Cancer Epigenetics: Modications, Screening, and Therapy

Annu. Rev. Med. 2008.59:267-280. Downloaded from www.annualreviews.org by CINVESTAV on 05/28/13. For personal use only.

Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones
Department of Urology, Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California 90089; email: jones p@ccnt.hsc.usc.edu

Annu. Rev. Med. 2008. 59:26780 First published online as a Review in Advance on October 15, 2007 The Annual Review of Medicine is online at http://med.annualreviews.org This articles doi: 10.1146/annurev.med.59.061606.095816 Copyright c 2008 by Annual Reviews. All rights reserved 0066-4219/08/0218-0267$20.00

Key Words
DNA methylation, histone modication, CpG islands

Abstract
Deregulation of gene expression is a hallmark of cancer. Although genetic lesions have been the focus of cancer research for many years, it has become increasingly recognized that aberrant epigenetic modications also play major roles in the tumorigenic process. These modications are imposed on chromatin, do not change the nucleotide sequence of DNA, and are manifested by specic patterns of gene expression that are heritable through many cell divisions. We review these modications in normal and cancer cells and the evolving approaches used to study them. Additionally, we outline advances in their potential use for cancer diagnostics and targeted epigenetic therapy.

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INTRODUCTION
DNMT: DNA methyltransferase Nucleosome: the core unit of chromatin, composed of 147 base pairs of DNA wrapped around a histone octamer. It was initially viewed as a structural component of chromatin, but now its composition and position are considered important in the control of gene expression HDAC: histone deacetylase

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During normal development, somatic cells that are descended from a single progenitor, and contain a similar genotype, differentiate to acquire diverse functions and features by expressing and repressing different sets of genes. This process is brought about by modications that affect how the genetic material is packaged and utilized without changing its nucleotide sequence. Importantly, these epigenetic modications are maintained through cell division. The involvement of these modications in cancer states has been increasingly recognized and is the subject of this review.

THE INTERPLAY OF EPIGENETIC MODIFICATIONS

Epigenetic modications can be generally divided into three interacting processes: DNA methylation, histone modication, and chromatin remodeling. DNA methylation is catalyzed by at least three DNA methyltransferases (DNMTs) that add methyl groups to the 5 portion of the cytosine ring to form 5 methyl-cytosine. During S-phase, DNMTs, found at the replication fork, copy the methylation pattern of the parent strand onto the daughter strand, making methylation patterns heritable over many generations of cell divisions. In mammalian genomes, this modication occurs almost exclusively on cytosine residues that precede guaninei.e., CpG dinucleotides. The term CpG applies to both methylated and unmethylated dinucleotides; the p refers to the phosphate moiety that connects deoxycytidine and deoxyguanosine. CpGs occur in the genome at a lower frequency than would be statistically predicted because methylated cytosines can spontaneously deaminate to form thymine. This is not efciently recognized by the DNA repair machinery, so C-T mutations accumulate during evolution. As a result, 99% of the genome is CpG depleted. The other 1% is composed of discrete regions

that have a high (G+C) and CpG content. These regions are called CpG islands (1, 2). CpG islands are mostly found at the 5 regulatory regions of genes, and 60% of human gene promoters are embedded in CpG islands. Although most of the CpG dinucleotides are methylated, the persistence of CpG islands suggests that they are not methylated in the germ line and so did not undergo CpG depletion during evolution (3). Around 90% of CpG islands are estimated to be unmethylated in somatic tissues (4), and the expression of genes that contain CpG islands is not generally regulated by their methylation. However, under some circumstances CpG islands do get methylated, resulting in long-term gene silencing. DNA methylation is essential for normal development, as mice lacking any one of the enzymes responsible for placing the mark die in the embryonic stages or shortly after birth (5, 6). As a silencing mechanism, it plays a role in the normal transcriptional repression of repetitive and centromeric regions, X chromosome inactivation in females, and genomic imprinting (7). The silencing mediated by DNA methylation occurs in conjunction with histone modications and nucleosome remodeling, which together establish a repressive chromatin structure (Figure 1A). The functional link between DNA methylation and histone modications was initially established by studies showing that histone deacetylases (HDACs) are recruited to methylated DNA by methyl-CpG binding proteins (8, 9). Histones, which are the building blocks of nucleosomes, undergo numerous post-translational modications that regulate chromatin structure, gene expression, and DNA repair (10). The most studied histone modications are methylation and acetylation of lysine residues. Until recently, histone methylation was considered a permanent mark placed on chromatin. However, several histone demethylating enzymes have been discovered in recent years (10), and both acetylation and methylation are now

