1. Introduction
Cancer growth and metastasis involves coordinate changes in programs of gene expression of multiple genes. Since genomic expression programs are regulated by the epigenome it stands to reason that epigenomic changes plays a critical role in oncogenesis. Understanding the mechanisms responsible for these changes would enhance our understanding of oncogenesis as well as provide us with diagnostic and therapeutic targets. Two different but tightly linked components comprise the epigenome. The first component is the chromatin, which packages the genetic information in either accessible or inaccessible configurations [1, 2]. Recent studies demonstrate that different modifications of histone tails and core histones, which are the building blocks of the nucleosome, the basic unit of chromatin, determine whether a DNA associated with these nucleosomes is either active or inactive [1, 2]. Some extensively studied modifications are methylation [3] and acetylation of lysines at tails of H3 and H4 histones. Histone acetylation for example is required for active chromatin configuration, is catalyzed by histone acetyltransferases and is targeted to specific genes by site specific trans-activators which interact with cis acting regulatory sequences [4]. The state of histone acetylation is dynamic and is determined by equilibrium of acetylation catalyzed by histone acetyltransferases (HAT) and deacetylation catalyzed by histone deacetylases (HDAC) [5]. HATs and HDACs are recruited to target genes by either activator or repressor complexes [5]. In addition to histone modifications, the positioning of nucleosomes on regulatory regions of genes is modulated by energy dependent chromatin remodeling complexes [6]. The different histone modifications create a "histone code", which dynamically programs and maintains gene expression profiles and is responsive to different developmental, physiological, environmental and pathological cues [2]. A second level of the epigenome is the DNA methylation pattern. Cytosines residing in the dinucleotide CGs are modified by a methyl moiety [7]. The transfer of the methyl group from the methyl donor S-adenosylmethionine (SAM) onto DNA is catalyzed by DNA methyltransferases (DNMTs) concurrently with DNA replication [8]. A number of DNMT genes are known in vertebrates. DNMT1 is responsible for maintaining the DNA methylation pattern during cell division including cancer cells [9] while DNMT3A and DNMT3B are believed to be responsible for de novo methylation of specific sequences during early development [10, 11] as well as maintenance of the DNA methylation of specific repetitive sequences [12, 13]. Not all CGs are methylated and the distribution of methylated CGs is cell-specific generating a tissue specific pattern of methylation [14]. Thus, the DNA methylation pattern is a covalent mark on the DNA of its cell type identity. Because of the chemical stability of DNA methylation it has been a longstanding accepted belief that the DNA methylation pattern is a fixed characteristic of genomes in mature cells and therefore should not play a role in the dynamic changes in gene expression programming throughout adulthood. However, we have proposed that DNA methylation could be enzymatically reversed by demethylase activity [15] and that the DNA methylation pattern is a balance of methylation and demethylation [16].
It is well established that there is a tight correlation between the state of methylation of regulatory regions of genes and their state of activity [7, 14, 17]. Methylated regulatory regions of genes are associated with inactive deacetylated chromatin while unmethylated DNA is associated with hyperacetylated open chromatin, which is transcriptionally active [17, 18]. The relation between DNA methylation and chromatin structure was originally believed to be unidirectional; DNA methylation determines chromatin structure. A molecular explanation for this relation has been provided by the discovery of methylated DNA binding proteins such as MeCP2 which bind methylated DNA and recruit histone deacetylases and histone methyltransferases to regulatory regions of genes resulting in chromatin inactivation [19, 20]. However, recent data suggests that there is a bilateral relation between DNA methylation and chromatin structure. Inactivation of chromatin leads to recruitment of DNA methyltransferases and DNA methylation to regulatory regions of genes [21] and histone acetylation and chromatin activation bring about demethylation [22, 23].
Since cancer involves a programmed activation and inactivation of genes it is perhaps not surprising that both hypermethylation of certain genes, which suppress tumorigenesis [24-26] and global hypomethylation [27, 28] take place in cancer . The central question is how could both processes of hypermethylation and hypomethylation co exist in the same cell? Could we distinguish the mechanisms responsible for these two processes? Could we dissociate these processes therapeutically by selectively targeting either DNA methylation or demethylation without affecting the opposed process? Since hypermethylation of tumor suppressor genes has been the focus recently, most attention in the field has been directed at DNA methylation inhibitors as potential activators of tumor suppressor genes. Since DNA methylation inhibitors are being tested now as potential anticancer agents it is imperative upon us to determine the impact of these inhibitors on hypomethylation and activation of genes, which might promote tumorigenesis. A full understanding of the mechanisms involved in these two processes hypermethylation and demethylation is required for proper utilization of agents targeting the DNA methylation machinery in anticancer therapy [16].

