Genetics,Cytogenetics,and Epigenetics of Colorectal Cancer

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[Epigenetics 3:4, 193-198; July/August 2008]; ?2008 Landes Bioscience

Colorectal cancer (CRC) is the most common cancer in non-smokers posing a significant health burden in the UK. Observational studies lend support to the impact of environmental factors especially diet on colorectal carcinogenesis. Significant advances have been made in understanding the biology of CRC carcinogenesis in particular epigenetic modifications such as DNA methylation. DNA methylation is thought to occur at least as commonly as inactivation of tumor suppressor genes. In fact compared with other human cancers, promoter gene methylation occurs most commonly within the gastrointestinal tract. Emerging data suggest the direct influence of certain micronutrients for example folic acid, selenium as well as interaction with toxins such as alcohol on DNA methylation. Such interactions are likely to have a mechanistic impact on CRC carcinogenesis through the methylation pathway but also, may offer possible therapeutic potential as nutraceuticals.

Introduction

In the UK, colorectal carcinoma (CRC) is the most common gastrointestinal tract malignancy with almost 30,000 new cases diag-nosed per annum and an average 40% five year survival although in some parts of Europe and United States this approaches 60%.1-4 This difference has been attributed in part due to variation in stage of presentation of the disease. Globally, the majority of cases occur in developed countries, particularly North America and Western Europe 5 with a world wide annual mortality of 600,000.2 Since the change to a Western dietary pattern in Japan, the incidence and mortality rates of CRC have increased markedly.6 Observational studies lend support to the influence of environmental factors which demonstrate an increase of colorectal cancer in migrants from low to high-risk countries, compared to age and sex matched controls.7 Geographical variation of CRC between east and west may also partly be explained by local customs—one such example is

consumption of green tea. This popular beverage in the Far East has been shown in murine models to possess anti-carcinogenic proper-ties.8 The importance of environmental exposure (especially diet) is further highlighted by the fact that only a small proportion of cancers can be attributed to germ line mutations. Doll and Peto 9 in 1981 initially concluded that diet caused cancer in up to 70% of the population in the USA and other industrialised countries. However a more realistic and generally accepted figure is probably up to a third in the variance of cancer incidence between populations can be attributed to habitual variation in diet, as highlighted in the recent World Cancer Research Fund report (WCRF/AICR 2007).10

Hence, this review is timely and will endeavour to examine the exisiting literature and summarize the influence of common dietary intakes such as alcohol, folate, selenium, green tea and phytoestro-gens on CRC risk with particular emphasis on mechanisms related to DNA methylation. These dietary substrates (alcohol, folate and selenium) were chosen as they have been shown in epidemiological studies to be associated with colon cancer.32-32,52,60 Although the role of phytoestrogens and green tea 51 in cancer prevention remains controversial, these substrates nevertheless represent proof of prin-ciple of dietary factors influencing DNA methylation. In addition, we introduce a novel concept of the ‘Methylation Health and Carcinogenesis pendulum model’ and the role of diet in maintaining this equilibrium.

DNA Methylation and Colorectal Cancer (CRC)

Epigenetic alterations (genetic alterations that do not alter the DNA sequence) such as DNA methylation, are among the most common molecular alterations in human cancers including colorectal cancer.11 The addition of a methyl group to the carbon-5 position of cytosine residue is the only common covalent modification of human DNA.12 It occurs almost exclusively at cytosines that are followed immediately by guanine (CpG dinucleotides). The majority of the genome displays a depletion of CpG dinucleotides and those that are present are nearly always methylated. Conversely, small stretches of DNA known as CpG islands (usually located within the promoter regions of human genes), whilst rich in CpG dinucleotides, are nearly always free of methylation. It is this methylation within the islands that has been shown to be associated with transcriptional inactivation of the corresponding gene.13 The methylation patterns are controlled by DNA methyltransferases (DNMT) as well as CpG

Review

A review of dietary factors and its influence on DNA methylation in colorectal carcinogenesis

R.P . Arasaradnam,1-3,* D.M. Commane,1 D. Bradburn 2 and J.C. Mathers 1

1Human Nutrition Research Centre; School of Clinical Medical Sciences; Newcastle University; Newcastle UK; 2Department of Surgery; Northumbria Healthcare NHS Trust;

