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Mutation Research 693 (2010) 53–60

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

journa l homepage: www.e lsev ier .com/ locate /molmutCommuni ty address : www.e lsev ier .com/ locate /mutres

eview

pigenomics of human colon cancer

. Javier Carmona, Manel Esteller ∗

ancer Epigenetics and Biology Program (PEBC), Bellvitge Institute for Biomedical Research (IDIBELL), 08907 L’Hospitalet, Barcelona, Catalonia, Spain

r t i c l e i n f o

rticle history:eceived 7 October 2009eceived in revised form 13 July 2010

a b s t r a c t

DNA methylation exerts critical effects on gene-expression regulation in normal cells, and alterations onthe normal methylation patterns characterize, and likely contribute, to cancer development and progres-sion. The complete understanding of the distinctiveness, role and dynamics of the cancer cell epigenome

ccepted 26 July 2010vailable online 5 August 2010

eywords:NA methylationpigenomics

has been hindered so far by technical limitations, and new, whole-genome approaches are required.Here we review the methods that have permitted us glimpses of the colon cancer methylome and out-line future directions that will eventually lead researchers to identify the altered epigenetic patterns incancer cells.

© 2010 Elsevier B.V. All rights reserved.

olon cancerenome-wide

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532. Besieging the colon cancer epigenome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543. Face to face with the epigenome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

. Introduction

Disruption of the epigenetic landscape is considered to be a com-on hallmark of many widespread diseases and is a feature of all

ancers so far examined [1]. Research in the last few decades hasielded important advances with the exploration of the epigeneticlterations that occur in different types of tumour and stages ofancer development. Patterns of histone modifications have beendentified as a frequent trait in human cancer [2] and have proved toe of prognostic relevance [3]. MicroRNAs (miRNAs) – the headlin-rs of non-coding regulatory RNAs – have also proved to be relevantlayers, acting both as tumour suppressors and oncogenic, and offerany possibilities in the field of biomarkers and for the design of

argeted therapies [4]. Finally, DNA methylation changes associated

all human genes. It constitutes an essential mechanism that ispresent in normal “healthy” cells. As a regulatory system, thisprocess contributes to the maintenance of genome integrity bykeeping endoparasitic sequences inactive through heavy methy-lation; it maintains tissue-specific expression patterns and definesgenomic imprinting. However, neoplastic transformation distortsthis normal epigenetic state. Aberrant methylation of gene pro-moters can mimic genetic mutation or deletion by abolishingthe expression of genes involved in all cellular pathways, andit is of particular significance in the context of inactivationof tumour suppressor genes, thus allowing malignant progres-sion [5–9]. Interestingly, a mutually relationship between geneticmutations and DNA methylation has largely been observed, sug-

ith cancer are now much more intensively studied than in pastears.

Reversible methylation of cytosines takes place within CG din-cleotides clustered at the promoter region of around 60% of

∗ Corresponding author.E-mail address: mesteller@iconcologia.net (M. Esteller).

027-5107/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.mrfmmm.2010.07.007

gesting that these two molecular events provide comparableselective advantages to affected cells. An example of this effectcomes from the evidence that promoter hypermethylation canact as a “second hit” in many hereditary neoplasms that har-

bour a germline mutation with no identifiable second somaticmutation [9].

Colon cancer has become a paradigm in epigenetic research.Over 25 years ago, Feinberg and Vogelstein reported an exten-sive loss of DNA methylation in colon cancer cells [10], thereby

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pening up a rich seam of research that has since yielded seminalontributions to our understanding of cancer.

It is well recognized that epigenetic abnormalities arise in thearliest steps of colorectal cancer (CRC) development. Aberrantethylation patterns have been identified in preneoplastic lesions

ncluding dysplastic aberrant crypt foci, which are considered pre-ursors of colon cancer, and in hyperplastic polyps, which werehought to be benign in nature, and have been proposed to be aisk factor for CRC development as precursor lesions for the ser-ated adenocarcinoma pathway [11,2]. Abnormal loss of imprintingLOI) of IGF2 is also of special relevance since it is the only alter-tion that has so far been associated with both cancer and normalissue of cancer patients. Strong associations were found betweenOI in peripheral blood lymphocytes and LOI in the colon, and its associated with a five-fold greater incidence of suffering col-rectal neoplasia [13,14]. Although it has not been widely assessedy researchers, candidate-gene approaches indicate that aberrantpG methylation occurs early in cancer, albeit there are signifi-ant differences between the genes affected depending on the typef lesion examined (Table 1 ). Aberrant promoter methylation islready evident at aberrant crypt foci which will progress to morealignant lesions as adenomas/adenocarcinomas, where methy-