2. Regional hypermethylation in cancer
Gene inactivation plays an important role in cancer by removing normal impediments on uncontrolled growth. The most established mechanism for removing cell growth breaks during cancer progression is by alteration of tumor suppressor genes [29]. Both germ-line and somatic alterations have been identified in critical regulatory genes in familial and sporadic cancers [29]. Numerous studies have suggested that a frequent mode of silencing these genes in tumors is by epigenomic alterations, which involve both chromatin and DNA methylation changes [30]. A large list of genes, which includes classical tumor suppressor genes as defined by germ line mutations as well as other genes that were shown to suppress tumor cell growth by biological experiments but with no known mutation in tumors, were found to be epigenetically silenced in cancer [31, 32]. Most of the epigenetically silenced genes in tumors are also hypermethylated in CG sequences located in the 5' regulatory regions. The combinations of genes, which are silenced in different cancers, is diverse and varies from tumor to tumor type and with tumor stages [33]. Recent data suggests that the profile of methylated genes in cancers might constitute a molecular signature, which could be used to classify and stage tumors [34-36]. Methylation of certain specific genes could be used as a diagnostic marker of cancer. For example, p16 methylation can be detected in DNA in sputum in 100% of patients with squamous cell lung carcinoma up to 3 years before clinical diagnosis [37]. Since p16 is completely unmethylated in normal cells, hypermethylation of the gene in a small number of cancer cells is positively detected even over a high background of normal unmethylated cells using methylation specific PCR (MS-PCR), which exclusively amplifies methylated alleles [38].

3. Causal relations between hypermethylation of tumor suppressors and cancer; the therapeutic potential of DNMT and DNA methylation inhibitors
In contrast to genetic alterations, epigenetic changes could be modified pharmacologically. If DNA methylation plays a causal role in the silencing of tumor suppressor genes then inhibitors of DNA methyltransferase should bring about demethylation and activation of these genes leading to cell growth arrest [39, 40]. 5-aza-deoxy-cytidine (5-aza-CdR) a cytosine analogue, which is phosphorylated to the trinucleotide derivative and is incorporated into DNA, traps DNMTs during replication leading to synthesis of nascent DNA in the absence of DNMT which is consequently demethylated [41]. 5-aza-CdR was shown to activate p16 and other methylated tumor suppressor genes [42]. Similarly, it was shown that either antisense or siRNA knockdown of DNMT1 lead to demethylation and expression of methylated tumor suppressor genes [9, 43]. A straight-forward interpretation of these experiments had been that DNA methylation plays a causal role in suppression of these genes and therefore inhibition of DNA methyltransferase DNMT has been proposed as a therapeutic approach to anticancer therapy [39]. Antisense DNMT1 oligonucleotides were shown to inhibit tumor cells in vivo in mice [44, 45]. Both antisense oligonucleotide inhibitors of DNMT1 and the nucleoside analog 5-aza-CdR are now being tested in different clinical trials, have shown evidence of some anti-tumor effects and changes in DNA methylation in vivo [46].
However, concluding that this data implies that DNA methylation plays a causal role in tumorigenesis and that demethylation of tumor suppressors is the main mechanism by which DNMT inhibitors inhibit tumorigenesis might be an oversimplification. While it is clear that methylation of a gene can suppress its transcription, it is important to determine whether methylation of genes such as p16 is the primary event involved in their silencing. Recent data suggests that chromatin structure inactivation is the primary cause of silencing of p16 and that DNA methylation follows rather than precedes initial inactivation of chromatin associated with p16 [21]. De novo methylation might therefore be a mechanism for stable suppression of genes following chromatin inactivation. Earlier events in the transformation cascade result in marking of p16 for chromatin inactivation, which leads to de novo methylation. For example, it has recently been shown in the mouse that the RAS-responsive zinc-finger transcription factor, (RREB) can suppress p16 promoter in BALB/c mice whose promoter bears a recognition RREB recognition element [47]. The human p16 promoter contains an RREB consensus sequence [47]. A repressor such as RREB might target p16 for inactivation, which is then followed by recruitment of proteins involved in chromatin inactivation. Proteins involved in chromatin modification and inactivation such as HDACs and histone methyltransferases were shown to associate with DNMTs [48-50] and might therefore recruit DNMTs to inactive genes resulting in their methylation (Fig. 1). It was recently shown for example that the leukemia-promoting PML-RAR fusion protein induces gene hypermethylation and silencing by recruiting DNA methyltransferases to target promoters [51].

