in DNA methylation patterns (42). Functional loss of DNMTs has also been associated with disease in some cases (45-47). Loss of promoter hypermethylation and re-activation of tumor suppressor gene function may explain why knocking out the gene for DNMT1 led to hypomethylation and decreased gastrointestinal neoplasia (48, 49), while disruption of DNMT1 and 3b genes in colon cancer cells led to loss of DNA methylation and reduced cell growth (50). The enzymes themselves have recently been shown to directly repress gene transcription (51-54), suggesting an uncoupling of promoter silencing by the DNMTs and DNA methylation. It should be kept in mind that no specific mutations in DNMTs have been identified, DNMT gene amplification has not been described, and an increase in DNA methylation resulting from a loss of DNMT function has not, as of yet, given rise to cancer. However, recent data suggests that DNMTs may need to act cooperatively to induce the abnormal promoter methylation (49, 55-57).

Chromatin plays a key role in regulating transcriptional activity, and chromatin remodeling and DNA methylation in cancer have recently been linked (57-60). Histones, the major components of chromatin, can be acetylated and deacetylated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. These enzymes are gene activators and repressors (61, 62), and histone acetylation/deacetylation is important in transcriptional regulation (63). These histone modifications and DNA methylation appear to be functionally coupled in fungi (64) and plants (65) and this relationship is beginning to be established in mammalian systems (60). DNA methyl-CpG binding domain proteins (MBDs) and DNMT1 may recruit HDACs to methylated promoters, which, in turn, deacetylate histones, to maintain chromatin in the repressed state (52, 60). Together with the observations that MBD-containing co-repressor complexes associate with methylated CpG islands (66), it has been suggested that histone modifications are secondary to DNA methylation. However, mutations in a putative methyltransferase specific for histone H3 lysine 9 result in loss of cytosine methylation in fungus (64), providing the first evidence that histone methylation can initiate DNA methylation. How histone H3 methylation recruits a DNA methyltransferase has not been resolved. Furthermore, in plants, it has recently been shown that CpG island methylation depends on histone H3 methyltransferase (65), suggesting that histone methylation can actually direct DNA methylation. Finally, a specific “histone code” has been proposed to target methylation to DNA (61). Modifications of histones, such as by deacetylation, methylation or phosphorylation, may be somehow required to initiate or maintain DNA methylation, perhaps by recruiting proteins, such as MBDs, transcriptional activators or repressors, and these interactions then determine which genes are expressed or silenced. Based on this observation, it has been suggested that histone modifications are the primary event in initiating gene silencing whereas CpG island hypermethylation, a subsequent event, establishes a permanent state of gene inactivation. Future research will reveal how chromatin remodeling contributes to epigenetic regulation of gene expression and perhaps control of the CpG island methylation machinery.

The role of CpG island hypermethylation in cancer initiation and progression

It has been demonstrated quite convincingly that CpG hypermethylation and subsequent inactivation of important genes, such as tumor suppressor genes, can provide a selective growth advantage to cancer cells (5). Nonetheless, the issue of what comes first, epigenetic alterations or cancer, is intriguing. In colorectal cancer hypermethylation of a number of genes has been shown to start in normal mucosa and early- and pre-neoplasia, and CpG island methylation increases as a function of age (67-69). Higher levels of methylation appear to increase the risk of colon cancer (70, 71), but whether the cells harboring these changes will actually become cancer is an open question. Nevertheless, there is substantial evidence that CpG island hypermethylation occurs early in the neoplastic process. For example, hypermethylation of the p16INK4a gene is frequently seen during metaplastic progression in Barrett's esophagus (72, 73), lung (74), cervical (74) and gastric carcinoma (75, 76). Hypermethylation of 14-3-3 sigma is an early event in breast cancer (77). The association of promoter hypermethylation of VHL (78), MLH1 (79, 80), and BRCA1 (81, 82) with familial cancers are additional examples of epigenetics being causative, but the possibility that hypermethylation of these genes might be late events in the nonfamilial cancers cannot be overlooked.

De novo methylation usually takes place at the outskirts of a promoter CpG island and progressively spreads into the core of the island (83). It is not clear why protection of CpG islands and the neighboring sequences from abnormal DNA methylation is lost in cancer cells. Studies suggest that some sequences in the promoter region may serve as docking sites to attract repression complexes, including HDACs, MBDs, DNMTs and others, from which the exonic sequences become hypermethylated and their histones become hypoacetylated and methylated (5, 61, 84, 85). Recognition sequences within and/or around CpG dinucleotides may also play a role in the association of proteins to methylated cytosines (60, 86, 87). The interference of these proteins in the binding of transcription factors that prefer unmethylated sequences was recently reported (88), suggesting that transcription factor abundance may compete with promoter methylation machinery and this may also be relevant to the initiation of CpG island hypermethylation in cancer. Non-promoter CpG islands also appear to be more susceptible to aberrant methylation than their nearby respective promoter sequences, and methylation can begin in exonic regions and then spread to CpG islands in other locations, including promoter regions (89). The density or number of methylated CpG dinucleotides can exert long distance transcriptional repression (88). Because of their heritable nature, hypermethylated CpG islands leave molecular footprints from which the event of epigenetic progression can be reconstructed during tumorigenesis (see illustration in Fig. 1) and therefore, are useful markers for molecular classification of different tumor types.

