REVIEW

The Epigenome as a Target for Cancer Chemoprevention

Levy Kopelovich, James A. Crowell, Judith R. Fay

Affiliations of authors: Chemopreventive Agent Development Research Group, National Cancer Institute, National Institutes of Health, Bethesda, MD (LK, JAC); CCS Associates, Mountain View, CA (JRF).

Correspondence to: Levy Kopelovich, PhD, Chemopreventive Agent Development Research Group, Division of Cancer Prevention, National Cancer Institute, National Institutes of Health, 6130 Executive Plaza North, Rm. 2117, Bethesda, MD 20892 (e-mail: kopelovl{at}mail.nih.gov)


    ABSTRACT
 Top
 Abstract
 Introduction
 Epigenetic Changes in High-Risk...
 Cancer Prevention by Targeting...
 Conclusions
 References
 
Epigenetic events, a key driving force in the development of cancer, are alterations in gene expression without changes in the DNA coding sequence that are heritable through cell division. Such changes occur throughout all stages of tumorigenesis, including the early phases, and are increasingly recognized as major mechanisms involved in silencing tumor suppressor genes. Epigenetic changes can be reversed by the use of small molecules and, thus, such changes are promising targets for cancer chemopreventive drug development. This review examines the basis for targeting the epigenome as a prevention strategy, focusing on understanding the epigenetic changes that occur before the development of frank malignancy, when chemopreventive intervention will have the maximal impact.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Epigenetic Changes in High-Risk...
 Cancer Prevention by Targeting...
 Conclusions
 References
 
Two changes integral to epigenetic transcriptional control are DNA methylation and covalent modification of histone proteins (1,2). In cancer cells, genome-wide hypomethylation is accompanied by hypermethylation of short CpG-rich regions known as CpG islands. These islands, found in the promoter region of about half of genes, are generally unmethylated in normal cells. However, hypermethylation of CpG islands has been observed in nearly every type of human tumor, with unique patterns of individual gene methylation exhibited by each tumor type (3,4). Hypermethylation of promoter regions is associated with transcriptional silencing and is at least as common as mutation as a mechanism for inactivation of classic tumor suppressor genes in human cancers (1). Furthermore, a number of candidate tumor suppressor genes that are not commonly inactivated by mutation are transcriptionally silenced by this mechanism (1). Hypermethylation and silencing of genes involved in DNA repair (e.g., MGMT and hMLH1) can set the stage for tumor development by predisposing to mutations (5,6). A broad spectrum of other genes are also aberrantly methylated in cancers, including those associated with the cell cycle (e.g., retinoblastoma, p16INK4a, p15INK4b, and p14ARF), signal transduction (e.g., RASSF1, LKB1/STK11, and APC), carcinogen metabolism (e.g., GSTP1), apoptosis (e.g., DAPK and CASP8), hormone response (e.g., ER, PGR, AR, and RAR{beta} ), angiogenesis (e.g., THBS1), and invasion and/or metastasis (e.g., TIMP3 and CDH1) (7,8).

The second major layer of epigenetic transcriptional control that has been widely studied is modification of histone proteins. These proteins serve as building blocks to package eukaryotic DNA into repeating nucleosomal units that are folded into higher order chromatin fibers. Histones undergo elaborate post-translational modifications on their amino-terminal tails, including acetylation, methylation, phosphorylation, and ubiquitination. Acetylation has been the most extensively studied and is controlled by histone acetyltransferases and histone deacetylases. Acetylation is associated with nucleosome remodeling and transcriptional activation, whereas deacetylation is associated with transcriptional repression via chromatin condensation. In addition to their histone-modifying activity, histone deacetylases may also control gene expression by deacetylating transcription factors and contributing to cell cycle regulation (2,9).