Figure 1. A model explaining the coexistence of global hypomethylation and regional hypermethylation.
Different oncogenic signals such as the one triggered by Ras activate specific transacting repressors (RREB is shown as an example) as well as induce demethylase activity. Repressors such as RREB target tumor specific genes such as p16. Some of these repressor recruit histone deacetylases (HDAC) to promoters resulting in chromatin inactivation and silencing of the gene (indicated by an X over the horizontal arrow representing transcription). HDACs then recruit DNMTs to the gene resulting in hypermethylation of the gene (CH3). The inactive chromatin renders the gene inaccessible to demethylase and protects its hypermethylated state although its levels are also induced in tumor cells.

4. Global hypomethylation in cancer
Multiple observations in numerous tumors have established that tumor tissue is globally hypomethylated relative to its normal counterpart [28, 58]. This global hallmark of cancer cell's DNA has been confirmed by either measurements of the general abundance of methylated cytosines in genomic DNA [58] or by looking at repetitive sequences with methylation sensitive enzymes [59, 60]. Three types of mechanisms were proposed to explain the role that global hypomethylation might play in cancer. First, hypomethylation was proposed to result in activation by demethylation of protooncogenes such as MYC or Ha-RAS [61-64]. Although there are documented examples of hypomethylated oncogenes in tumors, there is no evidence that activation of an oncogene in cancers is mediated by hypomethylation. Second, global hypomethylation was proposed to predispose the cells to chromosomal instability [59, 65, 66]. Third, hypomethylation is proposed to increase metastasis [68, 69, [67, 68].
The fact that that the opposing processes of regional hypermethylation and global hypomethylation coexists in the same cell suggests that different enzymes determine them. The global hypomethylation in cancer can not result from lack of DNMT activity or lack of methyl donors since many genes are hypermethylated and there is no evidence that DNMT levels are decreased in cancer, on the contrary several reports show elevated and deregulated levels of DNMT1 in cancer [69-71]. An alternative hypothesis is that there is a global increase in demethylation activity in cancer cells, which drives global hypomethylation [16, 57]. How can one reconcile the increased demethylation activity with the fact that many genes are hypermethylated? One possible answer is that the regional hypermethylation results from local changes in chromatin structure as discussed above, which protects specific genes from the high levels of demethylation activity [72]. Recent data suggests that proteins, which inhibit histone acetylation can protect a sequence from active demethylation [23]. Regional inactivation of chromatin is proposed to lead therefore to regional hypermethylation and maintenance of methylated status of certain genes (Fig. 1).
In summary, both global hypomethylation and regional hypermethylation confer a selective advantage upon cancer cells by targeting different sets of genes with opposing roles in cellular transformation. Regional hypermethylation targets the silencing of genes, which suppress tumorigenesis while global hypomethylation probably targets activation of genes, which are required for different stages of the transformation process.