ation is observed to increase – in detriment of gene expressionin the genes affected. Analysis of more severe stages have not

een performed so extensively, suggesting that such variations inNA methylation patterns affect cancer cells at initial phases thatill contribute, along with other epigenetic and genetic defects,

o define their final fate. This aberrant methylation is thereforeesponsible, at least in part, for further fostering tumour pro-ression [11,12]. Nevertheless it remains controversial whetherancer-related methylation of certain sequences occurs due to theresence of consensus motifs recruiting DNA methylation machin-ry, as has been previously suggested [15,16], or if, by contrast, itappens randomly but certain phenotypes are under greater selec-ive pressure than others to progress to malignant transformation.

Many attempts have been made to identify the methylationatterns appearing at the various stages of colorectal cancer pro-ression, although the different techniques employed, the unevenriteria for selecting samples and the biased validation methodsave yielded little consensus about the number of loci definingach step of cancer development. Over ten years ago, Toyota ando-workers identified a series of methylation markers whose sta-us defined the CpG island methylator phenotype (CIMP) [17]. Thisroposal emerged as a new pathway for colorectal tumourigenesis,

n addition to the classic mutator or chromosomal instable (CIN) andicrosatellite instable (MSI) categories, standing for a subset of spo-

adic colorectal tumours bearing excessive cancer-specific promoterypermethylation [17]. This molecular subgroup of tumours claimedo group up to 75% of sporadic CRC with MSI, and was initiallyharacterized for exhibiting concordant tumour-specific promoterypermethylation in a series of markers (CDKN2A, hMLH1, THBS1,INT1, MINT2, MINT31). However, CIMP is still a controversial topic

18,19] that will be clarified as research on the field provides newoncluding remarks to support or prove CIMP wrong. The upcomingpigenomic wave will help to refine tumour classification, and willllow the introduction of tailored therapies based on the differentolecular origins.

. Besieging the colon cancer epigenome

Many different strategies are available for assessing the epi-enetic patterns of normal and cancer cells. So far, most studiesddressing aberrant methylation in colorectal and other cancersave focused on genes already known to be involved in cancerathogenesis, especially those for which no genomic mutation had

Research 693 (2010) 53–60

been identified [20]. Using the candidate-gene approach, a plethoraof genes inactivated by promoter hypermethylation in a giventumour has been recognized (Table 1). Although to date, no plat-form has been able to interrogate the methylation status of the 28million CpG dinucleotides of the human methylome at single-baseresolution, it is likely that the number of hypermethylated geneswill grow as the sensitivity and coverage of epigenomic technolo-gies improve, and as unbiased integrative analyses are undertaken.

The first reports to consider DNA methylation events in agenome-wide manner (i.e., by studying a limited number ofgenomic sites that are representative of the genome) reliedon restriction enzyme-based methods suitable for comparinglarge series of samples and the simultaneous identification ofhypomethylation and hypermethylation events. Table 1 gathers arepresentative list of frequent targets of aberrant DNA methylationat different stages of colorectal cancer development, which havebeen identified either by candidate-gene approaches or followinggenome-wide epigenomic approaches [9,17,21–43].