Northumberland, United Kingdom; 3University Hospital Coventry & Warwick; Coventry UK

Key words: DNA methylation, colorectal cancer, diet, nutraceuticals, alcohol, folate, selenium

*Correspondence to: R.P . Arasaradnam; Newcastle University; Human Nutrition Research Centre 8; John St.; Coalville, Leicestershire LE67 8L UK; Email: r.p.arasaradnam@https://www.360docs.net/doc/8b12500540.html,

Submitted: 05/01/08; Accepted: 06/26/08

Previously published online as an Epigenetics E-publication:

https://www.360docs.net/doc/8b12500540.html,/journals/epigenetics/article/6508

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methyl binding proteins (MBD), the latter of which is involved in ‘reading’ methylation marks.13,14

In CRC, as with other tumors, loss of genomic methylation is a frequent early event and correlates with disease severity and metastatic potential.15 Likewise, aberrant promoter hypermethylation which usually occurs at CpG islands is also thought to be an early event in CRC as it is detect-able in early precursor lesions.16 Feinberg and Vogelstein were the first to report changes in DNA hypomethylation in tumors compared with normal tissue.17 However, this concept was further complicated by the observation that most cancer types have both global hypomethylation and hyper-methylation in the gene promoter regions.17-19 Hypermethylation in promoter regions is associ-ated with transcriptional silencing which is at least as common as inactivation of tumor suppressor genes through DNA mutations.20,21 Virtually all pathways in colorectal carcinogenesis, for example loss of control of cell cycle regulation (p16INK4a , p14ARF ), silencing of DNA mismatch repair genes (MLH1, O 6-MGMT ), possible loss of function of apoptosis genes (DAPK, APAF-1) and altered carcinogen metabolism (GSTP1) involves promoter gene hypermethylation.22-24 Global hypomethyla-tion which has been observed in both colorectal cancer and adenomas 25,26 is thought to be able to induce regional de novo hypermethylation 27 as well as expression of oncogenes.28 However, there is still paucity of evidence to suggest a ‘cross talk’ between global and promoter gene methylation and it is likely that these events are independent of each other.

The ‘Methylation Equilibrium’ Concept

Unlike germ line mutations (which are heritable from one cell to its daughter), epigenetic changes such as DNA methylation are potentially reversible. Because gene expression can be re-established by demethylation of promoter regions,29 this offers the opportu-nity for the role of diet in cancer prevention. Hence any imbalance

between promoter hypermethylation and global hypometylation may potentially be reversed in order to achieve ‘normal’ methylation patterns i.e., ‘methylation equilibrium status’. In other words, it is plausible that the genome default methylation status is to maintain normal methylation patterns and thus gene expression, which, in disease is subsequently altered. We hypothesize that certain dietary factors may contribute directly to this equilibrium by preventing or encouraging either promoter hyper or global hypomethylation. This is in essence an extension of the ‘Health Pendulum’ concept.30 The Health Pendulum concept simply states that an individual can shift from the normal healthy state to a disease state as a result of external

environmental pressures of which diet is an important factor. In Figure 1, the pendulum is held in ‘methylation equilibrium’ when in a state of good health. However, the pendulum can be displaced from equilibrium either to the right or left which could lead to colon carcinogenesis for example either through genome hypo methyla-tion if displaced to the left or gene promoter hypermethylation if displaced to the right. The arrows indicate movement away from equilibrium, resulting in displacement either right or left depending

on dietary intake (deficient or excess), phenotype and age of the individual. The implication being that with a balanced intake, the pendulum will remain in equilibrium and hence the ‘methylation equilibrium’ maintained.Dietary Intake Alcohol. Several studies have shown an association between higher alcohol intake and colorectal cancer particularly for distal rather than more proximal disease.31,32 It has been suggested that the carcinogenic effect of alcohol within the colon is mediated through its adverse effect on folate status. Alcohol in murine studies has been shown to reduce methionine synthase (MS) levels and consequently reduced s-adenosylmethionine (SAM—universal methyl donor) and elevated homocysteine levels.33,34 The ensuing genomic hypomethy-lation is likely to play a causal role in CRC carcinogenesis.