5. Causal role of hypomethylation in breast cancer metastasis and its therapeutic implications
The protease uPA, which is highly expressed in metastatic breast cancer and is required for invasiveness is methylated in noninvasive breast cancer cells and the breast cancer cell line MCF-7 and is hypomethylated in the invasive breast cancer cell line MDA-231. Treatment of MCF-7 cells with the DNA methylation inhibitor 5-aza-CdR resulted in demethylation and activation of uPA as well as increased invasiveness in vitro and metastasis in vivo [68]. Other prometastatic candidate genes were shown to be hypomethylated in invasive cancer and to be induced by demethylating agents; S100A4, a calcium-binding protein whose expression is associated with poor patient survival in breast cancer patients and induces metastasis in rodent models [73, 74], heparanase which degrades heparan sulfate proteoglycans and is preferentially expressed in metastasizing tumors [75], and testis-cancer specific antigens such as members of the MAGE family [76] whose expression correlates with aggressive and invasive breast cancer [77, 78]. These data invoke the possibility that DNA hypomethylation agents might induce metastasis. This concern is supported by data showing increased metastatic potential of cancer cell lines treated with 5-azacytidine and induction of prometastatic genes by 5-azacytidine [79-81]. This must be of concern in any future attempt to use DNA hypomethylating agents in anticancer therapy [16, 57]. It might be possible however to bypass this adverse consequence of demethylating agents by using agents which knock down DNMT1 and trigger cell arrest resulting in an attenuated demethylation response. For example, when human non small cell lung carcinoma A549 cells are treated with the demethylating agent 5-aza-CdR, the expression of testis-cancer antigen MAGE is induced while DNMT1 knock-down with antisense oligonucleotides results in growth arrest in the absence of induction of MAGE [55].
In addition to is role in metastasis; DNA hypomethylation was proposed to promote chromosomal instability, a hallmark of cancer. This hypothesis is supported by genetic evidence from mice and humans. Mouse embryonic stem cells nullizygous for the major DNA methyltransferase (Dnmt1) gene exhibited elevated mutation rates [65]. The genetic basis for a human syndrome, which exhibits chromosomal instability and pericentromeric hypomethylation (ICF), is a mutation in the gene encoding a DNA methyltransferase DNMT3B [11, 65, 82]. DNA demethylating agent 5-azacytidine was also shown to cause chromosomal instability, however it is not clear whether this effect is caused by its demethylation activity [59, 83] or other nonspecific DNA damaging activity since 5-azacytidine can cause genetic effects even in yeast which do not bear methylated cytosines [84].
Another line of experiments which supports a causal relation between hypomethylation and cancer comes from the modulation of dietary methyl intake in rodents [63, 85, 86]. These studies suggested a protective effect of high methyl diets against cancer in animals [87] and that hypomethylating diets promoted carcinogenesis [63, 85, 86]. There is also some human epidemiological data suggesting that low folate intake associated with high alcohol might increase the risk of colorectal cancer [88-90]. High alcohol intake reduces SAM concentrations in the liver [91]. Folate is required for the synthesis of tetrahydrofolate a cofactor required for synthesis of methionine, which is the precursor of SAM the methyl donor in DNA methylation reactions. Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism. Two common enzyme activity-reducing nucleotide polymorphisms (677C --> T/Ala222Val and 1298A --> C/Glu428Ala) have been described in MTHFR however although association of these mutations and cancer risk were found in some studies in colorectal cancer [92, 93] and premenopausal breast cancer [94] other studies in cervical cancer [95] and breast cancer [96] failed to show an association. The overall picture is still inconclusive and more studies are required. In summary, although indirect and incomplete, nevertheless the dietary data support the model that global hypomethylation can promote cancer. They might also offer potential dietary strategies for protection from global hypomethylation.
Recent genetic data also supports the hypothesis that global hypomethylation might promote cancer. Mice bearing hypomorphic DNMT1 alleles introduced by homologous recombination exhibit an increased incidence of lymphomas [97].

6. Inhibition of global hypomethylation as a potential therapeutic strategy in cancer

Figure 2. Demethylation inhibitors inhibit and 5-aza-CdR induce the activity of metastatic genes: a model.
The methylation status of metastatic genes such as uPA is in steady state equilibrium, which is a balance of methylation catalyzed by DNMTs and demethylation catalyzed by demethylases. In metastatic cancer cells that express high demethylase activity the balance is tilted towards demethylation. Inhibition of demethylase activity with inhibitors such as SAM blocks demethylation and tilts the equilibrium towards the left resulting in methylation, silencing of the genes and inhibition of metastasis. In nonmetastatic cancer cells genes required for metastasis are methylated and silenced resulting in inhibition of metastasis. Treatment of such tumors with the DNA methylation inhibitor 5-aza-CdR might result in blocking maintenance methylation (DNMT) during replication and allowing the methylation equilibrium to tilt toward the right resulting in hypomethylation, activation of metastatic genes and induction of metastasis.