Functional alterations of proteins in the histone acetyltransferase family (e.g., CREB-binding protein and p300) via mutation or the action of viral proteins are associated with certain cancers. Functional mutations and/or loss of heterozygosity in the CREB-binding protein gene are linked with precancerous Rubinstein-Taybi syndrome and hepatocellular carcinoma, and p300 is associated with glioblastoma and breast and colorectal cancers. Chromosomal translocations of both CREB-binding protein and p300 also occur in leukemias. Aberrant transcriptional repression mediated by histone deacetylases is associated with several hematologic malignancies, most notably, acute promyelocytic leukemia (2,10).

The two layers of epigenetic control, DNA methylation and histone acetylation, are integrally linked. Methylation is catalyzed by a family of DNA methyltransferases. DNA methyltransferases recruit histone deacetylases, leading to histone deacetylation and transcriptional repression. Methylated DNA is also recognized by a family of methylated DNA-binding proteins, which recruit histone deacetylases and ATP-dependent chromatin remodeling proteins, resulting in a tightly condensed chromatin structure and gene inactivation [for review, see (2)]. Additional links between the "histone code" and the "cytosine methylation code" are increasingly evident (1). A recent study (11) suggests that DNA methylation acts to lock in rather than initiate epigenetic silencing.


    EPIGENETIC CHANGES IN HIGH-RISK TISSUES: TARGETS FOR CHEMOPREVENTION
 Top
 Abstract
 Introduction
 Epigenetic Changes in High-Risk...
 Cancer Prevention by Targeting...
 Conclusions
 References
 
Epigenetic changes described in preinvasive lesions and/or high-risk tissues with the potential to serve as targets for chemoprevention are discussed below.

Colorectum

Aberrant epigenetic regulation during tumorigenesis has been studied extensively in the colon (Table 1). Changes in DNA methylation appear to play two distinct roles during colorectal carcinogenesis. The first is a progressive, age-related methylation that silences a subset of genes in normal colorectal tissue (i.e., type A methylation). These age-related changes, first observed for the ER gene (12), have also been described for CSPG2, EGFR, IGF2, MYOD1, N33, PAX6, and RAR{beta}2 genes. It has been hypothesized that type A methylation in the normal colon contributes to the increased risk of colorectal cancer associated with aging (1315).


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Table 1. DNA methylation in the colon during aging and colorectal cancer development*

 
Apart from age-related methylation, a number of genes are uniquely methylated in cancers and/or preinvasive lesions. These "type C" genes include APC, CACNA1G, CALCA, H1C1, MGMT, TIMP3, and WT1 (13,16,17). Methylation of hMLH1 is reportedly both age-related and tumor-specific (5,13). The frequency of methylation observed in cancers is lower for type C genes (10%–50%) than for type A genes (30%–100%). Type C methylation occurs in a subset of colorectal tumors that are defined as having the CpG island methylator phenotype. Colon cancers with the CpG island methylator phenotype include the majority of tumors with sporadic mismatch repair deficiency and KRAS mutations. Colon cancers that do not express the CpG island methylator phenotype develop along a more classic genetic instability pathway, with a high rate of p53 mutations and chromosomal changes. The CpG island methylator phenotype is observed in about 50% of colonic adenomas with synchronous cancers and in 25% of adenomas in the absence of cancer and is more common in adenomas with increased malignant potential. However, methylation is not associated with multiple adenomas from the same individual, suggesting that this epigenetic change is not a field defect in these lesions (13,16,17). Hypermethylation of the DNA repair gene MGMT also occurs frequently in adenomas and predisposes these tumors to KRAS (18) and p53 (19) mutations. The classic colorectal gatekeeper APC gene can also be inactivated epigenetically in adenomas (20). Interestingly, the candidate tumor suppressor HLTF gene, a member of the SWI/SNF (mating-type switching/sucrose nonfermenting) family of chromatin remodeling proteins, is methylated in high-risk adenomas and cancers (21). Thus, the epigenetic machinery itself may be a target of epigenetic regulation.

