Hum Genet (2002) 111 : 115–127 DOI 10.1007/s00439-002-0783-6
REVIEW
George A. Garinis · George P. Patrinos · Nick E. Spanakis · Panayiotis G. Menounos
DNA hypermethylation: when tumour suppressor genes go silent
Received: 4 March 2002 / Accepted: 30 May 2002 / Published online: 16 July 2002 © Springer-Verlag 2002
Abstract The phenotype of the cancerous cell may arise either from genetic alterations that disrupt gene function through sequence modifications (mutations) or epigenetic events that may alter the heritable state of gene expression (i.e. without changing the actual sequence of the genome). Whereas mutations in certain tumour suppressor genes are most often thought of in association with their inactivation during cancer initiation or progression, epigenetic alterations such as DNA methylation appear to be tightly linked to the sequential non-reversible events of normal tissue differentiation and organogenesis. This highlights a link between tissue differentiation and tumourigenesis with respect to the stable nature of certain epigenetic changes. In the case of tumourigenesis, both genetic and epigenetic mechanisms of altered gene expression often go hand in hand; not surprisingly, biallelic inactivation of a given tumour suppressor gene may occur via a combination of mutational and epigenetic events and is entirely consistent with the Knudson two-hit hypothesis of tumourigenesis. This review summarizes recent developments within the field of DNA methylation, highlighting its association with the transcriptional silencing of tumour suppressor genes in a variety of human cancers.
G.A. Garinis (✉) · G.P. Patrinos · N.E. Spanakis · P.G. Menounos Laboratory of Research, Nursing Military Academy, Athens, Greece Present address: G. A. Garinis Erasmus University Rotterdam, Faculty of Medicine, MGC Department of Cell Biology and Genetics, PO Box 1738, 3000 DR Rotterdam, The Netherlands, e-mail:
[email protected], Tel.: +31-10-2760597 Present address: G. P. Patrinos Erasmus University Rotterdam, Faculty of Medicine, MGC Department of Cell Biology and Genetics, Rotterdam, The Netherlands
Nomenclature Gene symbols used in this article follow the recommendations of the HUGO Gene Nomenclature Committee (Povey et al. 2001).
Methyltransferases and the regulation of the DNA methylation machinery DNA methylation in human cells is restricted to the covalent addition of a methyl group to the 5’ position of the cytosine ring within a CpG dinucleotide and to a lesser extent in CpNpG (Bird 1992). The process of methylation is mediated by at least three DNA methyltransferases (Dnmt1, 3a and 3b) that catalyse the transfer of a methyl group from S-adenosyl-L-methionine (methyl donor) to cytosine or adenine bases in DNA. These enzymes attach inheritable information to the DNA, information that is not encoded in the nucleotide sequence. Physiologically, methyltransferases are believed to function in the longterm silencing of the non-coding DNA in the genome. However, methylation may also change the interactions between proteins and DNA, leading to alterations in chromatin structure that will affect the rate of transcription and therefore will co-ordinate mRNA expression in normal tissues and overexpression in tumours (Robertson et al. 1999). A direct link between the copying of genetic and epigenetic information comes from the finding that Dnmt1 associates directly with the DNA replication machinery and can be visualized at replication foci during S phase (Araujo et al. 1999). Additionally, the N-terminal domain of Dnmt1 may form complexes with the transcription factors pRB and E2F, suggesting a possible role in transcriptional repression (Robertson et al. 2000). Inhibition of DNA methyltransferases in normal human fibroblasts has recently been shown to induce an irreversible senescentlike phenotype, including cell morphology, DNA content, overexpression of the inhibitor of certain cyclin-dependent kinases p21 and induction of type I collagenase (Young
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and Smith 2001); interestingly, in this study, Dnmt activity decreases as cells approach the end of their proliferative potential. This suggests that Dnmt activity is an integral part of the mechanisms by which cells count the number of cell divisions completed and that it initiates a signal that could result in the senescent phenotype. Although further studies are needed, the hypothesis that the mitotic clock could be linked to progressive demethylation seems highly attractive. From various lines of evidence, it is known that the methylation pattern of the cancerous cell is associated with a broad genomic hypomethylated state that is often accompanied by a more regional and locus-specific hypermethylated pattern. Therefore, certain investigators have speculated on the possible role of methyltransferases in tumour initiation. Recently, Jackson-Grusby et al. (2001) have demonstrated that the loss of Dnmt1 causes celltype-specific changes in gene expression that impinge on several pathways, including the expression of imprinted genes, cell-cycle control, growth factor/receptor signal transduction and mobilization of retroelements. Alternatively, the possibility exists that demethylating enzymes may also be involved in tumourigenesis (Wolffe et al. 1999), possibly through the action of 5-methylcytosine DNA glycosylase, which removes the methylated cytosine from DNA, leaving the deoxyribose intact (Jost et al. 1995).