Techniques such as restriction landmark genomic scanning(RLGS), which was the first large-scale method for investigat-ing methylation; differential methylation hybridisation (DMH),which was the first array method to be optimized for identifyingnovel methylated loci in cancer; and amplification of intermethy-lated sites (AIMS) are among the first examples of large-scaleepigenomic techniques. All of them took advantage of restric-tion enzymes that are sensitive to methylated cytosines, whoseuse enables the methylation patterns of different cell populationsto be compared. They provided reliable information about DNAmethylation in normal colon and primary colon tumour tissues[44,45] and in genetically manipulated colon cancer cell lines [28],and defined differential DNA hypermethylation and hypomethyla-tion signatures in CRC by interrogating particular sequences [45].Nevertheless, these ingenious approaches suffer many technicallimitations, including (i) laborious and time-consuming protocols;(ii) their dependence on inaccurate linker ligation and linker PCRamplification, as is the case with DMH, for example; (iii) the limitednumber of sequences that can be interrogated due to the deficientor biased activity of restriction enzymes; (iv) the high rate of false-positive and false-negative results; and (v) the low reproducibilitydue to the great complexity of the methods. Nevertheless, despitethe numerous pitfalls, studies based on these techniques succeededin identifying a surfeit of abnormally methylated loci in cancer, andprovided a picture of particular CpG methylation patterns shared bymany tumour types and of markers exhibiting distinct tumour-typespecificity.

Genetic unmasking strategies opened up a new line of investi-gation in the search for new hypermethylated loci. Specifically, thedisruption of the two major DNA methyltransferases (DNMT1 andDNMT3b) in the colorectal cancer cell line HCT-116 [46] provided auseful tool for identifying differentially methylated loci on a globalgenomic scale, by comparing the methylation profile of the dou-ble knock-out (DKO) with the unmodified HCT-116 cell lines. Ourlaboratory adopted this method to reveal targets of epigenetic inac-tivation in cancer, combining two different techniques previouslydescribed in this article differential methylation hybridisation(DMH), which uses a CpG island microarray enriched in single-copygenes, and amplification of intermethylated sites (AIMS), a PCR-based assay for characterizing anonymous DNA sequences withdistinct methylation patterns [28]. This study identified new targetsof epigenetic inactivation in colon cancer affecting many cellu-lar pathways, including the cadherin member FAT, the homeobox

genes LMX-1 and DUX-4, as well as others whose role in transfor-mation had not been previously characterized [28]. Our group alsoexplored how the microRNA scenario was distorted in colorectalcarcinogenesis in association with aberrant DNA methylation ofthe CpG islands in which they were embedded [47]. By comparing

F.J. Carmona, M. Esteller / Mutation Research 693 (2010) 53–60 55

Table 1Frequent targets of aberrant methylation processes in colorectal cancer. Most of the methylation events identified so far in CRC, have resulted from candidate-gene approaches.However, new epigenomic approaches are identifying new targets of aberrant DNA methylation. Furthermore, current available data suggests that most alterations in thenormal DNA methylation patterns occur at early stages of colorectal cancer development (precursor lesions and adenoma), while only few have been found to happen atmore advanced stages (adenocarcinoma and metastasis).

Histological grade Approach followed Gene symbol Description Role in colorectal tumourigenesis

Precursor lesions Candidate-geneapproach

CDH13 H-cadherin, T-cadherin Aberrant methylation of CDH13 was reported inaberrant crypt foci (ACF), and significanthypermethylation is consistently observed inadenomas. It is a regulator of the dynamic cellularadhesion–deadhesion processes, and its inactivationthrough hypermethylation contributes to thedissemination of cancer cells [17,18]

CRBP1 Retinol binding protein1

Encodes a carrier protein involved in the transport ofretinol (vitamin A alcohol), necessary for growth,reproduction, differentiation of epithelial tissues, andmay contribute to the loss of retinoic acidresponsiveness [18]

DAPK Death-associatedprotein kinase

DAPK hypermethylation was already observed in colonmucosa adjacent to intraepithelial neoplasia orcarcinoma. Its silencing contributes to the early stepsof tumour progression in colorectal carcinoma throughthe inactivation of gamma-interferon inducedprogrammed cell death [19]

IGF2 Insulin-like growthfactor H

Loss of imprinting at this locus is a risk factor for CRCpredisposition. It enhances the expression of VEGF andaffects cell proliferation [20]

MGMT O6-methylguanineDNA methyltransferase

Involved in repairing DNA damage caused byalkylating agents. Cancer-associated hypermethylationcontributes to colorectal field cancerization, andcorrelates with G-to-A mutation in the KRAS oncogene.Hypermethylation-induced inactivation is observed inhistologically normal-appearing mucosa of cancerpatients, and unmethylated in colonic epithelium ofunaffected individuals [8]

TPEF/HPP1 Transmembraneprotein containingepidermal growthfactor and follistatindomain