Importantly, recent evidence also suggests that epigenetic changes occur at the preadenoma stages of colorectal cancer. Altered patterns of DNA methylation are observed in early preinvasive aberrant crypt foci. Methylation is more frequent in dysplastic aberrant crypt foci and in lesions associated with sporadic cancers than in familial cancers (22). Some hyperplastic polyps, now thought to represent preinvasive lesions developing along an alternative pathway from the classic adenoma-carcinoma sequence (23), may arise from epigenetic field defects (24).

Aberrant methylation patterns are also observed in patients with ulcerative colitis, a chronic inflammatory condition of the large intestine which predisposes to cancer (Table 2). Changes include altered methylation of type A genes in the normal-appearing mucosa and in high-grade dysplastic areas (25), as well as of type C genes, such as the tumor suppressors p14ARF and p16INK4a (26,27).


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Table 2. DNA methylation in patients with ulcerative colitis (UC)

 
Genetically reducing expression levels of the predominant DNA methyltransferase Dnmt1 inhibits intestinal tumor development in mouse models of familial adenomatous polyposis (28) and hereditary nonpolyposis colon cancer (29); in the former, complete suppression of polyp formation was achieved. In both studies, Dnmt1 mutant mice exhibited a reduced frequency of CpG island hypermethylation in normal intestinal mucosa and tumors. It should be noted that, in addition to maintenance of DNA methylation, Dnmt1 can directly inhibit transcription (30), which may also contribute to the effects observed.

Breast

Quantitative and gene-specific epigenetic changes are also observed early in the progression of breast cancer (Table 3). In a study of four genes, the putative tumor suppressor gene RASSF1A was the most frequently and heavily methylated locus. Methylated alleles were present to a similar extent in intraductal papillomas and epithelial hyperplasias, as well as in ductal carcinoma in situ, but never in normal breast tissue (31). The gene for the negative cell cycle regulator 14-3-3{sigma} was hypermethylated in intraductal papillomas, epithelial hyperplasias, and ductal carcinoma in situ; however, it was also hypermethylated in lymphocytes and stromal tissue (31,32). By contrast, aberrant methylation of the CCND2 gene was restricted to ductal carcinoma in situ, with increased methylation associated with higher histologic grade tumors; however, p16INK4a was only rarely methylated in ductal carcinoma in situ lesions. When intraductal and invasive tumor cells were compared, the methylation status of p16INK4a, 14-3-3{sigma}, and RASSF1A genes was generally similar; however, variable quantitative changes in methylation of the CCND2 gene were detected (31). Others have confirmed methylation of the CCND2 gene in ductal carcinoma in situ (33,34) and in ductal fluid collected by routine operative breast endoscopy (33). The estrogen receptor {alpha} and CDH1 genes are also methylated in about 30% of ductal carcinomas in situ, which increases to about 50% and 60% in invasive and metastatic lesions, respectively (35). A direct association between breast cancer tumor suppressor genes BRCA1 and BRCA2 and the epigenetic machinery has been found. BRCA1 interacts with histone-modifying enzymes (36,37) and components of the chromatin-remodeling machinery (38) and can mediate large-scale chromatin decondensation (39). BRCA2 has intrinsic histone acetyltransferase activity (40).


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Table 3. DNA methylation during breast cancer development*

 
Of particular interest is the epigenetic silencing of the RAR{beta} gene during breast cancer development. The antiproliferative and differentiative effects of retinoids, well-recognized chemopreventive agents, are mediated largely through interactions with members of the nuclear retinoic acid receptor family (41). Loss of RAR{beta} expression is an early event during breast carcinogenesis (42), which is mediated, at least in part, by epigenetic mechanisms (33). RAR{beta} is methylated in invasive cancers and ductal carcinoma in situ but is not methylated in normal breast tissue. Overall methylation of at least one of three genes (RAR{beta}, CCND2, or TWIST) was found in 48 of 50 invasive breast cancers and in eight of 14 ductal carcinomas in situ but was not found in healthy breast tissue. Methylation of RAR{beta} was also detected in ductal fluid collected by routine operative breast endoscopy from invasive ductal carcinoma in situ and atypical ductal hyperplastic lesions. Fluid from routine operative breast endoscopies contained methylated RAR{beta}, CCND2, or TWIST alleles in 17 of 20 invasive lesions, in two of seven ductal carcinomas in situ, and in two of six atypical ductal hyperplasias. Importantly, two high-risk women with healthy mammograms whose ductal lavage fluid contained methylated markers and cytologically abnormal cells were subsequently diagnosed with breast cancer (33).