How does methylation affect mammalian gene expression? The first direct link between methylation and gene expression was revealed more than two decades ago when a mouse-human somatic cell hybrid clone, deficient in hypoxanthine-guanine phosphoribosyltransferase (HPRT) and containing a structurally normal inactive human X chromosome, was isolated. The hybrid cells were treated with 5-azacytidine and tested for the reactivation and expression of human X-linked genes. The frequency of HPRT-positives clones after 5-azacytidine treatment was 1000-fold greater than that observed in untreated hybrid cells. Later on, the W7 mouse thymoma cell line that does not express the metallothionein-I (MT-I) gene in the presence of either cadmium or glucocorticoids (unlike most other cell lines) was used as a model system for studying the role of DNA methylation on MT-I gene expression (Mohandas et al. 1981; Compere and Palmiter 1981). Subsequently, Becker et al. (1987) demonstrated that DNA methylation could interfere with protein-DNA interactions. Since then, several methyl CpG binding proteins (MBD family) have been shown to compete efficiently with sequence-specific DNA-binding transcription factors for the same promoter sequences, when these sequences are methylated (Bird and Wolffe 1999; Leonhardt and Cardoso 2000). On the other hand, the sequence-specific DNA-binding transcription factor Sp1 is able to bind DNA and activate transcription even when the binding site is CpG-methylated, demonstrating that not all DNAbinding proteins are directly sensitive to DNA methyl-
ation (Holler et al. 1988). Methylation might also influence transcription indirectly through chromatin condensation, which is the packing of DNA into higher-order three-dimensional protein-nucleic acid structures, often associated with gene inactivity. Chromatin condensation may be generated by the recruitment of histone deacetylases to specific sequences via protein-protein interactions by the aforementioned methyl-CpG binding factors (Johnson and Turner 1999). Histone acetyl transferases and deacetylaces remodel chromatin by increasing or decreasing the net charge of the histones themselves, thus altering their affinity for negatively charged DNA. This process could alter gene expression by making DNA either more or less accessible to transcriptional activators or repressors. In summary, both direct and indirect methylation-dependent mechanisms may act synergistically in silencing gene expression. Alternatively, a defect in the molecular machinery that links methylation and replication (Dnmt1) could be responsible for the observed changes in global methylation status (hypomethylation) observed in tumour cells.
Methylation, tumour suppressor genes and cancer Under physiological conditions, methylation is associated with the distinct, but mechanistically related, processes of X chromosome inactivation (silencing of one X chromosome but not the other in all human female cells; Wolf and Migeon 1982), genomic imprinting (silencing or activation of a particular gene inherited specifically from one parent or the other; Barlow 1995) and transcriptional silencing of repetitive DNA sequences (Kochanek et al. 1995). Interestingly, although the vast majority of known human genes contain CpG islands, most are actually unmethylated (Delgado et al. 1998). Despite this, most investigations into DNA methylation and its effects on gene expression have focused on promoter CpG islands rather than on the regions in which the majority of methylation is found, such as certain CpG islands within the coding part of genes and some observed methylated regions outside the promoter regions that have previously been shown to serve as silencers of cryptic transcriptional start sites. In cancer research, DNA methylation is often regarded as an epigenetic mechanism that blocks gene expression (Fig. 1). Indeed, it is noteworthy that methylated genes can either exhibit increased or decreased levels of transcription, depending on whether the methylation inactivates a positive or a negative regulatory element (Jones and Takai 2001). To date, aberrant methylation of normally unmethylated promoter 5’-CpG-rich areas (CpG islands) is the most commonly studied epigenetic mechanism associated with the transcriptional silencing of known and suspected tumour suppressor genes. To date, several studies have shown that methylation-associated silencing inactivates certain tumour suppressors as effectively as mutations and is one of the cancer-predisposing hits described in Knudson’s two hit hypothesis (Fig. 1). The frequency of this process, the variety of the genes involved and the large repertoire of the carcinomas previously shown to harbour
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Fig. 1 Schematic representation of tumour suppressor gene inactivation. For certain tumour suppressor genes, initially in the wildtype state (Nl), hypermethylation (Meth) progresses primarily in a monoallelic form and subsequently can expand to both alleles of the gene, causing abrogation of gene function. Alternatively, tumour suppressor genes suffering from germline mutations (Mt; depicted as a black box within the gene) can cause, in association either with promoter hypermethylation or with loss of heterozygosity (LOH), sporadic cancers. Arrowhead Transcriptional initiation site