There is evidence (at least in rodents) that alcohol induces genomic DNA hypomethylation but not methylation of specific genes such as p53.35 In the Netherlands Cohort Study,36 higher frequency of promoter methylation of specific genes involved in colorectal cancer carcinogenesis (APC-1A, p14ARF , p16INK4a , hMLH1, O 6-MGMT and RASSF1A ) was observed in those with low folate/high alcohol compared with high folate/low alcohol consumers. This finding would

Figure 1. The Methylation ‘Health and Carcinogenesis’ Pendulum Model. In health the pen-dulum is held in equilibrium dependant on individual phenotype and age. In the presence of external pressures such as diet, this pendulum can be swung to promote carcinogenesis either through genomic hypomethylation or promoter gene hypermethylation. Alcohol consumption in excess can shift the pendulum either way. Similarly folate when depleted or when supple-mented in the presence of putative precursor lesions such as colonic aberrant crypt foci (ACF) or adenomas can swing the pendulum away from health. Conversely selenium depletion may result in both genomic hypomethylation and aberrant promoter methylation.

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suggest that the mechanistic link between high alcohol intake and increased CRC risk seems to be mediated through low folate status.Phytoestrogens. Phytoestrogens which include coumestans, isoflavones and lignans are plant derived oestrogen-like compounds. They are naturally occurring in many foods including fruits, legumes such as soy and rice.37 Phytoestrogens have several biological actions including anti-oestrogenic, anti-inflammatory and anti-carcinogenic effects.38,39

In vitro studies have suggested that phytoestrogens antagonise 17β-oestrodiol and compete for binding to oestrogen receptor (OR).40,41 CRC risk is known to be influenced by oestrogen expo-sure although the specific mechanism remains unknown.42 The ESR gene (oestrogen receptor gene) shows age related methylation and is methylated in colorectal adenoma 43 and sporadic colorectal neoplasia,44 suggesting that ESR methylation may predispose to colorectal neoplasia. In cell line studies, aberrant hypermethylation of the promoter region of the ESR gene has been shown to result in transcriptional silencing.45 Hence no OR will be available for binding thereby increasing overall circulating oestrogen levels and cancer risk. Clearly phytoestrogens will not have any beneficial effects once ESR gene is hypermethylated and it may be that its beneficial effects occur prior to ESR gene methylation. In rodents a proposed protec-tive mechanism of phytoestrogens has been promoter methylation of H-ras proto-oncogenes, although interestingly, no methylation was observed in either c-myc or c-fos proto-oncogenes.46 Whilst the bioavailability for these individual compounds is low, consump-tion in combination with other polyphenols, and possibly histone deacetylase inhibitors, may produce an enhanced effect on DNA methylation.47 Suffice to say current evidence would suggest that the protective role of phytoestrogens are heterogeneous, depending on target tissue 48 with little evidence to support direct influence on DNA methylation at least in the colonic epithelium.

Tea. Green tea, which is a popular beverage particularly in the Far East, has been shown in murine models to possess anti-carcinogenic properties.8 The major (polyphenol) ingredient, (-) epigallocatechin-3-gallate (EGCG) is a potent inhibitor of catechol-O-methyltransferase (COMT) activity.49 Both COMT and DNA methyl transferase (DNMT) belong to the same family of SAM dependant methyltransferases. Inhibition of DNMT by certain drugs such as 5-aza-deoxycytidine has been shown in mice to inhibit cancer growth, induce apoptosis and reduce tumor volume.50

In an in vitro experiment using HT 29 cells, (where molecular modelling was used to show that EGCG fits into the catalytic pocket of DNMT1) prevention of carcinogenesis is through competitive inhibition of DNMT1.51 The authors also showed resultant reversal of methylation in p16INK4a (tumor suppressor gene), retinoic acid receptor (RARB ), O 6-methylguanine methyltransferase (MGMT ) and hMLH1 genes. Thus green tea (EGCG) may inhibit DNMT causing CpG demethylation and reactivation of previously methyla-tion silenced genes. Moreover, high concentrations of EGCG are not required, 20 micromol being sufficient, which can be achieved in saliva and stomach. EGCG thus provides a good example of a food component with the capacity for reversal of aberrant methylation—at least in vitro.