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A
Normal
Exon Exon Repeat

nonmethylated Cytosine

Cancer

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Exon Exon Repeat

methylated Cytosine

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Normal
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nucleosome repressive complex (e.g. PcG)

x
Cancer
Figure 1

methylated DNA binding protein H3K4 methyl mark H3K9/K27 methyl mark acetylation mark

Epigenetic patterns in normal and cancer cells. (A) DNA methylation. In normal cells, nearly all of the CpG dinucleotides are methylated whereas CpG islands, mostly residing in 5 regulatory regions of genes, are unmethylated. In cancer cells, many CpG islands become hypermethylated, in conjunction with silencing of their cognate genes, while global hypomethylation, mostly at repetitive elements, occurs. (B) Chromatin and histone modication. Active genes are associated with acetylation of histone tails, methylation of lysine 4 on histone H3 (H3K4), and nucleosome depletion at their promoters. The promoters of silenced genes (drawn here in conjunction with DNA hypermethylation) become associated with nucleosomes, lose acetylation and H3K4 methylation marks, and gain repressive methylation marks such as lysine 9 or 27 on histone H3, which recruit repressive complexes. Methylated DNA binding proteins link methylated DNA with the histone modication and nucleosome remodeling machineries (not shown).

considered reversible modications catalyzed by enzymes having opposing activities. In general, regions silenced by DNA methylation also show hypoacetylation and hypermethylation of specic histone lysine residues, such as lysine 9 or 27 in histone H3 (10). In contrast, promoters of actively transcribed genes show hyperacetylation of histones H3 and H4, and methylation of lysine 4 of histone H3 (H3K4) (11, 12). DNA methylation and histone modications function in close interplay with nucleosome remodeling and positioning complexes that bind specic histone modications, such as trimethylated H3K4 (13, 14) and methyl CpG binding proteins (15), and move nucle-

osomes on DNA by ATP-dependent mechanisms. Nonmethylated CpG island promoters are usually hypersensitive to nucleases and are relatively depleted of nucleosomes, whereas methylated promoters have nucleosomes on them and are nuclease resistant (16, 17, 17a) (Figure 1B).

CANCER: A MODIFIED EPIGENOME

When a general role for DNA methylation in gene silencing was established more than 25 years ago (18), it was proposed that aberrant patterns of DNA methylation might play a role in tumorigenesis (19). Initial studies
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MicroRNAs: small, noncoding RNA molecules, approximately 22 nucleotides long that bind to the mRNA of target genes to negatively control their expression. MicroRNAs have essential roles in normal development and their expression patterns are linked to cancer development Methylomes: Distinct DNA methylation proles in tumors, tissues, or different cell types CpG island methylator phenotype (CIMP): a trait exhibited by a subset of tumors that show an exceptionally high frequency of methylation of distinct CpG islands

