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)
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ABSTRACT |
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INTRODUCTION |
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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.
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EPIGENETIC CHANGES IN HIGH-RISK TISSUES: TARGETS FOR CHEMOPREVENTION |
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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 RAR2 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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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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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 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
, 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
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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Prostate
The GSTP1 gene, which codes for the drug detoxification enzyme glutathione S-transferase , 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
2, RAR
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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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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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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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 EpsteinBarr virus infection (63).
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CANCER PREVENTION BY TARGETING THE EPIGENOME |
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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 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 -difluoromethylornithine (79) and organosulfur (80) and selenium (81) compounds.
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CONCLUSIONS |
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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 genesin 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 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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Manuscript received March 21, 2003; revised October 1, 2003; accepted October 8, 2003.
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