Prostate

The GSTP1 gene, which codes for the drug detoxification enzyme glutathione S-transferase {pi}, is hypermethylated in the vast majority of prostate cancers and in a large number of preinvasive prostatic lesions (4345) (Table 4); in one study (43), it was found in up to 70% of prostatic intraepithelial neoplasias. No mutations or deletions have been reported in the GSTP1 gene, suggesting that methylation is a major mechanism of gene inactivation. Loss of GSTP1 expression is closely associated with promoter hypermethylation in cancers; however, this tight association is lost in prostatic intraepithelial neoplasias (45). The discrepancy between gene expression and promoter hypermethylation may reflect higher levels of GSTP1 methylation in cancers than in preinvasive lesions (46). Methylation of other genes, including RAR{beta}2, RAR{beta}4, RASSF1A, CDH13, APC, CDH1, and FHIT, has been detected in cancerous tissues (47), but the status of these epigenetic changes in preinvasive lesions is unknown.


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Table 4. DNA methylation during prostatic cancer development*

 
Esophagus

Hypermethylation of the p16INK4a tumor suppressor gene is a common event in both esophageal adenocarcinoma and in preinvasive Barrett's esophagus (Table 5). Methylation is detected in metaplasias and is similar in all grades of dysplasia, suggesting that this epigenetic alteration occurs very early in tumor progression (4851). In a recent study (52) using bioinformatic algorithms to identify genetic and/or epigenetic lesions that provide a selective advantage (as compared with those that "hitchhike" but are neutral for tumor progression), hypermethylation of the p16INK4a promoter provided a clear and strong advantageous effect on cells early in the progression of Barrett's esophagus.


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Table 5. DNA methylation during esophageal cancer development

 
An in-depth study (53) of the methylation status of 20 genes in different stages of Barrett's esophagus and/or associated adenocarcinoma identified distinct "epigenetic fingerprints" in different histologic stages of the disease. Some genes were minimally informative because the frequency of hypermethylation was less than 5% (p14ARF, CDH1, p15INK4b, GSTP1, hMLH1, COX2, and THBS1), completely absent (CTNNB1, RB1, TGFBR2, and TYMS1), or ubiquitous (HIC1 and MTHFR), regardless of tissue type. Genes with an intermediate frequency of hypermethylation (in all tissue types combined) were more informative, with methylation ranging from 15% (p16INK4a) to 60% (MGMT) of samples. This group could be further subdivided into three classes, defined by the absence (p16INK4a, ER, and MYOD1) or presence (CALCA, MGMT, and TIMP3) of methylation in normal-appearing esophageal and stomach tissue or by the infrequent methylation of normal esophageal mucosa accompanied by methylation in all normal stomach samples (APC). Different stages of disease were characterized by unique epigenetic changes in each gene class. Aberrant methylation occurred at numerous loci in the same tissue, suggesting a generalized deregulation of methylation control, but a discrete group of tumors with the CpG island methylator phenotype was not identified. Importantly, normal and metaplastic tissues from patients with associated dysplasia or cancer had a higher incidence of hypermethylation than similar tissues from patients without further evidence of progression, suggesting that hypermethylation may identify esophageal lesions at risk for progression.