dense methylated promoter CpG islands all reflect the critical role of epigenetic mechanisms in propelling cancer initiation and progression. Aberrant methylation of normally unmethylated 5’-CpG-rich areas has been demonstrated, among others, in the human adenomatous polyposis coli gene (APC), the breast cancer susceptibility gene (BRCA1), genes for tumour suppressors p16Ink4a, p15Ink4b and p14Arf, the epithelial-cadherin gene (E-cadherin), the fragile histidine triad gene (FHIT) and the human MutL homologue gene (hMLH1; Table 1). Below, we have chosen to describe those genes that have been most extensively studied in the past and that have been shown to undergo both genetic and epigenetic changes in a variety of carcinomas. The APC gene The APC gene is located at 5q21 and codes for a 300-kDa protein that interacts with β-catenin, a protein involved in cellular adhesion and motility (Su et al. 1993). Together
with axin, GSK-3 beta and β-catenin, APC forms a tetrameric complex, resulting in the regulation of β-catenin stabilization (Behrens et al. 1998). Germline mutations in APC at 5q21–22 result in the dominantly inherited APC syndrome (Boardman et al. 2001). APC somatic mutations comprise an early event in colorectal tumourigenesis and data regarding the somatic mutation rate (20%–30%) have been presented (Iwama 2001). Both types (germline and somatic) of mutations are mainly concentrated in the 5’ half of exon 15 (Gayther et al. 1994), often resulting in a truncated non-functional protein. Methylation of the promoter region of this gene constitutes an alternative mechanism of gene inactivation in colon cancers and other tumours of the gastrointestinal tract. The APC gene was shown to be hypermethylated in 18% of primary sporadic colorectal carcinomas (n=108) and adenomas (n=48) by Esteller et al. (2000a). In that study, wherever APC methylation was observed, no expression of the APC gene transcript was detected. In 95% of the cases examined, methylation affected only the wildtype allele of APC and was not observed in tumours from familial adenomatosous polyposis patients who had germline mutations. In addition, aberrant methylation of the 1A promoter of the APC gene and loss of its specific transcript was also shown to be frequently present in breast (44%) and non-small-cell lung carcinomas and cell lines (53%) and, to a lesser extent, in small-cell lung carcinoma cell lines (26%; Virmani et al. 2001a), suggesting that methylation-associated transcriptional silencing of APC does not occur in a tumour specific manner.
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Table 1 Tumour suppressor genes, genes with a putative tumour suppression function and tumour-associated genes with demonstrated CpG hypermethylation-associated transcriptional silencing in sporadic carcinomas Gene abbreviation
Gene
Tumour type
Function
References
p14Arf
p14 Alternative reading frame
Colorectala
p15INK4b
Haematological malignancies Solid tumoursa
Esteller et al. 2000b, Fulda et al. 2001 Li et al. 1995, Tien et al. 2001
BRCA-1
Cyclin-dependent kinase inhibitor 4b Cyclin-dependent kinase inhibitor 4A Breast cancer gene 1
Cyclin-dependent kinase inhibitor Cyclin-dependent kinase inhibitor Cyclin-dependent kinase inhibitor DNA damage repair
FHIT
Fragile histidine triad
E-Cad
Epithelial cadherin
APC HMLH1 STK11 VHL p73 MGMT
Adenomatosis polyposis coli Human MutL homologue Serine/threonine kinase Von Hippel-Lindau
p16INK4a
GSTP1 pRb TIMP3 DAPK1 ER ALX3
p53 RASSF1A
O-6-methylguanine-DNA methyltransferase Glutathione S-transferase pi Retinoblastoma Tissue inhibitor of metalloproteinase 3 Death-associated protein kinase 1 Oestrogen receptor Aristaless-like homeobox 3
RARb2 COX-2
RAS association domain family 1A Retinoic acid receptor b2 Cyclo-oxygenase 2
Casp 8
Caspase 8
HTR1B
5-hydroxytryptamine receptor 1B Endothelin 1
Edn-1 a
Breast, ovarian
Esophageal, cervical, breasta Breast, gastrica
Oxidation, DNA damage
Colon, gastric Colon, gastrica Colon, breast Renal Lymphomas Colon, gastric, lymphomaa Breast, prostate a Glioblastoma Colon, renala
Interacts with b-catenin Mismatch repair Serine-threonine kinase Angiogenesis Cell cycle checkpoint DNA damage repair
Uterine cervix Bladdera Neuroblastoma
Liver Nasopharyngeal, ovarian, renal Nasopharyngeal Gastrointestinal Primary PNET/ medulloblastoma Lung Lung
Epithelial intercellular adhesion
Oxidation, DNA damage Cell cycle checkpoint Inhibits tissue metalloproteases Interferon-induced apoptotic kinase Receptor Human orthologue of the hamster homeobox gene Alx3 Cell cycle checkpoint Ras effector homologue Retinoic acid receptor beta Conversion of arachidonic acid to prostaglandins Protease involved in the apoptotic process 5-hydroxytryptamine receptor Potent vasoconstrictor
Belinsky et al. 1998, Garcia et al. 1999, Shim et al. 2000 Magdinier et al. 1998, Catteau et al. 1999, Sakamoto et al. 2001 Shimada et al. 2000 Zochbauer-Muller et al. 2001b, Machado et al. 2001, Wheeler et al. 2001, Si et al. 2001, Garinis et al. 2002 Esteller et al. 2000a Myohanen et al. 1998 Trojan et al. 2000 Prowse et al. 1997 Ping Siu et al. 2002 Oue et al. 2001 Lin et al. 2001 Nakamura et al. 2001 Esteller et al. 2001 Dong et al. 2001 Habuchi et al. 2001 Wimmer et al. 2002
