Identification of driver and passenger DNA methylation in cancer by epigenomic analysis

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with limited knowledge of specific genes and genomic regions associated with

hypomethylation. However, a cancer-causing role of DNA hypomethylation is clearly

suggested by studies in mice carrying hypomorphic alleles of DNA methyltransferase genes,

i.e. Dnmt1 (Gaudet et al., 2003). These mice develop malignancies, in particular lymphomas

and hepatocellular carcinoma but the effect of Dnmt1 loss can be complicated and may either

support or inhibit tumor development (Laird et al., 1995; Gaudet et al., 2003; Yamada et al.,

2005). The mechanisms how DNA hypomethylation is tumor-predisposing are unknown but

it is conceivable that reactivation of methylation-silenced repetitive DNA elements and

increased genomic instability are involved (Ehrlich, 2002). Most of the literature available on

epigenetic factors in initiation and progression of tumorigenesis is dealing with

hypermethylation of CpG islands or gene promoters and so is this review.NIH-PA Author Manuscript

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NIH-PA Author ManuscriptAberrant DNA methylation in cancer – starting from single gene studiesDNA methylation of promoter CpG islands is strongly associated with gene silencing and isknown as a frequent cause of loss of expression of, for example, tumor suppressor genes aswell as other genes involved in tumor formation. Much of what is known today about theimportance of DNA methylation in cancer was gained earlier through small-and moderate-scale analysis of gene promoters in different tumor types. The very first methodologiesemployed for the analysis of DNA methylation depended on initial digestion of DNA withmethylation-sensitive restriction endonucleases followed by Southern blotting (Bird andSouthern, 1978). Later on, sodium bisulfite sequencing and other methods based on that sameconcept became the methods of choice for single gene analysis (Frommer et al., 1992).Initial focus on DNA methylation in tumors was centered on the question of methylation-induced silencing of known tumor suppressor genes. During tumorigenesis, both alleles of atumor suppressor gene need to be inactivated. This can occur by chromosomal deletions orloss-of-function mutations affecting the gene's coding sequence. Alternatively,hypermethylation of CpG islands spanning the promoter regions of tumor suppressor genes

(for example, RB, CDKN2A, VHL, APC, MLH1, RASSF1A and BRCA1) can lead to gene

silencing and thus can be an integral mechanism in tumorigenesis equivalent to gene loss or

mutation (Issa, 1999; Costello et al., 2000; Dammann et al., 2000; Jones and Baylin, 2002;

Herman and Baylin, 2003; Nephew and Huang, 2003). Since hypermethylation generally leads

to permanent inactivation of gene expression, and is thought to be less reversible than altered

histone modifications, this epigenetic alteration is considered a key pathway for long-term

silencing of genes. To give some examples on one particular type of tumor, we focus on lung

cancer. Several specific CpG-island-associated gene methylation events were frequently

observed including, for example, CDKN2A, RASSF1A, RARβ, MGMT, GSTP1, CDH13, APC,

DAPK, TIMP3, along with many other genes (Zochbauer-Muller et al., 2001; Toyooka et al.,

2003; Yanagawa et al., 2003; Franklin, 2004; Topaloglu et al., 2004; Dammann et al.,

2005b; Kim et al., 2005). Genes altered by DNA methylation include those involved in

important cellular pathways such as cell cycle regulation (e.g. CDKN2A, CHFR), proliferation

(e.g. CDKN2A, CXCL12), DNA repair (e.g. MGMT), apoptosis (e.g. DAPK, caspase 8, FAS,

TRAILR1), RAS signaling (RASSF1A, NORE1A), invasion (e.g. cadherins, ADAMTS1, TIMP3,

PTGER2, laminin family) and Wnt signaling (APC, DKK1, SFRP genes). Some of these

pathways affected by epigenetic change are those described as the hallmarks of cancer

(Hanahan and Weinberg, 2000). Other studies of non-small cell lung cancer (NSCLC)

identified many additional hypermethylated genes (e.g., ARPC1B, DNAH9, FLRT2, G0S2,

IRS2, RUNX3, PKP1, SPOCK1, UCHL1, OTX1, BARHL2, MEIS1, and OC2) (Bowman et

al., 2006; Rauch et al., 2007; Rauch et al., 2008; Jin et al., 2009). In the literature, the

methylation frequency (i.e., the percentage of tumors analyzed that carry substantially

methylated alleles) generally ranges from only a few percent for some genes to more than 80%

for other genes. The reported methylation frequencies, even for the same genes, often differ

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substantially depending on the study population, tumor histology, and/or methodology used to

assess CpG island methylation.