found evidence for a decrease in the total 5-methylcytosine content in tumor cells (20), and the occurrence of global hypomethylation in cancer was rmly established in subsequent studies. Hypomethylation occurs mainly at DNA repetitive elements and might contribute to the genomic instability frequently seen in cancer (20). Hypomethylation might also contribute to overexpression of oncogenic proteins and was shown to be associated with loss of imprinting of IGF2 (insulin growth factor 2), leading to aberrant activation of the normally silent maternally inherited allele. This was found to be associated with an increased risk for colon cancer (21). The mechanisms underlying global hypomethylation patterns are currently unknown. Aberrant hypermethylation at normally unmethylated CpG islands occurs parallel to global hypomethylation (Figure 1A). The CpG island promoter of the Rb (Retinoblastoma) gene, found to be hypermethylated in retinoblastoma, was the rst tumor suppressor shown to harbor such a modication (22). This discovery was soon followed by studies showing promoter hypermethylation and silencing of other tumor suppressor genes such as VHL (von HippelLindau) in renal cancer (23), the cell cycle regulator CDKN2 A/p16 in bladder cancer (24), the mismatch repair gene hMLH1 in colon cancer (25), and many others. On the basis of these ndings, it was proposed that epigenetic silencing of tumor suppressor genes by DNA methylation can serve as an alternative hit to mutation and/or deletion in Knudsons two-hit carcinogenesis model (26). This led to the notion that nding hypermethylated genes would result in the discovery of new tumor suppressors. An example is ID4, a proposed tumor suppressor, which was found to be hypermethylated in hematological malignancies but for which no mutations were detected in tumors (27). The development of large-scale unbiased methods for detecting methylation, such as restriction landmark genomic scanning (RLGS) and array-based techniques (see below), led
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to a urry of studies reporting numerous hypermethylated genes in cancer (see Reference 28 for a partial list). It is now established that aberrant hypermethylation at CpG island promoters is a hallmark of cancer. Notably, not only protein-coding genes undergo these modications; CpG island promoters of noncoding microRNAs were shown to be hypermethylated in tumors, possibly contributing to their proposed roles in carcinogenesis (29, 30). What is the origin for the deregulated methylation patterns in cancer? Initially it was suggested that like genetic mutations, de novo hypermethylation events are stochastically generated, and that the nal patterns observed are a result of growth advantage and selection (30a). However, several observations made in recent years should be noted: First, hypermethylation events are already apparent at precancerous stages, such as in benign tumors and in tumor-predisposing inammatory lesions (31, 32). Second, there seem to be dened sets of hypermethylated genes in certain tumors. These differential methylation signatures, or methylomes, may even differentiate between tumors of the same type, as was recently shown for the CpG island methylator phenotype (CIMP) in colon cancer (33). Third, although many hypermethylated genes have tumor-suppressing functions, not all are involved in cell growth or tumorigenesis. Furthermore, some of these genes are not expressed in the corresponding normal tissue, so their methylation does not result in their de novo silencing in the cancer cells (34; E. Nili Gal-Yam, G. Egger, A. Tanay, P. A. Jones, unpublished data). Thus, although the hypothesis of stochastic methylation and selection is probably true for some cases, the observations detailed above suggest that these patterns may be generated by upstream-acting programs that have gone wrong. Evidence for such a program involving the Polycomb group complexes (PcGs) is emerging. PcGs are protein complexes responsible for maintenance of long-term silencing of genes, which is

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mediated by methylation of lysine 27 of histone H3 at the repressed regions. The enzyme that catalyzes this modication is EZH2, which is known to be upregulated in tumors and involved in tumor progression (35). In embryonic stem cells, repression of a large set of developmental genes mediated by PcGs is thought to maintain these cells in a pluripotent state (36, 37). Several studies have recently shown that these genes are prone to be hypermethylated in cancer, suggesting a functional link between the two repressing systems and lending support to the idea of an epigenetic stem cell signature in cancer (3840). Future studies that analyze global methylation patterns after manipulation of PcG components are needed to provide further insights into the role of this system in aberrant DNA methylation. As discussed above, silenced hypermethylated promoters are generally associated with hypoacetylation of lysine residues on histones H3 and H4 and hypermethylation of lysine 9 or lysine 27 on histone H3, which mediate the formation of a repressive chromatin structure (Figure 1B). Global histone modications are also altered in cancer: Leukemias, colon cancers, and cell lines derived from them exhibit

loss of acetylation at lysine 16 and trimethylation at lysine 20 of histone H4. These changes seem to occur at hypomethylated repetitive elements (41). The mechanisms responsible for alterations of these global patterns are mostly unknown but may involve the disruption of the enzymes responsible for these modications (28).