Aberrant hypermethylation of this gene has beenreported in precursor lesions as hyperplastic andadenomatous polyps, as well as in dysplasia of thecolon mucosa associated with ulcerative colitis [21]

WIF1 Wnt inhibitory factor-1 Tumour suppressor gene which inactivationcontributes to the aberrant activation of Wnt signalingprogram. It has been proposed as a valuable biomarkerin plasma for early detection of CRC [22]

Epigenomic approach DUX-4 Double homeoboxprotein 4

Mediates cell death in development processes [24]

FAT1 Cadherin-relatedtumour suppressor

Adhesion molecule important in developmentalprocesses and cell communication [24]

HOXD1 Homeobox D1 Sequence-specific transcription factor that is involvedin differentiation. Its inactivation occurs inpremalignant lesions and accumulates duringcarcinogenesis [23]

LMX-1 LIM homeoboxtranscription factor 1

Homeodomain protein that binds an A/T-rich sequencein the insulin promoter and stimulates transcription ofinsulin [24]

RASGRF2 Ras protein-specificguaninenucleotide-releasingfactor 2

Implicated in Ras-mediated signaling anddevelopment [23]

SFRP1 Secretedfrizzled-related protein

SFRP family genes are soluble modulators of Wntsignaling. Epigenetic silencing of SFRP genes occurs atprecursor lesions leading to aberrant activation of theWnt pathway [25]

SLC5A8 Solute carrier family 5(iodide transporter),member 8

RLGS revealed hypermethylation of this sodiumtransporter was common in colon adenomas, andcould be also detected in microscopic colonic aberrantcrypt foci [39]

Adenoma Candidate-geneapproach

APC Adenomatouspolyposis coli

Tumour suppressor gene, antagonist of the Wntsignaling pathway. Involved in cell migration andadhesion, transcriptional activation, and apoptosis [26]

COX-2 Prostaglandin-endoperoxide synthase2

Involved in inflammation and mitogenesis, tumourangiogenesis acting through VEGF, and metastasis [27]

Cyclin A1 Cyclin A1 Binds to important cell cycle regulators, such as Rbfamily proteins, transcription factor E2F-1, and the p21family proteins [28]

56 F.J. Carmona, M. Esteller / Mutation Research 693 (2010) 53–60

Table 1Continued.

Histological grade Approach followed Gene symbol Description Role in colorectal tumourigenesis

GATA4 GATA binding protein Potential biomarker in stool. Suppresses colonyformation, proliferation, migration, invasion, andanchorage-independent growth of colorectal cancercells [29,40]

GATA5 GATA binding protein Suppresses colony formation, proliferation, migration,invasion, and anchorage-independent growth ofcolorectal cancer cells [29]

HIC-1 Hypermethylated incancer-1

Modulates the transcriptional stimulation of genesregulated by Wnt/beta-catenin signaling. Inactivationin cancer leads to abnormal cell proliferation at earlystages [30]

hMLH1 mutL homolog 1 Implicated in DNA repair, its inactivation causesmicrosatellite-unstable tumours, mainly in sporadiccases. A higher frequency of aberranthypermethylation is observed in patients with a familyhistory of CRC [31]

p14ARF Cyclin-dependentkinase inhibitor 2A

Tumour suppressor gene, stabilizer of the tumoursuppressor protein p53 and involved in cell cyclecontrol [32]

p16INK4a Cyclin-dependentkinase inhibitor 2A

Tumour suppressor gene, negative regulator of cellgrowth and proliferation in the G1 phase of the cellcycle, and is an important regulator of the angiogenicswitch. Hypermethylation of these gene was detectedin a high percentage of adenomas [33]

RARB2 Retinoic acid receptor Tumour suppressor gene that mediates thegrowth-inhibitory action of retinoic acid. Its silencingpromotes cell growth and tumour progression [34]

RASSF1A Ras association(RalGDS/AF-6) domainfamily member 1

Tumour suppressor gene that inhibits theaccumulation of cyclin D1, and thus induce cell cyclearrest. Methylation of this gene is an early event inCRC. Hypermethylation was related to wild-type KRAS,except for MSI + CRC in which concomitant events areobserved [32]