Pogribny and James 2002 Kwong et al. 2002 Kwong et al. 2002 Kikuchi et al. 2002 Zuzak et al. 2002 Takai et al. 2001 Takai et al. 2001
Tissue other than the one normally affected
In colorectal cancer cell lines, APC mRNA expression levels have recently been shown to be repressed through methylation of a CpG island located in the 5’ untranslated region of the APC gene (Sakamoto et al. 2001). Interestingly, in these cell lines, no major genetic alterations, such as mutations, amplifications or deletions of the gene have been detected. Epigenetic alterations occur independently of APC protein truncation and the methylation status of a non-APC related gene such as the hMLH1. These findings suggest that epigenetic and genetic alterations may occur independently and that, apart from the previously
demonstrated protein truncation by point mutation within the coding region of the APC gene, epigenetic alteration suppressing APC gene expression may contribute to oncogenesis and the progression of colorectal cancer. The BRCA1 gene BRCA1 serves as one of the best paradigms for selective methylation in discrete regions upstream of the gene promoter and seems to be restricted so far to breast and ovar-
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ian carcinomas (Magdinier et al. 1998; Catteau et al. 1999). The 5’ end of the BRCA1 gene is embedded in a large CpG island of 2.7 kb in length. Epigenetic studies carried out by Magdinier et al. (2000) demonstrated that the BRCA1 CpG island was regionally methylated in a variety of cell lines and in fetal and cancer breast tissues but remained unmethylated in gametes. These authors investigated the potential role of methyl CpG binding proteins in the regulation of BRCA1 expression and showed that the regional methylation of the 5’-end CpG island of BRCA1 was associated with reduced gene expression in human somatic cells (Magdinier et al. 2000). In the same study, chemically induced hypomethylation increased BRCA1 expression levels in cells and, in cell extracts, in vitro methylation was able to induce MeCP2-dependent repression of the BRCA1 putative promoter located at the 5’-end of the exon 1a. Recent studies have identified epigenetic alterations, by cytosine methylation, as being highly associated with histone hypoacetylation and chromatin condensation in the inactivation of gene transcription. Rice and Futscher (2000) have tested whether this association occurs in BRCA1 gene silencing. Chromatin immunoprecipitation studies revealed that the aberrantly methylated BRCA1 promoter of BRCA1-negative breast cancer cells was associated with hypoacetylated H3 and H4 histones at all 75 CpG dinucleotides analyzed compared with the nonmethylated promoter of normal and breast cancer cells expressing BRCA1. Interestingly, this hypermethylated promoter and hypoacetylated histone state correlated with a restrictive chromatin configuration and BRCA1 transcriptional repression. The gene for tumour suppressor p16Ink4a The p16 protein is encoded by the CDKN2 gene and functions as an inhibitor of cyclin dependent kinase 4 and 6 (CDK4/6). Phosphorylation of the retinoblastoma protein (pRb) by CDK4/6 represents a vital step in cell cycle progression. Down-regulated expression of p16Ink4a due to genetic aberrations, such as deletions and/or point mutations has previously been shown in a variety of carcinomas and may be responsible for the imbalance between cyclin D and its inhibitor p16Ink4a (Gazzeri et al. 1998; Ashizawa et al. 2001). However, it is noteworthy that p16Ink4a point mutations have not been reported in certain primary colorectal carcinomas. In addition, no abnormalities have been documented (truncations) in the Rb protein itself. However, p16Ink4a protein expression is undetectable in certain tumour samples and cancer cell lines. In an attempt to provide an explanation for the above inconsistencies, several studies have focused on epigenetic mechanisms that could alter the transcriptional status of certain candidate genes within the pRb pathway. Since then, hypermethylation of the p16Ink4a tumour suppressor gene 5’-CpG islands and subsequent transcriptional silencing has been implicated as an additional mechanism of p16Ink4a gene inactivation in diverse types of cancer (Garcia et al. 1999; Shim et al. 2000). In breast cancer,