DETECTION OF EPIGENETIC MODIFICATIONS DNA Methylation

Various approaches exist to study DNA methylation at specic loci (Figure 2). The oldest approach relies on the use of methylation-sensitive restriction enzymes (MSREs), which distinguish between methylated and nonmethylated sites. These were initially used in conjunction with Southern blotting to analyze methylation status at candidate genes. This technique is labor-intensive, requires large quantities of high-quality DNA not readily obtained from tumors, and depends on the existence of the enzymes specic recognition sites. Nevertheless, MSRE-based techniques are also being

DNA methylation

Histone modifications

Bisulfite Conversion
C G T C A C G G

Restriction

Immunoprecipitation

Expression
untreated 5-aza-CdR

Chromatin IP (ChIP)

Bisulfite Treatment
U G T U A C G G

Sonication

RNA Reverse Transcription PCR

Crosslink, Sonication

IP

IP

methyl group

Figure 2 Approaches for detection of epigenetic marks. DNA methylation can be detected by three main approaches: one based on bisulte conversion, which changes the nucleotide sequence depending on the methylation state of cytosines; another based on methylation-sensitive restriction enzymes, which differentially digest methylated and unmethylated DNA; and a third based on pulldown of methylated DNA by 5 -methylcytosine binding proteins. Alternatively, specic activation of genes after treatment with the demethylating agent 5 -aza-2 deoxycytidine identies potentially methylated genes that need to be conrmed by direct analyses. Histone modications are usually detected by chromatin immunoprecipitation. These approaches, initially used to detect modications at candidate regions, have also been adopted for genome-wide studies (see text for details).
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PCR T G T T A C G G

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Oligonucleotide tiling arrays: microarrays on which overlapping oligonucleotides, usually 2550 base pairs long, are printed, covering contiguous regions of the genome. Used to interrogate enrichment of genomic regions that are bound by specic factors or modications

adopted for large-scale analyses, as detailed below. Methods based on bisulte conversion provide the most accurate methylation detection at the genomic-sequence level. Bisulte treatment of DNA results in deamination of nonmethylated cytosines to uracils while methylated cytosines are not altered (42). This change in the nucleotide sequence, reecting the initial methylation pattern, can be interrogated by various methods. Genomic bisulte sequencing, performed after PCR amplication and cloning of the region of interest, is considered the gold standard for methylated cytosine detection; this method gives the exact methylation status for each CpG site. However, because of the large amount of locus-specic amplication and sequencing involved, this is currently not the preferred method for high-throughput methylation analyses. Methylation-specic PCR (MSP) or its quantitative derivatives, such as Methyl-light (42a), amplify converted DNA using primer sets that are specic either for the methylated or unmethylated DNA (43). These sensitive techniques have become the most common methylation detection tools using a candidate gene approach, and they allow for the analysis of small quantities of DNA derived from archived tissue. However, as only totally methylated or totally unmethylated molecules are amplied in these techniques, the exact pattern of methylation is not reected in the result. Additionally, owing to their high sensitivity, rigorous negative (unmethylated) and positive (totally methylated) controls should be used. Other methods based on bisulte-converted DNA, such as MS-SNuPE or pyrosequencing, have been adapted from the eld of single nucleotide polymorphism (SNP) detection; these enable the accurate quantication of methylation at discrete CpG sites within a given region (44, 45). With the realization that aberrant methylation patterns are common in cancer and the advent of genomic technologies to detect them, the eld has moved from candiGal-Yam et al.