TSP1 Thrombospondin-1 Angiogenesis inhibitor. Reported to behypermethylated in a small percentage of adenomas,but increasing along with histological grade,subsequently impairing the TGF-beta signalingpathway [35]

Adenocarcinoma Candidate-geneapproach

DKK-1 Dickkopf-1 Extracellular Wnt inhibitor hypermethylated inadvanced colorectal neoplasms [36]

HLTF Helicase-liketranscription factor

Encodes a chromatin remodelling factor withgrowth-suppressive effect in cancer cells [37]

Epigenomic approach miR-34b/c Tumour suppressor microRNA down-regulatesoncogenic target genes such as C-MYC, E2F3 and CDK6.Inactivation of this microRNA correlates with lymphnode metastasis [38]

miR-148a Tumour suppressor microRNA targets TGIF2 oncogene.Inactivation of this microRNA correlates with lymph

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NA expression profiles of HCT-116 wild type and the DKO cell linee explored the presence of DNA methylation-associated silenc-

ng of miRNAs in colon cancer cells. Genetic screening revealedhat 18 of the 320 human miRNAs included in the array showed

inimal basal expression in the HCT-116 wild-type cell line thatas increased greater than three-fold in the DKO. In particular, theethylation of miR124a was shown to be effectively repressed in

ancer cells, which in turn enabled CDK6 upregulation, a bona fidencogenic factor. The study provided an illustrative example of howhese regulatory molecules were subject to epigenetic silencing47] (Table 1).

Other methods have exploited the plasticity of epigeneticodifications. Pharmacological unmasking of methylated-inactive

enes by use of the deoxycytidine analogue 5-aza-2′-deoxycytidine5-aza), which is widely employed as a DNA methylation inhibitoro induce gene expression by covalent trapping of DNMT, and ofistone deacetylase inhibitors, such as trichostatin A (TSA), in con-

unction with global gene-expression analysis, have proved to be

node metastasis [38]Hypermethylation-associated inactivation of thismicroRNA correlates with lymph node metastasis [38]

valuable strategies. The CRC epigenomic approach guided us andothers to identify upregulated genes that were kept in a “dor-mant” state in cancer cell lines [41,48,49]. Suzuki et al. identifiedgenes inactive in untreated cells whose reactivation was responsiveeither to 5-aza combined with TSA, or to TSA alone. This indicateda predominant role for DNA methylation in transcriptional repres-sion, although it could also be achieved by histone deacetylationin completely unmethylated promoters [49]. The study also sup-ported the concept of “field effect” observed in histological normalmucosa, in which premalignant alterations were found in a wideregion of the colon, as is reflected by the tendency of certain CpGislands to undergo hypermethylation in normal-appearing mucosa.Nevertheless, it is important to note that due to the pleiotropic

effects produced by these epigenetic drugs, many genes not directlyaffected by aberrant methylation – or not even epigenetically reg-ulated – were transcriptionally over-activated, owing to the factthat upstream transcription factors were turned on, for instance.This leads to quite a high frequency of false-positive results [50].

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ubsequent studies have identified a substantial panel of genesisplaying cancer-specific promoter hypermethylation in colorec-al and other tumours [48,51,52], and also in tumour suppressor

icroRNAs whose inactivation by hypermethylation contributeso the development of metastases [41] (Table 1).

In spite all the achievements attained with the aforementionedtrategies, a comprehensive, large-scale understanding of the can-er epigenome remains elusive [53]. This turns to be of specialomplexity since there is no one unique cancer epigenome, butifferent settings depending on tumour types and histologicalrade. The first group of approaches based on the digestion ofenomic DNA with methylation-sensitive restriction enzymes, lim-ted methylome profiling to the particular sequence motifs bearingpecific restriction sites. On the other hand, the employment ofenetic and pharmacological unmasking approaches lacked speci-city in finding functionally relevant hypermethylated genes withssociations with cancer. In order to overcome these constraints,everal techniques involving the use of specific antibodies orethylation-binding proteins have been designed (Fig. 1B).Specifically, direct immunoprecipitation of methylated DNA