because of the reported scarcity of p16Ink4a mutations, the observed high frequency of hypermethylation could offer an alternate explanation to the frequently observed absence of p16Ink4a protein (Brener and Aldaz 1995; Rush et al. 1995; Berns et al. 1995; Quesnel et al. 1995). However, in certain tumours, the detected frequency of methylation varies considerably between the various studied cases, probably because of the inter-individual variability in methylation topology; this is also reflected by the reported methylation frequencies in normal colonic epithelial versus control white blood cell DNA (Nguyen et al. 2001). It is reasonable to assume that (1) the use of different restriction enzymes in Southern blot experiments, (2) the design of different bisulphite sequencing primer sets for the same promoter regions, (3) the fact that several studies may not include specimens solely from primary untreated cancer patients (inter-individual variability), (4) the mixed population of cells of different origins (intra-individual variability), and (5) demographic diversity could account for the observed discrepancy in methylation frequency. Inevitably, since methylation topology is not identical in different individuals, intra- and inter-individual heterogeneity will exist. Intra-individual heterogeneity in DNA methylation can be dealt with efficiently by comparing microdissected paired tumour and normal samples. However, intra-individual variability of methylation of a certain promoter in different tissues does not reflect inter-individual variability between the various studied cases. Inter-individual variability comprises an additional difficulty that can be addressed in part if bisulphite sequencing is coupled with mutation screening studies and detailed expression analysis both at the mRNA and protein levels of the gene of interest in a relatively large number of primary paired normal and tumour specimens. Sequencing of the bisulphite-treated DNA often reveals common methylated areas (consensus sequences) among different individuals, whereas expression studies may provide evidence regarding the significance of the observed methylation towards gene inactivation. The genes for tumour suppressors p15Ink4b and p14Arf The genes for tumour suppressors p15Ink4b and p14Arf are located at 9p21 and may comprise alternative targets of DNA hypermethylation. p15Ink4b is positively regulated by TGF-β in certain cells and also inhibits the cyclindependent kinases CDK4/6 (Li et al. 1995; Reynisdottir et al. 1995). p14Arf and p16Ink4a share an exon and their gene products are generated from different reading frames (Quelle et al. 1995). p14Arf has been implicated in a regulatory feedback loop with p53 and MDM 2 and therefore comprises one of the few links between the major pRb and p53 pathways. Because of the close proximity between the genes for p14Arf, p15Ink4b and p16Ink4a, it is reasonable to assume that the frequency of hypermethylation would be approximately the same for these three gene loci. Although the p16Ink4a and p14Arf methylation data reported by Belinsky et al. (1998) and Esteller et al. (2000b) in lung and colorectal carcinomas, respectively, exhibit
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similar frequencies, it is noteworthy that p14Arf has been shown to be methylated, in one of these studies, in a p16Ink4a-independent manner. Moreover, in chronic and acute myelogenous leukaemia, p15Ink4b and p16Ink4a promoter CpG islands exhibit different levels and patterns of methylation (Nguyen et al. 2001). One possible explanation for the frequently seen discrepancies described above might be the interference of transcription-initiation complexes and transcription factors with DNA methyltransferases in promoter-associated CpG islands (Meehan et al. 1989; Boyes and Bird 1992). The chromosomal 9p21 locus may provide an excellent model for studying selective hypermethylation mechanisms in a tissue- and gene-specific manner. The FHIT gene The FHIT gene bridges the region at 3p14.2 and encodes a polypeptide of 147 amino acids with 70% similarity to a core region of the Schizosacharomyces pombe diadenosine tetraphosphate (AP4A) hydrolase with a putative (although unsubstantiated) role in AP4A metabolism (Huang et al. 1995). Although specific in vivo roles of the human AP4A have not yet been defined (Barnes et al. 1996), it may act as an intracellular mediator by regulating the ability of cells to adapt to metabolic stress, such as heat, oxidation and DNA damage (Segal and Le Pecq 1986). FHIT allelic deletions and reduced or absent FHIT protein expression have been observed in a variety of tumours including lung, kidney, bladder, gastric, cervical carcinomas, suggesting a putative tumour suppressor function (Sozzi et al. 1998; Garinis et al. 2001). A mechanism whereby the loss of FHIT activity could contribute to malignancy is through the alteration of AP4A metabolism, which may result in the stimulation of DNA synthesis and inappropriate cellular proliferation. In breast and lung carcinomas, methylation at the FHIT locus has been shown to be a frequent event and is associated with the loss of both FHIT mRNA expression and protein detection (Zochbauer-Muller et al. 2001a); moreover, in the same study, the methylation status at the FHIT was reversible with 5-aza-2’-deoxycytidine. These findings are consistent with the tumour suppressor gene behaviour of FHIT and point to additional mechanisms of FHIT inactivation in certain human cancers. However, aberrant methylation of FHIT has been reported only in oesophageal (Shimada et al. 2000), cervical (Virmani et al. 2001b), breast and lung (ZochbauerMuller et al. 2001a) carcinomas. Since few reports have demonstrated FHIT methylation thus far, further investigation is needed to examine whether FHIT transcriptional repression may also occur in other carcinomas and to estimate the extent of FHIT inactivation in the absence of genetic perturbations.