date gene approaches to methods that detect methylation on a large scale in an unbiased manner. In restriction landmark genomic scanning (RLGS), the DNA from tumor and healthy tissue is cleaved by methylationsensitive enzymes, radiolabeled, separated by two-dimensional gel electrophoresis with further enzyme digestion, and autoradiographed. Comparison between the normal and tumor gels reveals spots with differential intensity, representing differential methylation and/or copy number at specic loci. Although only 1000 CpG islands can be interrogated in this manner, this was one of the rst techniques that compared global methylation proles in a large number of tumor samples, and a nonrandom and type-specic pattern of promoter hypermethylation was found in tumors (46). Methods relying on microarray technologies have further advanced the study of genomic methylation. An early example was the differential methylation hybridization method (DMH), in which DNA is cleaved by MSREs, labeled, and hybridized to a CpG island array. A differential hybridization signal between normal and tumor DNA reects differential methylation at a specic CpG island (47). More recently developed techniques rely on the ability of proteins or antibodies to bind specically to methylated DNA (48, 49). The methylated DNA immunoprecipitaion (MeDIP) technique, for example, utilizes antibodies that specically recognize 5-methylcytosine to immunoprecipitate methylated DNA, resulting in its enrichment in the sample. Coupling this method with oligonucleotide tiling arrays covering the majority of human promoters (50) or the complete Arabidopsis thaliana genome (51) resulted in the rst high-resolution methylomes to date and promises to be a powerful tool for genome-wide methylation detection in various applications. An alternative approach to detect aberrantly methylated regions relies on the treatment of cells with demethylating compounds such as 5-aza-2 deoxycytidine, which results in the demethylation and transcriptional

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upregulation of specic genes (52). The use of these compounds in conjunction with expression microarrays enables large-scale screening for differentially expressed genes in treated compared to nontreated cells. An advantage of this approach is that it detects functionally relevant changes in methylation, which are assumed to affect the tumorigenic process, rather than simply the hypermethylation itself. However, as elevated expression of a gene after drug treatment could be due to indirect effects of the drug, the actual methylation status of the identied genes needs to be conrmed by other methods such as those described above. Another drawback is that the actual experiments can only be performed on cultured cell lines, which do not necessarily reect the situation in the tumors themselves.

quence reads aligning to a specic genomic locus dened as enrichment at this locus (53). Advantages of this approach are relative ease of analysis, unbiased results, and the fact that the nucleotide sequence of the pulled down fragments is precisely known. Furthermore, rapid developments in sequencing techniques may eventually render ChIP sequencing cheaper and more timely than conventional ChIP-chip (54).

EPIGENETIC DIAGNOSTICS
Early detection and risk assessment remain high priorities in oncology. Ideal tumor markers would have high sensitivity and specicity and be present in sufcient amounts to reveal minimal disease in peripheral samples. Detection of hypermethylated DNA is considered a promising diagnostic tool in cancer because aberrant methylation events are abundant in tumors, occur early in the tumorigenic process, and different cancers exhibit specic hypermethylation patterns. Because they are DNA markers, they are more stable than RNA or proteins. Furthermore, whereas detection of other DNA aberrations such as point mutations often requires examination of different sites within a gene in various patients, promoter hypermethylation usually occurs over the same region of a given gene, simplifying the design of a detection assay. During the past decade, many studies have detected tumor-derived free circulating hypermethylated DNA in plasma or serum of patients with cancer. Additionally, hypermethylated DNA was obtained from various body uids of cancer patients, such as urine, stool, saliva, bronchoalveolar lavage (BAL), sputum, mammary aspiration uid, pancreatic juice, peritoneal uid, and vaginal secretions (55). Many of these samples can be obtained with minimal invasiveness and thus are suitable for large population screening. Most of these studies were performed using the highly sensitive bisulte-based MSP methods and provide a basis for future clinical trials using DNA methylation markers in cancer detection and
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HISTONE MODIFICATIONS
The detection of histone modications largely relies on the existence of high-quality antibodies that recognize specic modication on various amino acid residues of histones. Western blots and immunostaining can be used to detect global levels or localization patterns for these modications in the nucleus. The now commonly used chromatin immunoprecipitation (ChIP) technique enables researchers to measure the enrichment of specic histone modications at dened genomic regions. This technique can be scaled to global studies, mainly by combining it with microarray technology (ChIP-chip). ChIPchip can be used to study modications at dened genomic entities such as promoters or CpG islands, or in contiguous genomic regions or even whole chromosomes using recently developed oligonucleotide tiling arrays. A drawback to ChIP-chip is the inability to study repetitive elements, as their inclusion in the arrays will interfere with hybridizations and skew the results. Additionally, a bias may be introduced by the amplication performed to obtain the large amounts of DNA needed for hybridizations. ChIP-derived DNA can also be sequenced, with the number of se-