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NIH-PA Author ManuscriptThe choice of methylation targets analyzed in the numerous single gene studies has often beenbased on existing knowledge of the presumed function of a particular gene or gene familymember, or it was the result of a more or less serendipitous discovery of a particular methylationevent. In most cases, CpG islands overlapping the 5' gene ends or promoters of genes havebeen analyzed. More recent unbiased genome-wide studies, however, have revealed commontumor-associated methylation of CpG islands outside of promoter regions, and it is still unclearwhether or not such methylation changes have biological consequences and what exactly theseconsequences are for tumor formation. Interestingly, there are often cancer-specificallymethylated CpG islands not associated with any known genes at all. These CpG islands mayrepresent remote regulatory elements or may represent functionally relevant sequencesassociated with non-coding RNAs.Aberrant DNA methylation in cancer – genome-wide studiesA much better understanding of the role of DNA methylation in cancer, either as a marker ofdisease or as an active driver of tumorigenesis, will likely be gained from genome-wide studiesof this modification in normal and malignant cells. This goal has become more reachable withthe recent introduction of large-scale genome analysis methodologies. These techniques havebeen adopted in various ways to allow for investigation of DNA methylation of many geneloci simultaneously (Table 1). In this section, we review several technological advances ingenome-wide methylation profiling.One of the earliest large-scale methylation profiling techniques developed was methylation-sensitive representational difference analysis (MS-RDA) (Ushijima et al., 1997; Smith andKelsey, 2001; Ushijima and Yamashita, 2009). Genomic DNA is predigested using themethylation-sensitive restriction enzyme HpaII, and a mixture ratio of tester and driver DNAs

is optimized to detect differences in methylation status of single copy genes between two tissue

samples. Restriction endonuclease digestion-based DNA methylation analysis was modified

by Huang and colleagues and developed as differential methylation hybridization (DMH) on

array platforms by combining restriction endonucleases and microarrays for high-throughput

analysis of the methylation status of CpG islands in human genomes (Huang et al., 1999; Yan

et al., 2001; Wei et al., 2002). This method utilizes a restriction enzyme MseI, which recognizes

TTAA, a sequence that is rarely present within GC-rich regions, and leaves most CpG islands

intact. MseI-generated fragments are ligated to defined synthetic linkers and are further

digested, for example, with BstUI, a methylation-sensitive restriction endonuclease. BstUI

recognizes and digests the sequence 5'-CGCG within CpG islands when they are unmethylated.

CpG islands, which are methylated, resist BstUI restriction digestion, and these methylated

fragments can be subsequently amplified by linker-dependent PCR. The resulting PCR

products are labeled with fluorescent dyes. To compare genome-level CpG island methylation,

equal quantities of BstUI-digested amplicons from two samples (e.g., normal and cancer) are

mixed and hybridized onto a microarray. The resulting ratio between the two dyes represents

the methylation difference between the two samples.

There are several other methods that are based on restriction endonuclease digestion, such as

classical restriction landmark genomic scanning (RLGS) (Hatada et al., 1991; Costello et al.,

2000), or HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) (Khulan et al.,

2006; Figueroa et al., 2009). Nouzova et al (Nouzova et al., 2004) and Lippman et al (Lippman

et al., 2005) developed a DNA methylation profiling technique by replacing BstUI or HpaII

with McrBC, an unusual restriction enzyme that recognizes and cleaves CpG-methylated DNA.