MeDIP) using a monoclonal antibody against 5-methylcytidine5mC) has turned out to be a suitable technique for the paral-el comparison of two populations in the search for differentially

ethylated loci, as in the case of HCT-116 wild type and DKO cells54]. In addition, coupling this application with standard bisulphiteenomic sequencing has enabled the identification of a large num-er of genes with hypermethylated-CpG islands in colon cancer andther tumour types [27,54,55]. Some limitations of this approachre the bias towards higher enrichment of CpG-rich sequencest the expense of CpG-poor sequences, and the difficulties ofaking MeDIP compatible with next-generation sequencing as a

tandard procedure [56]. MeDIP-sequencing (MeDIP-seq) yieldsamples of enormous complexity that require an excessive amountf sequence reading to give in sufficient coverage at unique genomicegions. Recently, a cross-platform algorithm for the quantitativenalysis of the MeDIP data generated using arrays (MeDIP-Chip)r genomic-sequencing platforms (MeDIP-seq) has been reported57], but its practical implementation on a routine basis remains toe established.

The advent of the bisulphite treatment of DNA, which convertsytosine residues to uracil, but leaves 5-methylcytosine residuesnaffected, was a fundamental contribution to cancer epigeneticsesearch [58–60]. The implementation of this technique in conjunc-ion with genome sequencing – bisulphite sequencing – or PCRmplification – methylation-specific PCR (MSP) – [61] allows theensitive and fast examination of DNA methylation of any sequence,nd requires only very small amounts of material. This revolution-ry contribution allowed epigenetic studies on CRC to be extended,nd key discoveries were made, including the epigenetic inactiva-ion of all four genes of the secreted frizzled-related proteins (SFRP)amily and other Wnt antagonists in CRC, including DKK-1 and

NT5A, which allow constitutive activation of the Wnt pathway29,39,62] and the inappropriate silencing of the so-called epige-etic gatekeepers GATA-4 and GATA-5 [43], and ˇ-catenin, whichacilitates the expansion of cancer cells and eases the way for geneticatekeeper mutations to appear [63]. Bisulphite sequencing of generomoters also permitted a detailed and high resolution study ofhe coverage of methylation at the CpG-rich promoters, which inurn opened up new avenues of investigation involving nucleo-ome positioning [64] and methylation profiling [65]. In agreementith these latter studies, the depiction of specific profiles is neces-

ary to refine the classification of tumours [66] in order to improveiagnosis and to develop tailored treatments.

Also, the promoter CpG island hypermethylation in differentuman cancers was comprehensively analyzed using bisulphiteequencing [65], which, to date, has been considered thegold-

Research 693 (2010) 53–60 57

standard technique for directly studying DNA methylation and forvalidating results obtained using other approaches.

The use of standardized procedures to scan the entireepigenome, the availability of well-classified samples and com-prehensive data management will clarify the degree and scopeof epigenetic disruptions in the transformation cascade, providingnew insights into cancer biology and endowing physicians withnew tools for the better treatment of their patients. From a clin-ical point of view, the discovery of methylation biomarkers withinformative value regarding cancer diagnostics, or predicting prog-nosis and response to therapy, represents a promising alternative tocurrent invasive procedures or imaging techniques, following timeand low-priced protocols. The most reliable techniques employedin a clinical setting are based on the methylation-specific PCR(MSP) principles, and the notable range of derivatives. The origi-nal MSP is an inexpensive technique that uses small amounts ofbisulphite-converted DNA to provide qualitative information aboutthe methylation status of a given region, and has been–and still is-widely used in research. At a clinical level, MSP is being imple-mented to conduct routine diagnostic tests. Such is the case fordetecting MGMT methylated promoter for patient stratification inglioblastoma multiforme, since patients with inactive MGMT due toaberrant hypermethylation have been associated with significantlygreater long-term benefit from treatment with alkylating therapiesthan patients with an unmethylated MGMT promoter [67]. DNAmethylation analyses are also of current use to diagnose Prader-Willi syndrome, a disease provoked by an imprinting disorder. Inthe case of colorectal cancer, methylation markers have not yetreached the clinic, but are being examined in large prospective trials– SYNE1 and FOXE1, among others.