Chromatin immunoprecipitation (ChIP): A commonly used method to detect binding of histones, modied histones, or other factors to specic genomic regions. Chromatin is cross-linked and sheared followed by pull down with specic antibodies to the histones and their bound DNA. This is further interrogated by PCR amplication of specic regions or microarray analysis (ChIP-chip)

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DNMTi: DNA methylation inhibitor(s)

surveillance. However, various confounding issues, such as the specicities of the markers for the different tumors, need to be claried. For example, many of the markers, such as RASSF1 and CDKN2A/p16, appear to be methylated in various tumors or preneoplastic conditions and are therefore not tumorspecic. Additionally, methods used for sample collection and methylation detection need to be standardized to achieve sufcient reproducibility of the results. Ideally, one marker could be used for the diagnosis of each tumor type. In prostate cancer, hypermethylation of GSTp1 may be promising in that respect (56). In other cases, highly dened panels of genes will probably be used for screening. One example of the latter is a prospective study in which sputum was collected from individuals who were at high risk for lung cancer but were cancer-free upon entering the trial. Methylation status of six genes predicted the occurrence of lung cancer within two years of trial initiation with a specicity and sensitivity of 65% (57). Although further optimization of this panel is needed to reach sufcient sensitivity and specicity, this study provides a proof of concept for the prospective use of methylation markers in early detection of cancer. DNA methylation markers can also be used for disease classication, and to predict prognosis and response to therapy. For instance, methylation of RASSF1A in many tumors, including lung, breast, and prostate cancers, has been shown to be associated with poor prognosis (58). In another example, neuroblastomas harboring the CIMP phenotype were highly correlated with poor prognosis (59). Metastatic potential can be predicted on the basis of the E-cadherin promoter methylation in breast and oral cancers. In terms of response to therapy, the most compelling example to date is the hypermethylation of the MGMT (O6 -methylguanine methyltransferase) promoter, which increases the sensitivity of glioblastomas to alkylating agents (60). In addition to the study of single genes, large-scale techniques are now generating
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tumor methylation proles, or methylomes, which can be used for molecular classication. Furthermore, high-throughput platforms that can analyze the methylation state of a large number of loci in a large number of samples have been developed. One such recently described technology adapts a highthroughput single nucleotide polymorphism (SNP) genotyping system to detect methylation based on genotyping bisulte-converted DNA (60a). By using this technology, 1500 CpG sites in 400 genes from 96 samples can be analyzed simultaneously. Studies using this technology identied panels of methylation markers that distinguished lung or bladder cancers from their normal counterparts at high specicity (61; G. Liang, E. Wolf, P. A. Jones, unpublished results). These panels are promising in terms of their implementation in DNA methylation analyses in large populations.

EPIGENETIC THERAPY
Because of their dynamic nature and potential reversibility, epigenetic modications are appealing therapeutic targets in cancer. Various compounds that alter DNA methylation and histone modication patterns are currently being examined as single agents or in combination with other drugs in clinical settings. Most DNA methylation inhibitors (DNMTi) that have been clinically tested belong to the nucleoside analog family. These drugs are converted into deoxynucleotide triphosphates intracellularly and are incorporated into replicating DNA in place of cytosine. Their main mechanism of action is probably through trapping of the methyl transferases at sites of nucleoside incorporation (3), which depletes the cells of enzymatic activity, resulting in heritable demethylated DNA. Because incorporation occurs during DNA synthesis, only replicating cells are demethylated by DNMTi (62), which may confer the preference for highly proliferating cancer cells. The hypomethylation that ensues over the following cell division