Lung

p16INK4a methylation is detected in the earliest cytologic stages of lung cancer (Table 6) and increases during disease progression (54,55). In one study (54), methylation increased from 17% in basal cell hyperplasias to 24% in squamous cell metaplasias to 50% in carcinomas in situ to 61% in squamous cell cancers and was associated with loss of expression in both tumors and precursor lesions. p16INK4a is also methylated in nonmalignant bronchial epithelium from current and former smokers and in sputum from high-risk individuals and patients with lung cancer (6,54,5658) but not in the bronchial epithelium of never smokers (56). Furthermore, p16INK4a methylation has been detected in sputum up to 3 years before the diagnosis of cancer (58) and in hyperplasias, adenomas, and adenocarcinomas from rats treated with tobacco-specific carcinogens (54).


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Table 6. DNA methylation during lung cancer development*

 
The strong association between p16INK4a methylation status in bronchial epithelium and corresponding primary tumors (56) and the high frequency of promoter methylation (up to 80% in cancers) [for review, see (59)] underscores the importance of this event for lung cancer development. Although p16INK4a methylation occurs early in lung tumorigenesis, the available data suggest that it is not predictive for cancer development in this tissue but is likely permissive for acquisition of additional genetic and/or epigenetic changes (6,55,56). Altered methylation of additional genes, including DAPK, MGMT, and FHIT, has also been implicated in the early stages of lung cancer progression (6,56,60).

Cancers Associated With Inflammation and/or Infection

In addition to being observed in ulcerative colitis, aberrant methylation is seen in other chronic inflammatory conditions which predispose to cancer. Methylation of the p16INK4a promoter is found in chronic hepatitis and cirrhosis associated with hepatitis B and hepatitis C viral infections, which are risk factors for liver cancer. Importantly, analysis of serial samples from patients with methylation-positive hepatocellular carcinoma detected the loss of p16INK4a gene methylation and protein expression in 18 of 20 patients at the stage of chronic hepatitis that precedes clinically detectable carcinoma (61).

Changes in methylation of host genes occur in infection-related cancers in addition to hepatocellular carcinoma. For example, in malignant mesothelioma, methylation of RASSF1A is linked with simian virus 40 infection (62), and in gastric cancer, methylation of multiple host genes is associated with chronic Epstein–Barr virus infection (63).


    CANCER PREVENTION BY TARGETING THE EPIGENOME
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 Abstract
 Introduction
 Epigenetic Changes in High-Risk...
 Cancer Prevention by Targeting...
 Conclusions
 References
 
Unlike tumor suppressor genes inactivated by genetic alterations, genes silenced by epigenetic mechanisms are intact and responsive to reactivation by small molecules. Many diverse genes hypermethylated in cancers can be reactivated with DNA methyltransferase inhibitors (64). These agents, all analogs of the nucleoside deoxycytidine (Fig. 1, A), inhibit the growth of cancer cells in vitro and in animal models (64). Four demethylating agents have been tested clinically: 5-azacytidine, 5-aza-2'-deoxycytidine (decitabine), 1-{beta}-D-arabinofuranosyl-5-azacytosine (fazarabine), and dihydro-5-azacytidine. The first two show some efficacy in treating leukemia but show little activity against solid malignancies; trials of the latter two were stopped because of lack of efficacy (65). 5-Azacytidine and 5-aza-2'-deoxycytidine are unstable in aqueous solution, necessitating fresh preparation and administration via injection (66); moreover, their therapeutic use is limited by toxicity (64).



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Fig. 1. A) DNA methyltransferase inhibitors. B) Representative histone deacetylase inhibitors.

 
The orally available nucleoside analog zebularine, originally developed as a cytidine deaminase inhibitor, is also a potent inhibitor of DNA methylation (67). Zebularine suppresses growth of human bladder carcinoma xenografts in nude mice in association with reactivation of silenced p16INK4a and may be less toxic than the aforementioned nucleoside analogs (66). Non-nucleoside drugs such as the antiarrthymic procainamide also induce reexpression of methylation-silenced genes (68).