Relevance of CpG island methylation to ovarian cancer Ovarian cancer has the highest mortality rate of the reproductive cancers and is the 4th leading cause of cancer death in women (90). Because ovarian cancer is often asymptomatic in its early stages, most patients are diagnosed at the late stages and 5-year survival for patients with advanced disease is less than 20% (90). There has been little change in ovarian cancer incidence and mortality over the past 5 decades.

We have taken a high-throughput screening approach for profiling methylation alterations of CpG islands in ovarian tumors and identifying candidate markers for diagnosis and prognosis of the disease. This novel microarray approach, called differential methylation hybridization (DMH), allows us to do a global analysis of DNA methylation in ovarian (91, 92) and other carcinomas (93, 94). By using DMH, we have shown that aberrant DNA methylation is a frequent epigenetic event in ovarian cancer, and we have also revealed tumor groups with distinctly different methylation profiles (92). Furthermore, patients defined by these groups differed markedly in the time until disease recurrence following chemotherapy; in addition, a higher degree of CpG island methylation was associated with early disease recurrence after chemotherapy (92). A select group of CpG island loci is also revealed in the study (92), which may be useful as epigenetic markers for predicting chemotherapy outcome in patients with ovarian cancer. Further, comparing epigenetic changes in early- versus late-stage ovarian cancer may provide a rationale on which to base the need for aggressive therapy. We have further refined DMH by using expressed CpG island sequence tags (ECISTs) for dual detection of CpG hypermethylation and gene silencing in cancer cells (95). ECISTs exist in the genome, and their GC-rich fragments can be used to screen aberrantly methylated CpG sites in cancer cells; in addition, the exon-containing portions can be employed to measure levels of gene expression simultaneously. Using an ECIST panel, hypermethylated loci were identified and at the same time their association with gene silencing was confirmed in breast and ovarian cancer cells. Our current approach and those of others (96, 97), for the reasons discussed above, is to use methylation inhibitors and HDAC inhibitors together to relieve transcriptional repression of methylated promoters. In an initial study, treatment of ovarian cancer cells with DNMT inhibitor 5-aza-2’-deoxycitidine (DAC, a cytidine analogue that sequesters DNA methyltransferase after its incorporation into genomic DNA) resulted in reactivation of some hypermethylated genes, but no change in expression levels of methylated genes was observed in cells treated with the HDAC inhibitor trichostatin A (TSA) (Huang et al., unpublished observation). However, treatment with DAC plus TSA resulted in the synergistic upregulation of many methylated-silenced genes, and several methylation-unrelated genes were also upregulated by the drug combination. We are currently using this approach for screening gene promoter function and activity of promoters in ovarian cancer.

Summary and therapeutic implications

The potential to reverse DNA methylation and re-express tumor suppressor genes and key control pathways in cancer cells presents attractive and exciting clinical possibilities. Experimental results have shown that re-expression of epigenetically silenced hMLH1 by treatment with the DAC sensitized cisplatin-resistant ovarian cancer cells to chemotherapeutic agents both in vitro (98) and in vivo (99). Reversal of the methylated estrogen receptor (ERa) gene in human ER-negative breast cancer cell lines resulted in re-expression of functional ER and breast cancer cells were then able to respond to the antiestrogen therapies (21, 96). Evaluation of DNMT inhibitors as a cancer chemotherapeutics is underway, and clinical trials that will allow for the examination of specific hypermethylated genes, which are reactivated by therapeutic DACs have started for solid tumors, including ovary tumors (100-102). HDAC inhibitors, alone or together with DNA methyltransferases inhibitors, may represent novel treatment approaches that could be combined with currently available chemotherapies. Methylation profiling of tumors could provide a more focused test for reactivation of methylation-silenced genes as therapeutic targets and form a rational basis for future treatment strategies designed to alter this fundamental process in cancer.

Acknowledgements

This work was supported in part by National Cancer Institute grants CA-85289 (K. P. N.) and CA-69065 and CA-86701 (T. H.-M. H.). T. H.-M. H. is a consultant to Epigenomics, Inc.

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Figure 1. An epigenetic model for cancer initiation and progression. Five promoter CpG islands, as well as their neighboring CpG sites, critical to tumorigenesis are depicted here. In the initial neoplastic step, protection of some CpG island loci from aberrant DNA methylation is lost. De novo methylation occurs at the flanking CpG sites and progressively spreads into the core of a CpG island, resulting in silencing of the corresponding gene. This methylation spread may occur later in some other loci important for certain stages of neoplasm. In general, the density of methylated CpG sites within a locus as well as the number of methylated loci increase in more advanced stages of cancer. : methylated CpG dinucleotide; : unmethylated CpG dinucleotide.

Table 1.Some scenarios of methylation imbalance and potential consequences of gene expression

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