reactivates various silenced tumor suppressor genes, which is proposed to undermine the antineoplastic properties of the drugs. The prototypes of DNMTi are 5-aza cytidine and 5-aza-2 deoxycytidine. Initially described as cytotoxic agents (63), they were later found to cause DNA demethylation and differentiation and to reactivate silenced genes at much lower doses than those initially used (62). These low doses are now used, mainly for hematological malignancies, leading to better responses and lower toxicity. Both drugs were recently approved by the U. S. Food and Drug Administration (FDA) for the treatment of myelodysplastic syndrome, a preleukemic disease (64). Zebularine is a new addition to the family of nucleoside analogs that has demethylating properties. The drug can be delivered orally, is less toxic than the 5-aza analogs, acts preferentially on cancer cells, and inhibits polyp formation in female APC/MIN-decient mice (65; C. Yoo, P. A. Jones, unpublished results). However, the need for high concentrations of zebularine and its limited bioavailability in primates have slowed its advancement into clinical trials (66). As discussed above, epigenetic silencing is tightly coupled with histone deacetylation. Various compounds that inhibit HDACs have demonstrated antitumor, growth inhibitory, proapoptotic, and prodifferentiation properties (67). One of the universal targets of HDAC inhibitors (HDACi) is the cell cycle inhibitor p21, which is consistently upregulated by treatment with these drugs in conjunction with histone hyperacetylation at its promoter (68). Several silenced proapoptotic genes, which are members of the death receptor pathway, are also targets of HDACi treatment in leukemic cells, resulting in their promoter hyperacetylation and upregulation (69). Notably, tumor cells are almost always more sensitive to HDACi activity than healthy cells (70). It should be emphasized that in addition to their effects on transcription, the antitumoral activity of HDACi is probably mediated by other mechanisms,

such as disruption of higher-order chromatin structure and DNA repair pathways (67). In the clinic, many phase I trials show that these drugs are well-tolerated, and one of the initial HDACi, suberoylanilide hydroxamic acid (SAHA), has recently been approved by the FDA for the treatment of T cell cutaneous lymphoma. More are being developed and tested in clinical trials for both hematological and solid tumors (71). As histone methylation is also a major player in establishing long-term silencing, drugs targeting the enzymes involved in this modication are being developed. For example, 3-Deazaneplanocin A (DZNep) was recently shown to deplete Polycomb group components, inhibit histone H3K27 methylation, and induce selective apoptotic cell death in breast cancer cells (72). In another study, the use of polyamine analogs inhibited the enzyme that removes the active H3K4 methylation mark, resulting in upregulation of aberrantly silenced genes in a cancer cell line (73). The specicities of these drugs and their potential clinical effectiveness need to be carefully established in further studies. As the interplay between epigenetic pathways is unraveled, the combination of epigenetic drugs with each other or with standard chemotherapies has become a focus of interest. HDACi and DNMTi show synergistic effects on transcriptional activation (74), and initial clinical trials using combinations of both have been promising (75). Further randomized trials are needed to prove their synergy in patients. Both classes of epigenetic drugs might sensitize cells to the action of biological agents such as all-trans retinoic acid, standard chemotherapeutics, or potential immunotherapies. Clinical trials using these combinations are ongoing (75). Despite the promise of epigenetic therapy, several concerns remain, mainly stemming from the nonspecicity of the drugs. Induction of genomic hypomethylation in mice caused chromosome instability and promoted tumor formation (76, 77), and the question
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HDACi: histone deacetylase inhibitor(s)