A limited number of preclinical studies have examined the ability of DNA methyltransferase inhibitors to prevent cancer. 5-Aza-2'-deoxycytidine markedly reduced tumor development in ApcMin/+ mice, diminishing intestinal tumor formation by 82%. In Apc-deficient mice also heterozygous for Dnmt1, inhibition of tumor formation with 5-aza-2'-deoxycytidine was greater than 98%. Early administration is essential for chemopreventive activity; inhibition was lost when treatment was initiated at 50 days rather than 7 days of age (69). In other studies, 5-aza-2'-deoxycytidine diminished the formation of aberrant crypt foci in the colons of selenium-deficient rats treated with carcinogen (70) and prevented lung tumor formation in mice treated with a tobacco-specific carcinogen (71). 5-Aza-cytidine also reversed the immortal phenotype of a subset of cultured oral dysplastic cells together with inhibition of telomerase activity and reexpression of silenced RAR{beta} and p16INK4a (72).

The ability of histone deacetylase inhibitors (Fig. 1, B) to modify the epigenome is also being explored. These drugs induce cell-cycle arrest, apoptosis, and/or differentiation in transformed cells in vitro and suppress the growth of a wide variety of solid tumor xenografts with minimal toxicity. Several structural classes of histone deacetylase inhibitors have been identified, including short-chain fatty acids (butyric and valproic acids), hydroxamic acids (suberoylanilide hydroxamic acid, m-carboxycinnamic acid bishydroxamide, trichostatin A, oxamflatin, and pyroxamide), tetrapeptides (depsipeptide), and benzamides (MS-275, which stands for N-(2-aminophenyl)-4-[N-(pyridin-3-yl-methoxycarbonyl)aminomethyl]benzamide and CI-994, which stands for N-acetyldinaline). Phenylbutyrate, valproic acid, suberoylanilide hydroxamic acid, pyroxamide, depsipeptide, MS-275, and CI-994 are in clinical trials, and development of new histone deacetylase inhibitors is an active area of research (2,9,73). Interestingly, only 2%–10% of genes are expressed after exposure to histone deacetylase inhibitors. Activated genes are largely associated with regulation of cell growth and survival, providing a rationale for the antitumor actions of the histone deacetylase inhibitors; however, the mechanism for selectivity of gene activation remains unknown (2,74).

Several studies (75,76) have also examined the chemopreventive activity of histone deacetylase inhibitors. Suberoylanilide hydroxamic acid decreased the incidence and multiplicity of N-methyl-N-nitrosourea-induced rat mammary tumors; unlike 5-aza-2'-deoxycytidine, inhibitory effects were manifest at stages after initiation. Suberoylanilide hydroxamic acid also inhibited hyperplastic nodule formation in response to 7,12-dimethylbenz[a]anthracene treatment in mouse mammary gland organ culture (Mehta R, et al.: unpublished results) and the multiplicity of carcinogen-induced lung tumors in mice (77). However, the agent did not inhibit formation of carcinogen-induced aberrant crypt foci in the rat colon when fed at 300 mg/kg diet (Reddy B, et al.: unpublished results). While the histone deacetylase inhibitor phenylbutyrate diminished development of aberrant crypt foci in rat colon (78), under the conditions tested, it did not inhibit the formation of colon tumors (Reddy B, et al.: unpublished results). Changes in epigenetic regulation may also contribute to the efficacy of a number of well-recognized chemopreventive agents, including {alpha}-difluoromethylornithine (79) and organosulfur (80) and selenium (81) compounds.