to morphogenesis and the maintenance of tissue structure (Takeichi 1995). Its relatively short intracellular domain interacts with the actin cytoskeleton through α- and β-catenins, whereas the extracellular domain associates with complexes at intercellular junctions (Wijnhoven and Pignatelli 1999). E-cadherin germline mutations resulting in the loss of gene expression or protein function have previously been shown to predispose individuals to familial gastric and colorectal cancer (Richards et al. 1999). However, somatic mutations within the E-cadherin locus occur infrequently (Berx et al. 1998). Loss of E-cadherin expression significantly increases the invasive and metastatic capacity of cancer cells in vitro (Takeichi 1991) and activation of E-cadherin results in the growth inhibition of tumour cell lines (Watabe et al. 1994). These findings are in agreement with the Knudson two-hit hypothesis and provide direct evidence for E-cadherin tumour suppressor behaviour. 5’-CpG promoter methylation has been demonstrated in association with the transcriptional silencing of E-cadherin gene expression in breast (Droufakou et al. 2001), lung (Zochbauer-Muller et al. 2001b), gastric (Machado et al. 2001), colorectal (Wheeler et al. 2001), oesophageal (Si et al. 2001) and bladder (Bornman et al. 2001) carcinomas. Methylation is often heterogeneous, as unmethylated alleles have been observed in several microdissected tumour samples with reported E-cadherin gene methylation. In agreement with these findings, tumour samples with E-cadherin promoter methylation often demonstrate heterogeneous and decreased membranous E-cadherin immunostaining. The heterogeneous nature of the E-cadherin gene methylation profile may reflect a plasticity of phenotype with respect to cell adhesion that is not possible when the gene is inactivated by a germline mutation (Graff et al. 2000). This plasticity could be regarded as a selective advantage that might contribute to metastatic progression, since E-cadherin re-expression could actually facilitate cell survival within metastatic deposits (Fig. 2). Interestingly, in view of the previously reported scarcity of allelic loss and/or mutations within the E-cadherin locus in primary sporadic colorectal carcinomas (SCRCs), we have demonstrated a consistent and uniform methylation-dependent decrease or absence of E-cadherin expression in ~80% of SCRCs, suggesting an epigenetically mediated loss-of-E-cadherin function (Garinis et al. 2002). This also suggests that the frequency of mutations for the E-cadherin gene is not associated with DNA methylation, as is known from a plethora of studies that show genetic alterations to occur independently of methylation and to comprise the only known inactivation mechanisms for a large repertoire of tumor suppressor genes.
The timing of methylation: “a chicken and egg” conundrum The E-cadherin gene E-cadherin belongs to a family of Ca++-dependent adhesion molecules that mediate intercellular contacts critical
For certain genes, an aberrant decrease or absence of expression may not be a consequence of somatic mutations or allele loss at all; in these cases, DNA hyperme-
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Fig. 2 Hypothetical model of the dynamic nature of E-cadherin promoter hypermethylation during metastatic progression. The loss of E-cadherin expression is proposed to be heterogeneous and may be modulated by the tumour microenvironment. In this model, a cell with the highest density of methylation (black) and the most diminished E-cadherin protein expression may have a selective advantage over the neighbouring cells to dissociate from the primary tumour (Tissue A) and disseminate to a distal secondary tissue site of a different organ (Tissue B). Subsequently, survival and growth pressure will result in reduced methylation, partial restoration of E-cadherin expression and hence increased intercellular adhesion
thylation may be the primary mechanism of gene inactivation (Fig. 1; Berx et al. 1998). This hypothesis is supported by the reported scarcity of mutations or loss of heterozygosity within certain gene loci; however, detailed expression studies are still necessary to show the uniform association of hypermethylation with decreased or absent gene expression. One possibility is that hypermethylation could begin in a monoallelic form with subsequent expansion to both alleles of the gene and the resultant complete abrogation of gene function. However, epigenetic mechanisms are often regarded as secondary events that are not responsible for initiating gene transcriptional silencing. Most of the densely methylated genes harbour genetic perturbations that would otherwise prevent normal gene expression and are presumed to have occurred before methylation-dependent gene silencing.
Methylation may also function synergistically with genetic mutations to promote tumourigenesis in a more indirect fashion. For example, Myohanen et al. (1998) have shown that the stable loss of tumour suppressor function can occur through a combination of an inactivating mutation in one allele and aberrant methylation of a non-mutated promoter in the other allele. Given that the mutated allele is fully inactivated, this finding supports the observation that hypermethylation may act as a direct mechanism of tumour suppressor gene inactivation and may contribute indirectly to the expression of a mutated phenotype. In the case discussed above, there is a clear sequence of events (a germline mutation followed by epigenetic inactivation of the second allele as one of the possible second events) regarding the progressive inactivation of tumour suppressor function. However, hypermethylation of the normally unmethylated hMLH1 mismatch repair gene is often tightly linked with microsatellite instability-positive colorectal cancers (Young et al. 2001). Hence, hypermethylation of the hMLH1 gene could indirectly give rise to a cascade of mutagenesis, since any given alteration (such as transcriptional silencing) of a DNA repair gene can also affect DNA integrity at the primary sequence. This example highlights a “chicken and egg” conundrum regarding the timing of epigenetic versus genetic perturbations. Currently, there is a lack of both evidence and consensus with regard to the timing of genetic versus epi-
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genetic perturbations in tumourigenesis and plausible nonmutually exclusive mechanisms can be proposed in either case. However, since the frequency and number of mutated tumour suppressor genes exceeds that of the hypermethylated DNA repair genes that have been demonstrated thus far in a variety of human cancers, we feel that genetic perturbations may indeed precede epigenetic alterations most of the time. Clearly, the timing of the methylation and gene inactivation process with respect to tumour formation remains to be properly defined.