Selenium. Interest in selenium and cancer prevention stemmed from early population based studies noting an inverse relationship between selenium status and carcinogenesis in particular colon

cancer in geographical areas where selenium was low in soil.52 The large Nutritional Prevention of Cancer T rial,53 which was a randomised double blind placebo controlled interventional trial provides the strongest evidence for the protective effect of selenium against colorectal cancer. Skin cancer was the primary focus of the trial and effects on CRC were secondary outcomes. The dose of selenium used was 200 mcg/day in the form of a yeast supplement. After a follow-up of 8,271 person years, the relative risk for all cancers in those supplemented with selenium was 0.5 with signifi-cant protection against CRC, lung and prostate.

Selenium is an essential trace element with both antioxidant and pro-apoptotic properties.54 In human fibroblasts, selenium in the form of Selenomethionine has also been shown to induce DNA repair.55 Other mechanisms whereby selenium deficiency may promote carcinogenesis can be seen in both cell culture (caco-2 cells) and animal studies. Both have demonstrated that in the colon, selenium deficiency causes global hypo methylation and in addition, promoter methylation of p53 and p16 genes.56 In addition, selenium supplementation has shown marked reduction in the number of aberrant crypt foci in the colon.57 In human colon cancer, selenium (sodium selenite) has been shown in to play a role in chemopre-vention by inhibiting DNA Methyltransferase (DNMT), thereby suppressing aberrant DNA methylation.58 Of note most studies have used selenomethionine as it is the major component of Se-enriched foods and has non-toxic properties.

Folic acid. Until recently, folate has been one of the nutrients most strongly implicated in terms of protection against CRC. An inverse relationship between folate intake and risk of colorectal adenoma was demonstrated in the Health Professionals Follow-Up study and in the Nurses Health study where the relative risk of adenomas was 0.66 for women and 0.63 for men in those with a higher intake of folate.59 Similarly in a review of 11 case control and cohort studies, a 40% risk reduction of CRC was seen in the highest consumers of folate.60 Conversely, recent results from the Polyp Prevention Group 61 has shown no reduction (RR 1.13 at 3–5 years follow up) in risk of adenoma recurrence with folic acid supplemen-tation (1 mg/day) even in susceptible individuals (low baseline folate status and those that consumed alcohol). Folate through its role in the one carbon metabolism is crucial for both DNA synthesis as well as methylation. Potential mechanisms for folate deficiency mediated carcinogenesis include (1) DNA damage 62 (uracil misincorporation), (2) increased cell proliferation,63 (3) aberrant global or promoter methylation,64 (4) MTHFR polymorphisms 65 and (5) possibly DNMT inhibition.66

Aberrant global and site specific methylation. In rodents, folate deficiency protects against whilst supplementation increases risk of development of cancer.67 Results from studies on the effect of folate deficiency on DNA methylation status are inconsistent, with some showing hypo methylation,68 no change 69 or hyper methylation.70 In the APC min mouse model, folate deficiency resulted in lower S-adenosyl methionine (SAM) levels—universal methyl donor, without affecting genomic DNA methylation status.66 Moreover, the effects of folate supplementation were less clear.71-73 In a recent randomised, double blind, placebo controlled intervention study of 31 patients with adenomatous polyps, Pufulete et al.74 has shown that supplementation with physiological amounts of folic acid (400 mcg/day) over a ten week period increased genomic DNA methylation in

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both leucocytes (significantly) and rectal mucosa (non-significantly) with an accompanying fall (significant) in plasma homocysteine. The validity of measuring methylation status in the leucocyte as a surrogate marker of methylation within the colono-cyte remains unknown. Methylation status within rectal mucosa has been shown to be increased in patients with colorectal adenoma 75-76 and CRC 77 when supplemented with high doses of folate (5–10 mg/day) over 3–6 months. In the study by