Sites on the DNA recognized by McrBC consist of two half-sites of the form (G/A)mC. These

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NIH-PA Author ManuscriptPage 4half-sites can be separated by up to 3 kb, but the optimal separation is ~50–100 base pairs. Thismethod was used to identify a number of hypermethylated regions in an acute promyelocyticleukemia cell line compared to normal peripheral blood mononuclear cells (Nouzova et al.,2004). Irizarry et al modified the McrBC assay and developed a comprehensive high-throughput array analysis for relative methylation (CHARM) (Irizarry et al., 2008). Theunmethylated or methylated fractions can be analyzed on microarray or high-throughputsequencing platforms for genome-wide identification of aberrant methylation. Methylated CpGisland amplification (MCA) coupled to microarrays also is based on methylation-sensitiverestriction enzymes. Target sequences are amplified by PCR using flanking primers followedby sequence analysis or microarray probing. MCA is a powerful approach for simultaneousidentification of differentially methylated genomic regions (Toyota et al., 1999; Estecio et al.,2007).Genome-scale DNA methylation analysis by bisulfite conversion of DNA has now becomepossible. In bisulfite conversion of DNA, treatment of DNA with sodium bisulfite convertsunmethylated cytosines to uracils, whereas methylated cytosines are not affected. The bisulfite-treated samples are then PCR-amplified and unmethylated and deaminated cytosines arereplaced by thymines during PCR. Then, these samples can be hybridized to microarrays forlarge-scale analysis of DNA methylation status (Gitan et al., 2002; Hou et al., 2004) or byIllumina sequencing (Lister et al., 2009; Gu et al., 2010; Laird, 2010). This approach still isexpensive when applied to whole mammalian genomes and requires substantial computationalresources. Variations of bisulfite-based approaches include analysis of sub-areas of the genome(Meissner et al., 2008; Ball et al., 2009), or a highly multiplexed PCR-based approach usingthe Illumina bead platform (Bibikova et al., 2006).A third general type of high-throughput approach in methylation analysis is based on affinity-purification of methylated DNA. Methylated DNA immunoprecipitation (MeDIP) utilizesnonspecific fragmentation of the genomic DNA followed by anti-5mC antibody precipitation

to enrich for methylated DNA fragments (Weber et al., 2005). The immunoprecipitated DNA,enriched in hypermethylated sequences, and total genomic DNA (as input) are labeled withfluorescent dyes Cy5 and Cy3, respectively, and cohybridized onto microarray chips oranalyzed by high-throughput sequencing. MeDIP is thus a valuable general fractionationapproach, compatible with different analysis platforms to query the level of methylation ingenomic sequences at a level of resolution of about 100 bp. One of the crucial factors in thisassay is the quality of the anti-5-methylcytosine antibody. Moreover, the MeDIP method ismost sensitive for densely methylated sequences, as DNA fragments with many contiguousmethylated CpGs are more efficiently precipitated. MeDIP requires effective DNA

denaturation before antibody binding.

Affinity purification of methylated DNA by a protein or peptide that can specifically bind tomethylated CpGs was initially reported by Cross et al (Cross et al., 1994). Among the methodsmost suitable for genome-wide mapping of DNA methylation, the methylated CpG islandrecovery assay (MIRA) represents an approach that is based on a methyl-CpG binding proteincomplex. MIRA depends on the fact that the methyl-CpG-binding protein MBD2B specificallyrecognizes methylated CpG dinucleotides (Hendrich and Bird, 1998) and that this interactionis strongly enhanced by the MBD3L1 protein (Rauch and Pfeifer, 2005; Rauch et al., 2006;Rauch et al., 2007), a heterodimerization partner of MBD2 (Jiang et al., 2004). Among allmethyl-CpG-binding proteins known, MBD2B has the highest affinity for methylated DNAand displays the greatest ability to distinguish between methylated and unmethylated DNA. Itrecognizes a wide range of methylated CpG sequences with little sequence specificity (Fragaet al., 2003). In our lab, lack of a defined sequence specificity of the MBD2B/MBD3L1complex was confirmed by cloning and random sequencing of MIRA-enriched DNAfragments. Pulldown of methylated fragments is most efficient when a minimum of two