Hypermethylation spreading and ageing DNA methylation seems a general process of gene regulation throughout growth and development and it is assumed that it is not the methylation itself but its result, extent and target specificity that are the relevant factors that offer the selective advantage for the tumour cell. This is supported by the observation that certain loci, occasionally with contiguous related genes, are epigenetically altered in clusters (Reik and Walter 1998). In addition, not all tumour suppressor genes become silent by DNA hypermethylation and aberrant hypermethylation cannot actually be used to pinpoint candidate genes with tumour suppressor activity. However, intriguing questions are why the methylation of promoter CpG islands arises at all and whether the first step of this process is a stochastic phenomenon or is facilitated by unknown factors. Several studies have described the dynamic character of the DNA methylation process in normal fibroblasts (as a function of growth and confluence) and in CpG islands of the active X chromosome. In the latter, transient DNA methylation has been demonstrated as a function of time. To date, ageing is the only well-documented predisposing factor associated with promoter hypermethylation in normal colon mucosa and other normal epithelial tissues (Jubb et al. 2001; Habuchi et al. 2001). However, to the best of our knowledge, specific genetic perturbations have not yet been demonstrated in normal tissues derived from ageing individuals. The oestrogen receptor, a well-documented tumour suppressor gene, was the first gene to be demonstrated with extensive promoter methylation in normal colonic enterocytes (de Carvalho et al. 2000). The function or selective advantage of this age-related promoterspecific methylation, if there is one, is currently unknown and certain genes do not appear to be methylated in normal tissues with respect to ageing (Issa et al. 1994). It is possible that methylation of specific promoters may be an example of antagonistic pleiotropy, which is somehow advantageous during growth and development (albeit by an unknown mechanism) but disadvantageous later in life. Since the frequency of colorectal neoplasia parallels that of progressive ageing, it has been proposed that age-related methylation may contribute to an acquired cancer predisposition (Ahuja and Issa 2000). Highly repetitive sequences such as the Alu repeats have also been shown to exhibit age-related methylation changes (Liu and Schmid 1993; Hellmann-Blumberg et
al. 1993). Such repetitive elements may act as focal centres of methylation that could participate in the initiation of methylation spreading. It is possible that several genes located in close proximity with these focal centres may acquire a higher susceptibility to methylation (Fig. 3). The proposed mechanism of this spreading relies on previous observations that methylated DNA serves as a better substrate for methylation spreading than fully unmethylated DNA (Constancia et al. 1998; Turker 1999; Yokochi and Robertson 2002). Interestingly, by using a quantitative assay, Nguyen et al. (2001) have analyzed the methylation levels of promoter and exonic CpG islands of the p15Inkb, p16Ink4a and PAX 6 genes in a series of haematological malignancies and colorectal cancers. These authors have shown that exonic CpG islands are more susceptible to de novo methylation than promoter islands and that methylation seeded in exonic regions may spread to other islands, including promoter regions. Moreover, hypermethylation of the exonic regions in the aforementioned genes was invariably observed. In contrast, promoter hypermethylation was tumour-specific. As Nguyen et al. (2001) have previously suggested, promoter-associated CpG islands are protected from DNA methyltransferases because of steric hindrance by transcription-initiation complexes and various transcription factors. However, CpG islands within the exonic regions of a gene are less protected from de novo methylation. Thus far, there are no data that demonstrate any association of exonic methylation with decreased mRNA or protein levels. Han et al. (2001) have shown that protein binding protects sites on episomes and in chromosomes from de novo methylation by the Dnmt3a methyltransferase in human cells. In brief, they have tested episomes containing the lac operator (O) sequences to determine whether the lacO sequence is a target of murine Dnmt3a. Interestingly, they found that Dnmt3a could methylate the lacO sequences on episomes; active transcription did not affect de novo methylation of the lacO sites by Dnmt3a in 293/EBNA1 cells. However, LacI could protect lacO sites on the episomes from de novo methylation in cells overexpressing Dnmt3a and IPTG could abolish this protection. Therefore, it is possible that a repertoire of proteins could actually interfere with the methylation spreading, by masking certain focal methylation centres that otherwise would confer a highly selective advantage to those cells that harboured them, possibly in a tumourspecific manner. It would be particularly interesting to examine whether methylation spreading occurs within the exons of genes previously shown to be associated with cell adhesion and tumour metastases (E-cadherin, nm23, etc.) and whether certain proteins could interfere with this process. The observation that less protected (exonic) CpG islands are more susceptible to methylation may partly explain the association of progressive ageing and regional hypermethylated status. Age-related methylation may result from the counterbalance between DNA methyltransferases involved primarily in de novo methylation and those with a preference for methylation maintenance (Liang et al. 2002; Lopatina et al. 2002). It is possible that, over
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Fig. 3 a Transcriptional repression of tumour suppressor genes by methylation of CpG islands. Arrow Transcription, crossed arrow no transcription. b Model for the two types of methylation spreading in cancer. Highly repetitive sequences such as Alu repeats may act as methylation foci (black circle) that could participate in the initiation of methylation spreading (top left). It is possible that methylation spreading can override a weakly protected promoter CpG island and hence render the promoter inactive (bottom left). On the other hand, certain genes are protected from DNA promoter hypermethylation by trans-acting factors, which may bind to Sp1 sites (Sp1) located at the boundaries of CpG islands (top right). Methylation may spread throughout the promoter region and eventually silence the gene, provided that this protective mechanism is rendered inactive (bottom right). Arrow Transcription, crossed arrow no transcription
time, some CpG islands become progressively less protected against methylation mechanisms because of the possibility that repetitive transcription of a gene may interfere with DNA-protein interactions that otherwise serve as defensive mechanisms against DNA methylation. As Han et al. (2001) have previously proposed, the concentration and affinity of DNA-binding proteins, their interactions with other proteins and the availability of DNAbinding sites can dramatically alter the methylation pattern in the cell. For example, certain genes are protected from DNA promoter hypermethylation by trans-acting factors, which may bind to Sp1 sites that encompass the boundaries of CpG islands (Herman and Baylin 2000). Given that this protective mechanism is rendered inactive, methylation may spread throughout the promoter region and eventually silence the gene (Fig. 3). Hence, DNA-protein interactions may play a crucial role in the changes of the CpG methylation pattern in mammalian cells.