Several new PCR-based methods for detecting DNA methylationusing bisulphite-treated DNA are now complementing traditionalPCR-based techniques by increasing the analytical sensitivity andproviding quantitative information. For instance, methylation-sensitive melting-curve analysis (MS-MCA) takes advantage of thedifferential resistance of DNA to melting, depending on the relativeGC content. In presence of a fluorescent intercalating chemical, thePCR is conducted and the dye is then integrated in the ds-DNA. Amelting analysis performed straight-on after the amplification willdifferentiate the products based on their sequence – which in turn isproportional to the methylation status of the original DNA – as twodifferent temperature peaks should be noticed [68]. In an ideal sit-uation, DNA fully or void of methylation will be clearly discernible,however heterogeneously methylated DNA can be more tricky toanalyze. An improvement of this technique, also based on melt-ing analysis of PCR amplicons, is the high-resolution melting-curveanalysis (MS-HRM). The improvement with regard to MS-MCA areincreased sensitivity, the capacity to adapt it for high-throughputanalyses, and better results studying heterogeneously methylatedDNA by employing limiting dilutions [69]. Alternative approachesbased on the fundamentals of MSP have also experienced technicaladvances. The MethyLight technology was developed as the quanti-tative version of the MSP technique [70], providing a high specificityand sensitivity in the detection of DNA methylation in a high-throughput fashion by using fluorescent DNA methylation-specificprobes. Research on biomarkers for cancer detection has takenadvantage of this technique to identify collections of DNA markersthat are frequently aberrantly methylated in tumours compared totheir normal counterpart tissue. Particularly in colorectal cancer,several groups have identified methylation biomarkers obtainedfrom faecal DNA or blood samples with informative diagnosticvalue in cancer detection [71–73]. It is likely that these methodolo-gies will eventually be incorporated into clinic for routine analysis.

They have already proven their accuracy and analytical sensitivity,and await for informative panels of biomarkers to be implementedfor specific, straightforward and cost-effective clinical tests.

58 F.J. Carmona, M. Esteller / Mutation Research 693 (2010) 53–60

Fig. 1. Different approaches to address the cancer epigenome. (A) Specific-sequence methylation analysis techniques such as Illumina methylation assay, interrogate particularCpG sites making the assumption that the island in which they are embedded display similar methylation. This bead array matrix permits the investigation of the methylationof more than 1,500 CpG sites along more than 800 genes for which a relation between cancer and DNA methylation has been spotted; another version broadens the analysisto more than 27,000 CpGs covering more than 14,000 genes and 110 microRNAs. A different highly sensitive and specific platform available is CHARM (comprehensive high-throughput array-based relative methylation), which is independent of bisulfite conversion of DNA, and gives quantitative measurements of DNA methylation across thegenome, excluding repetitive sequences, and extending the analysis beyond canonical CpG islands. The data provided by any of these platforms has to be carefully analyzedand validated through either bisulfite sequencing or pyrosequencing techniques. (B) Genome-wide approaches do not, a priori, delimit the sequences to be analyzed. ForM richm( witha s an ip g.

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ethyl-DNA-Immunoprecipitation (MeDIP), and MIRA techniques, there is an enMeDIP) or methyl-binding proteins (MIRA). This techniques can be then combinedppropriate tools for targeting the epigenome, although cost-effectiveness remainrocedures can be applied to reduce the complexity of the sample before sequencin

. Face to face with the epigenome

Thus far, studies that have attempted to decipher the epigenomef colon cancer cells have only provided local sketches of DNAethylation patterns at certain loci. With the development of new

equencing technologies, coverage and resolution are substantiallyncreased, and the large-scale analyses of normal and cancerousamples are providing laboratories throughout the world with newools to identify genes that have escaped detection by lower reso-ution or restricted approaches [58].

Recently, many array platforms have emerged that permithe interrogation of DNA methylation in a genome-wide context.xamples include the Illumina Goldengate and Infinium arrays [74]Fig. 1A), which were originally designed for genome-wide analysisut have been adapted for methylation analysis; the methylated-pG island recovery assay (MIRA) [75]; and the HELP assay (HpaIIiny fragment Enrichment by Ligation-mediated PCR) [76]. Their

fficiency in examining DNA methylation in diverse normal andancer tissues and cell lines varies due to technical variations andalidation to prove the results is still a must.