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methylated CpG sites are present (Rauch et al., 2006). In the MIRA procedure, sonicated

genomic DNA is incubated with the MBD2B/MBD3L1 protein complex. Unlike the MeDIP

technique, which requires single-stranded DNA for antibody recognition, MIRA works on

normal double-stranded DNA; in fact the complex does not bind to single-stranded DNA. The

CpG-methylated DNA is collected from the binding reaction via the GST-tagged MBD2B and

glutathione beads, linker ligated and then PCR amplified. These PCR amplified MBD-enriched

DNA fractions and total genomic DNA (input) are labeled with fluorescent dyes Cy5 and Cy3,

respectively, and cohybridized onto microarrays. The ratio of fluorescent intensity (Cy5 to

Cy3) indicates the methylation status at each particular sequence analyzed. The MIRA-

enrichment method has been proven to be compatible with several types of microarray

platforms and high-throughput DNA sequencing platforms and is highly sensitive requiring

only 100–200 ng of genomic DNA.NIH-PA Author Manuscript

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NIH-PA Author ManuscriptResults from DNA methylation profilingThe importance and widespread occurrence of CpG island hypermethylation in cancer isbecoming increasingly recognized. In initial studies examining a limited number of loci, it hasbeen estimated that between 0.5% and 3% of all genes carrying CpG-rich promoter sequencesmay be silenced by DNA methylation in several types of cancer (Costello et al., 2000; Shiraishiet al., 2002). Examining all or most CpG islands in the genome, recent reports indicate thatgenerally several hundred to even more than a thousand CpG islands can be methylated inindividual tumors (Rauch et al., 2006; Rauch et al., 2007; Dudley et al., 2008; Kuang et al.,2008; Omura et al., 2008; Rauch et al., 2008; Koga et al., 2009; Tommasi et al., 2009). Table2 summarizes some of the recent studies describing methylation profiling of cancer genomes.Genome-wide analysis of DNA methylation of lung squamous cell carcinoma (SCC) andmatching normal tissue DNA revealed a large number of lung SCC-specific hypermethylatedgenes. Chromosome tiling array analysis has indicated that all of them were CpG islands orCpG-rich regions, often overlapping or located in close proximity to promoter regions (Rauch

et al., 2008). Islands with different CpG densities can become hypermethylated in tumors. It

is clear that not all of these hundreds of methylated genes can be tumor suppressor genes. For

example, substantial subsets of the methylated genes were represented by a variety of

homeobox genes (Rauch et al., 2007). Homeobox gene-associated CpG islands were among

the most common stage I disease DNA methylation events identified so far, i.e. this methylation

event appears in almost every early stage tumor (Rauch et al., 2007; Rauch et al., 2008).

Genome-wide DNA methylation analysis identified CpG island methylation, for example in

proximity of the OTX1, NR2E1, PAX6, IRX2, OC2, TFAP2A, and EVX2 genes. These genes

are tumor-specifically methylated with very little methylation found in normal lung tissue or

in blood cell DNA (Rauch et al., 2008).

The frequent methylation of homeobox genes and other developmental genes regulated by the

Polycomb complex is a phenomenon observed in many different histological types of human

cancer (Rauch et al., 2006; Ohm et al., 2007; Rauch et al., 2007; Schlesinger et al., 2007;

Widschwendter et al., 2007), as exemplified by several studies, which we will discuss briefly.

Genome wide methylation profiling of ductal carcinoma in situ, a premalignant breast lesion

with a high potential to progress towards invasive carcinoma identified 108 significant CpG

islands that undergo aberrant DNA methylation in ductal carcinoma in situ and stage I breast

tumors, with methylation frequencies greater than or comparable with those of more advanced

invasive carcinoma (50% to 93%) (Tommasi et al., 2009). A substantial fraction of these

hypermethylated CpG islands (32% of the annotated CpG islands) was associated with several

homeobox genes, such as the TLX1, HOXB13, and HNF1B genes. Fifty-three percent of the

genes hypermethylated in early-stage breast cancer overlapped with known Polycomb targets

and included homeobox genes and other developmental transcription factors (Tommasi et al.,

Adv Genet. Author manuscript; available in PMC 2011 January 1.