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arises whether the use of hypomethylating drugs will also have carcinogenic effects. One study examining this has not found such effects, although the number of patients was small and the time period short (75). Furthermore, in other mouse models, inhibition of DNMTs prevented tumor development (78). As clinical use of these drugs increases, these concerns will be answered in the coming years. However, the search for more specic drugs targeting epigenetic modications is warranted.
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CONCLUDING REMARKS
With the recognition of the role of aberrant epigenetic processes in cancer and the rapid advent of new technologies to study them, this is an exciting time for the cancer epigenetics eld. National and international collaborations are forming to launch a human epigenome project (79). The ultimate aim of this project would be to map all epigenetic modications, resulting in a comprehensive

description of these in both normal and diseased cells. Additionally, a pilot project to the Cancer Genome Atlas Project was recently launched, which aims to systematically explore the entire spectrum of genomic changes involved in human cancer, including epigenetic changes such as DNA methylation (80). The data derived from these projects will be able to answer questions such as how many genes are actually affected by epigenetic aberrations in a given tumor. They will also shed further light on the underlying mechanisms. Although screening using epigenetic markers is a promising prospect, specic and sensitive screening panels are yet to be developed and tested in large prospective clinical studies. It is important to directly compare the efcacy of these panels with classic screening procedures and other evolving screening techniques based on proteomics, mRNA expression, or microRNA arrays. Knowledge of the prevalence and mechanisms of epigenetic modications will allow the design of rational intervention strategies to target them.

DISCLOSURE STATEMENT
The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

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Annual Review of Medicine

Contents
The FDA Critical Path Initiative and Its Inuence on New Drug Development Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 Reversing Advanced Heart Failure by Targeting Ca2+ Cycling David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13 Tissue Factor and Factor VIIa as Therapeutic Targets in Disorders of Hemostasis Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Therapy of Marfan Syndrome Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43 Preeclampsia and Angiogenic Imbalance Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61 Management of Lipids in the Prevention of Cardiovascular Events Helene Glassberg and Daniel J. Rader p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Genetic Susceptibility to Type 2 Diabetes and Implications for Antidiabetic Therapy Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95 Array-Based DNA Diagnostics: Let the Revolution Begin Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 113 Inherited Mitochondrial Diseases of DNA Replication William C. Copeland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 131 Childhood Obesity: Adrift in the Limbic Triangle Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 147 Expanded Newborn Screening: Implications for Genomic Medicine Linda L. McCabe and Edward R.B. McCabe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 163 Is Human Hibernation Possible? Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 177 Advance Directives Linda L. Emanuel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 187

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Genetic Determinants of Aggressive Breast Cancer Alejandra C. Ventura and Soa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 199 A Role for JAK2 Mutations in Myeloproliferative Diseases Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 213 Appropriate Use of Cervical Cancer Vaccine Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 223 A Decade of Rituximab: Improving Survival Outcomes in Non-Hodgkins Lymphoma Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 237 Nanotechnology and Cancer James R. Heath and Mark E. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 251 Cancer Epigenetics: Modications, Screening, and Therapy Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p 267 T Cells and NKT Cells in the Pathogenesis of Asthma Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p 281 Complement Regulatory Genes and Hemolytic Uremic Syndromes David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 293 Mesenchymal Stem Cells in Acute Kidney Injury Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 311 Asthma Genetics: From Linear to Multifactorial Approaches Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 327 The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in Human Disease Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas, and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 343 Advances in Antifungal Therapy Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p 361 Herpes Simplex: Insights on Pathogenesis and Possible Vaccines David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 381 Medical Management of Inuenza Infection Anne Moscona p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 397 Bacterial and Fungal Biolm Infections A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 415 EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 429 Adaptive Treatment Strategies in Chronic Disease Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 443
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Antiretroviral DrugBased Microbicides to Prevent HIV-1 Sexual Transmission Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 455 The Challenge of Hepatitis C in the HIV-Infected Person David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 473 Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection Luc Geeraert, Gnter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 487 Advancements in the Treatment of Epilepsy B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 503 Indexes
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Cumulative Index of Contributing Authors, Volumes 5559 p p p p p p p p p p p p p p p p p p p p p p p p 525 Cumulative Index of Chapter Titles, Volumes 5559 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 529 Errata An online log of corrections to Annual Review of Medicine articles may be found at http://med.annualreviews.org/errata.shtml

Contents

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