If hypomethylation plays a causal role in cancer, such as promoting metastasis, then inhibition of demethylation should inhibit metastasis (Fig. 2). Since the identity of the demethylating enzymes responsible for global hypomethylation in cancer is yet unknown, no specific inhibitors of demethylation are available. We have recently tried two different strategies to induce hypermethylation and silencing of prometastatic genes in cancer. First, we have recently shown that the methyl donor SAM inhibits active replication-independent demethylation of ectopically methylated DNA in HEK 293 cells [98]. SAM is generally believed to stimulate the DNA methyltransferase methylation reaction however our data suggested that its main effect was inhibition of active demethylation. We therefore tested whether treating highly invasive breast cancer cells MDA-231 with SAM would result in reversal of the hypomethylated state of the protease uPA and as a consequence silencing of its expression. Our unpublished data has shown that treating MDA-231 cells with SAM results in hypermethylation and silencing of uPA. SAM treated cells showed a drastic reduction in their invasiveness in vitro and metastasis in vivo. Since SAM might have effects other than increasing DNA methylation, we tested whether the antimetastatic effects of SAM are reversible by the DNA demethylating agent 5-za-CdR. The reversal of the 5-aza-CdR effect on metastasis supports the hypothesis that SAM inhibits metastasis by causing hypermethylation (see Fig. 2 for general model). We have previously suggested that MBD2b a methylated DNA binding protein encodes a demethylase activity [99]. Our suggestion was contested by a number of studies [100, 101], however some of our recent data support the claim that MBD2 is associated with DNA demethylase activity [22, 98, 102, 103]. We therefore tested whether antisense knockdown of MBD2 would result in silencing of uPA. Our unpublished data suggests that knock down of MBD2 results in hypermethylation and inhibition of uPA expression, inhibition of invasiveness in vitro and metastasis in vivo (Pakenshan et al., submitted).
Taken together this preliminary data supports the hypothesis that inhibition of hypomethylation might unfold to a principal therapeutic approach for the treatment of metastasis (Fig. 2). If hypomethylation coordinates the expression of multiple genes as suggested from the increasing list of hypomethylated genes involved in metastasis, then inhibition of hypomethylation is preferable to current approaches targeting specific proteins or proteases. Redundancy in the repertoire of metastatic genes might be a serious drawback of targeting single proteins. It is critical therefore to first characterize the proteins responsible for demethylation in metastatic cancer cells and to develop their specific inhibitors. It is also important to identify the repertoire of genes induced by demethylation and their role in metastasis.

7. Conclusions
Recent data suggests that the DNA methylation machinery might have different roles at different stages of cancer. It is therefore important to further understand the relative roles of methylation and global hypomethylation. Such an understanding is critical for taking advantage of the therapeutic potential of different components of the DNA methylation machinery as anticancer targets. Since both hypo- and hypermethylation are involved in cancer it is important not to provoke one process by inhibiting the reverse reaction. Hypermethylation is proposed to be critical for deregulated growth while hypomethylation is important for metastasis. In addition, DNMT inhibition could arrest cell growth and induce tumor suppressor gene expression without inducing global hypomethylation. It might be possible therefore to knock down the growth promoting effects of excessive DNMT without promoting the adverse effects of global hypomethylation. Inhibition of hypomethylation on the other hand might reverse metastasis. An important question is whether inhibition of demethylation could promote silencing of tumor suppressor genes by remethylation. Since inhibition of metastasis by inhibition of demethylation would be applied mainly to aggressive tumors that most probably have silenced their relevant tumor suppressor genes, this might not be a major concern. However this possible concern might be relevant if either dietary or pharmacological hypermethylation is attempted as a prophylactic measure. Although many obstacles and question remain in proper targeting of DNA methylation in cancer therapy, nevertheless this field offers the opportunity of modulating nodal machineries that coordinate the programming of tumor growth and metastasis.

Acknowledgements
The work from MS laboratory discussed in this paper was supported by the National Cancer Institute of Canada (NCIC)) and the Canadian Institute of Health Research CIHR. Work from SAR laboratory was supported by the CIHR.

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