In the past years methylation of non-canonical CpG islands haseen associated with tissue-specific expression patterns [77], and

ent in methylated DNA after the selective binding of 5-methylcytosine antibodyhigh-throughput sequencing techniques. (C) Next-generation sequencers providessue. There are multiple sources from which DNA can be obtained, and different

even more strikingly, DNA methylation profiling of chromosomes6, 20 and 22 revealed that a considerable number of genes ana-lyzed showed an inverse correlation between the methylation ofthese “non-canonical” CpG-rich regions and active transcription[78]. Following this line of evidence, a pilot study of the colon cancerhypermethylome was recently published by Feinberg’s labora-tory, employing a comprehensive high-throughput array for relativemethylation (CHARM) approach. They could identify an alterna-tive mechanism of DNA methylation-mediated regulation to thatof CpG island promoter regions, based on the existence of the so-termed “CpG shores” [79]. These are regions located between 200and 2000 kilobases away from the canonical islands that representrelevant regulatory regions for gene activity. Genes associated tothese CpG shores showed a clear inverse correlation between tran-scription and methylation levels of these newly identified regions,rather than with that of the gene promoter CpG island. Interest-ingly, the authors discovered that these shores represent more than

75% of the tissue differential methylation regions in the genomecompared to a 6% ascribed to the classic islands. Analyzing cancer-specific DNA methylation, investigators found similar rates of hyperand hypomethylation (44% and 56%, respectively), and impor-tantly, regions undergoing aberrant methylation in cancer show

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F.J. Carmona, M. Esteller / Mu

significant overlap with tissue-specific methylation regions. Theypermethylation spread to include CpG islands – in agreement tohat is generally reported in cancer – whereas hypomethylation

xpanded 2–3 kilobases away from the islands. These findings givenew twist on epigenetic research since new pathways affected bypG-shore methylation are likely to be identified offering furtherhances for the design of targeted therapies, and the depiction ofew biomarkers for a better clinical management of the disease.oon after the first report on non-canonical CpG islands methyla-ion, another article came out reporting methylation in a non-CGontext inherent to embryonic stem cells and absent in differ-ntiated cell types. These latest findings are surely raising thexpectations to see what comes out from the epigenomic analysesurrently under development.

In 2008, several next-generation sequencing platforms werentroduced onto the market: 454 FLX, the Illumina-Solexa Genomenalyzer, the ABI SOLiD system and HeliScope [80] (Fig. 1C). Theseew sequencing technologies are based on, for instance, pyrose-uencing using millions of picolitre-scale reactions, sequencing byynthesis and sequencing by ligation [81]. They are able to sequencep to 2 gigabases of DNA (compared with the human genome,hich is made up of ∼3.1 gigabases) in a single experiment. They

re proposed as the new tools with which to engage with thempending era of epigenomics. Furthermore, novel DNA sequenc-ng techniques under development in many research groupsround the world could eventually make it possible to sequencecomplete genome in less than one day for under $1000. The

ombination of these whole-genome sequencing techniques withmplemented epigenomic analysis will add a new dimension topigenetic research. Bayley and co-workers have developed a newethod for sequencing large amounts of DNA using a nanopore-

ased device coupled to an exonuclease enzyme [82]. This has theotential to accomplish genomic sequencing of DNA at single-baseesolution in a cost-effective manner, allowing methylcytosineesidues to be identified and thereby the accurate analysis of DNAethylation as it truly is. The concurrence of epigenomics with

ther “omics”, such as transcriptomics and proteomics, togetherith the unifying power of systems biology, will, before too long,rovide an integrated overview of the relevance of DNA methyla-ion in cancer.

In summary, it is evident that DNA methylation in disease andpecifically in cancer is still a dense and tangled affair that willequire huge efforts to unravel. Avant-garde technologies are incor-orating the latest discoveries in order to provide researchers withost-up-to-date instruments. Further studies are therefore needed

o gain insight into the distribution of DNA methylation, its role inancer pathogenesis and the existence of common patterns in thestablishment of aberrant epigenetic changes in cancer cells andther pathologies.

onflict of interest

None.

unding

This work was supported by Grants FIS PI08-1345, ConsoliderSD2006-49, CANCERDIP FP7-200620, S and Dr Josef Steiner Canceresearch Award. The study sponsors had no involvement in thetudy design; collection, analysis and interpretation of data; theriting of the manuscript; decision to submit the manuscript for

ublication.

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