Finally, it should be noted that DNA damage, such as that caused by reactive oxygen species, accumulates as a function of age and could play a role in triggering or accelerating CpG island methylation. However, current data indicate that 8-hydroxyl guanine, which results from a reaction of guanine with a radical, inhibits rather than enhances methylation in vitro (Turk et al. 1995).
Methods for studying DNA methylation Analysis of methylation patterns has traditionally been conducted by utilizing Southern analysis after cleavage with methyl-sensitive restriction endonucleases. Based on these methyl-sensitive enzymes, restriction landmark genomic scanning was established as a method enabling the simultaneous visualization of a large number of loci as two-dimensional gel spots (Hayashizaki et al. 1993). With this approach, the status of DNA methylation can efficiently be determined by monitoring the appearance or disappearance of spots following the use of specific methylation-sensitive restriction enzymes. Subsequently, the methylation-specific polymerase chain reaction (PCR) technique has been introduced, which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes (Herman et al. 1996). This assay entails the initial modification of DNA by sodium bisulphite, the conversion of all unmethylated, but not methylated, cytosines to uracil and a subsequent amplification with primers specific for methylated versus unmethylated DNA. Recently, the genome-wide methylation
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profiling microarray technique has been able to combine efficiently either the fragmentation by a methyl-sensitive restriction endonuclease followed by size fractionation (Tompa et al. 2002) or the methylation-specific PCR methodology described above with the high density array method (Gitan et al. 2002). Although, at present, the method of choice to be used to monitor DNA methylation is a matter of debate, it is generally accepted that bisulphite treatment and subsequent amplification of the altered sequence is more sensitive than methyl-sensitive endonucleases. However, both the use of methyl-sensitive restriction enzymes and PCR-based methylation analysis are practical methods for analysing a few genes at a time and therefore genome-wide profiling of DNA methylation (microarrays) seems very promising in this regard.
Conclusions The study of epigenetic mechanisms in the transcriptional silencing of tumour suppressor genes has added a new dimension to our understanding of tumourigenesis in a variety of human cancers. In combination with genetic aberrations, hypermethylation has been consistently shown to drive the process of tumour initiation. At present, this points to a clear need for the use of a carefully selected panel of CpG hypermethylated biomarkers to aid in the evaluation of tumour progression. Specifically, recent developments in the detection of p16 methylation in the plasma and serum of liver cancer patients (Wong et al. 1999) and the demonstration of the hypermethylation of tumour suppressor genes in the serum of lung cancer patients may prove to be promising strategies towards earlier detection and hence better prevention of cancer. In mice, a combined genetic and pharmacological reduction of DNMT had no significant toxicity but reduced the levels of APC-induced intestinal neoplasia (Laird et al. 1995). Antisense oligonucleotides may provide another approach to inhibit DNA methylation. So far, they have been directed against the DNMT1 mRNA, thereby reducing DNMT1 protein levels and inducing the demethylation and expression of the p16 tumour suppressor gene in human tumour cells, and inhibiting tumour growth in mouse models (for more information see www.methylgene.com). Alternatively, more efficient and possibly less toxic doses of 5-azacytidine and its abundant derivatives could be used for the reactivation of numerous epigenetically silenced tumour suppressor genes. Re-expression of genes that exhibit a methylation-associated transcriptional silencing can result in the suppression of tumour growth or sensitization to other anticancer therapies. For example, re-expression of caspase-8 by demethylation might be an effective strategy to restore sensitivity for chemotherapy or death-receptor-induced apoptosis in various tumours in vivo (Zheng et al. 2000). In addition, small molecules that reverse epigenetic inactivation are now undergoing clinical trials in cancer patients. Progress in any of these areas will reinforce the current interest in epigenetic mechanisms and may initiate a se-
ries of therapeutic clinical trials with a potentially positive impact on cancer treatment. Acknowledgements Most of our own work was supported by the Nursing Military Academy research budget. We are indebted to Dr. J. Mitchell for critical reading of the manuscript and for his detailed comments.
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