Kim et al.76 however, both groups (patients with adenomas and placebo) showed an increase in rectal mucosa DNA methylation at one year. The above studies have used varying doses of folic acid over varying time courses with methylation measured

in different tissues which may explain the inconsistencies observed. Furthermore, in the aged, altered colonic physiology may mean inconsistent response to folate such that beneficial effects of folate are lost even when folate status is replete.73,78 Thus there is a fine balance

between preventing and promoting carcinogenesis depending on folate status—Figure 2.DNA methyltransferase (DNMT). The role of DNMT in partic-ular DNMT1, in promoting CRC in the presence of folate deficiency remains inconclusive. This enzyme is critical in catalysing methyla-tion reactions of cytosine residues in DNA. Thus deficiency of this enzyme would be expected to alter methylation status. However, T rasler et al.66 have shown that in the APC min mouse model, folate deficiency in the presence of DNMT deficiency reduced tumor load but had no effect on global genomic DNA methylation or promoter methylation of two specific genes (p53 and E-cadhedrin ; p53 unlike E-cadhedrin is normally methylated at the promoter region in CRC tumorogenesis). Furthermore, the observation of reduced tumor load in the presence of folate deficiency was noted only after the devel-opment of adenomas in this model.79 No effect on tumor load was

noted if the amount of folate in the diet was altered prior to develop-ment of adenomas.

Whilst this study is supportive of tumor load reduction in the

presence of folate and DNMT1 deficiency, it does not support

the hypothesis that the sole mechanism is through its effects on

DNMT1. In fact studies with DNMT1 gene knock out models have

shown that it is still possible to obtain a viable offspring suggesting

that other DNMTs (DNMT3a or 3b) play a role in de novo methyla-tion crucial for development.80Issues surrounding folic acid and its effect on methylation and

colorectal carcinogenesis remain complex. Folate seems to act as

a double edged sword; protective against initiation of carcinogen-esis 60,64,68,71,81-83 but also promoting carcinogenesis, for example, increase in mortality from breast cancer during pregnancy with folate supplementation.84 The way we consider alterations in folate status within colonocytes and its effect on carcinogenesis may also be important. For example, alteration in folate form and distribution (qualitative alterations) in relation to certain geno-types might be more informative than total folate concentrations

(quantitative changes) alone.

85Conclusions Genotypic differences will naturally result in variable individual benefits from certain nutrients. The aged hypothesis of the protective effect of fruits and vegetables against CRC through their anti-oxidant properties and induction of apoptosis is clearly not the whole story. Epigenetic mechanisms are well defined in CRC carcino-genesis 11,16-18,22-26 and dietary intake of common nutrients have been shown to alter this.52,57,72-75 Thus, diet may act as a form of chemoprevention to influence DNA methylation status. Moreover, a balanced nutrient intake may contribute directly to maintaining the ‘methylation equilibrium’ by preventing either promoter hyper or global hypo methylation (Fig. 1). Clearly, effects of nutrient status cannot be defined based on its effects on genomic DNA methylation alone but rather taking into consideration the individuals genotype,

age, tissue specificity and critically in the colon, anatomical site (e.g.,

proximal vs. distal colon).

It is not clear however as to ideal duration of exposure of these

dietary components to affect DNA methylation status or the optimal

dose of these compounds required to exert a chemopreventitive

effect. Current evidence has not identified a unified mechanistic link

between diet and DNA methylation in CRC carcinogenesis which,

may simply reflect the heterogeneous nature of such substrates.

Future Work The impact of certain micronutrients on DNA methylation adds to our current understanding of possible mechanisms of how diet is linked with colorectal cancer. However, the specific mechanistic effects of diet on DNA methylation requires further study for example, the effect on promoter methylation of tumor suppressor genes and alteration in transcript levels of DNMT1,3a and 3b by varying concentrations of a particular micronutrient. Once these mechanisms

Figure 2. The proposed ‘see saw’ schematic illustrates the fine balance prevention and promotion of carcino-genesis. Known factors such as genotype, duration of exposure to folate, ageing and folate status (in box) can

slide one way or the other thereby either preventing carcinogenesis (through increasing nucleotide synthesis)

or promoting carcinogenesis through (global or aberrant promoter methylation). The presence or absence of putative pre-cancerous lesions such as aberrant crypt foci (ACF) may act as the fulcrum forcing the ‘box’ to slide one way or the other.

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have been elucidated, the next step would be human interventional trials using these substrates as nutraceuticals. Subsequent findings would then result in a paradigm shift moving us into an era of targeted modification of methylation patterns using diet.

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