    CONCLUSIONS
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 Abstract
 Introduction
 Epigenetic Changes in High-Risk...
 Cancer Prevention by Targeting...
 Conclusions
 References
 
It is clear that epigenetic changes are some of the earliest events observed during cancer development, making them excellent targets for chemoprevention. Populations that may benefit from this strategy include individuals who harbor genetic and/or acquired inflammatory or preinvasive lesions with aberrantly silenced DNA or who harbor infectious agents that alter the epigenome. A generalized increased cancer risk may also be associated with age-related changes in methylation patterns. In the latter, it will be important to determine specifically which age-related methylated genes predispose to cancer. The unique epigenetic fingerprint observed in patients with preinvasive esophageal lesions that progressed to more advanced lesions described above (53) underscores the potential use of epigenetic markers in risk assessment and early detection, and perhaps as surrogate end points in chemoprevention trials (82). The ability to detect aberrantly methylated DNA noninvasively (6,33,83,84) and the high degree of specificity of detection methods (85) make this approach particularly attractive. Moreover, a new microarray-based assay combining gene expression and epigenetic regulation offers the potential to identify the entire spectrum of genes silenced by epigenetic mechanisms during cancer development (86).

However, the observation that precancerous tissues display global DNA hypomethylation [e.g., (87,88)] suggests that a cautious approach must be undertaken in developing epigenetic drugs as cancer preventives. DNA hypomethylation has been associated with chromosomal instability, reactivation of transposable elements (such as retroviral elements), loss of imprinting, and activation of protooncogenes (89). Of concern are several recent reports of increased tumorigenicity and chromosomal instability in mice carrying Dnmt1 hypomorphic alleles (20,90,91). Mice bearing hypomorphic Dnmt1 mutations and lacking the Mlh1 mismatch repair gene, although protected against intestinal tumors, are at increased risk for lymphoma (29). The relevance of these studies to humans is unclear. Unlike mouse cells, human cancer cells lacking DNMT1 exhibit appreciable DNA methylation (92); indeed, DNMT1 and DNMT3b are both needed to maintain DNA methylation and gene silencing in human cancer cells (93). The rare recessive ICF (immunodeficiency, centromeric region instability, facial abnormalities) syndrome is caused by a mutation in the catalytic domain of the DNMT3b gene (94). Despite displaying increased chromosomal breakage, it is notable that patients with ICF syndrome do not appear to be at increased risk for cancer (95).

Given that preventive intervention with epigenetic drugs will likely require long-term administration to large populations at relatively low absolute risk for the development of cancer (96), establishment of acceptable chronic safety is essential. Combining demethylating agents and histone deacetylase inhibitors may provide a means to reduce the side effects associated with the former drugs. Synergy between 5-aza-2'-deoxycytidine and trichostatin A has been achieved in reactivating methylation-silenced tumor suppressor genes (97). Other promising strategies include the use of "synthetic lethality" (98) and the combined use of epigenetic drugs with agents that target reactivated genes—in effect using epigenetic drugs as cellular sensitizers (99). Restoration of response to retinoid signaling using this approach has been observed in acute myeloid leukemia (100) and in colon (101) and breast (102104) cancers. The latter finding is particularly appealing, given that methylation-induced silencing of RAR{beta} is an early event during breast carcinogenesis (33). This approach could be used to reactivate other genes silenced in precancerous tissues that are targets for established chemopreventive agents. For example, the ER gene, which is silenced in a subset of early breast lesions (35), might be targeted by epigenetic drugs in combination with selective estrogen receptor modulators (105).

Permutations of these approaches and continued advancement in understanding the mechanisms involved in epigenetic regulation and how they interact with genetic changes during tumor progression will facilitate development of newer, more efficacious, and safer chemopreventive agents. The observation that epigenetic changes occur across a broad range of tissues during the early phases of cancer development (Tables 17) makes targeting the epigenome a promising and widely applicable preventive strategy.


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Table 7. DNA methylation during cancer development: representative changes at miscellaneous target sites*

 


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 Abstract
 Introduction
 Epigenetic Changes in High-Risk...
 Cancer Prevention by Targeting...
 Conclusions
 References
 

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Manuscript received March 21, 2003; revised October 1, 2003; accepted October 8, 2003.


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