Bioscience & Chemistry Programme, Faculty of Health and

Wellbeing, Professional and Scientific Practice 3

A Review of the Effect of Methyl Donor Depletion on Global

DNA Methylation and Promoter Gene Methylation Patterns in

Models of Oral Cancer and Other Epithelial Carcinomas

By Amy Grayson

Submitted: 25th September 2015

Supervisors: Dr Vanessa Hearnden

Dr Craig Murdoch

Professor Hilary J Powers

Visiting Tutor: Dr Adrian Hall

1

Contents

1. Abstract 3

2. Introduction 4

3. Methyl Donor Depletion on Cancer Associated Genes

3.1. P16 6

3.2. P53 7

3.3. DAPK1 8

3.4. LINE-1 10

3.5. CDH1 11

3.6. RARβ 12

4. Conclusion 13

5. Acknowledgements 16

6. References 17

2

1. Abstract

It is known that DNA methylation provides a significant role in carcinogenesis of various

different cancers although there is debate over specific methylation patterns found in

cancerous cells. It is generally accepted that there is tumour suppressor gene promoter

hypermethylation but global hypomethylation in cancerous cells. DNA methylation requires

methyl donors obtained from the diet. Changes in the intake of dietary methyl donors is

thought to affect methylation patterns within normal cells, causing the initiation of

carcinogenesis. The results of this review found a general lack of knowledge and research

towards the association of methyl donor availability on oral carcinogenesis and other human

tissue cancers, however methyl donor availability has been argued to have a significant

effect on the promoter hypermethylation of p16 and p53 tumour suppressor genes in oral

cancers and DAPK, LINE-1, CDH1 and RARβ in other epithelial carcinomas. These results can

be used as a predictor of the effect of methyl donors on these specific genes in oral cancers.

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2. Introduction

Oral cancer is the 16th most common cancer worldwide, with more than 300,000 new cases

diagnosed in 2012 (CRUK, 2015). Oral cancer is also the 15th most common cancer in

Europe, with around 61,400 new cases diagnosed in 2012 (CRUK, 2015). Several factors are

commonly known to affect a person’s risk of oral cancer including; smoking tobacco or

chewing betel quid; excessive alcohol consumption; human papillomavirus infection;

inadequate intake of dietary methyl donors (Llewelyn et al, 2004; La Vecchia et al, 1997)

Dietary methyl donors are molecules which donate a methyl group (CH₃) to S-

adenosylmethionine (SAM) which in turn donates a methyl group to a DNA nucleotide and

methylate a specific region of DNA. There are four known methyl donors which are folate,

choline, betaine and methionine. All of these enter the body through specific dietary foods.

It is hypothesised that a low intake of methyl donors can initiate the development of

neoplasms through altering the gene expression in tumour suppressor genes and

proliferative genes involved in the cell cycle control and DNA replication. Conversely, it has

also been suggested that physiologically healthy levels of methyl donors are thought to be

pro-cancerous to already established neoplasms as they increase expression of proliferative

genes, causing increased growth of the tumour and progression of the cancer development.

There are two main types of methylation which alter gene expression and can lead to

carcinogenesis within cells. Global DNA methylation is the process by which a part of the

DNA within the CpG dinucleotide is methylated which causes silencing of that particular

gene expression. In cancer, global hypomethylation is known to contribute to the activation

of oncogenic genes and cause chromosome instability. The second mechanism is

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methylation of the promoter region of a specific gene. This gene is usually linked to

proliferation or cell cycle control. The promoter region which is methylated is at a specific

loci within the DNA and causes the silencing of a related gene located downstream of this

promoter.

Figure 1: Diagram showing the process of promoter methylation of a specific gene. When the promoter region is

unmethylated, the associated gene can be expressed however, if the promoter region is methylated, the

associated gene is inactivated by the methylation process and cannot be expressed. (National Cancer Centre

Research Institute, 2010)

There is currently very little literature regarding the effect of methyl donor deficiency on the

specific methylation patterns of distinct genes found in cancers however most papers that

do discuss this mainly focus on folate and folic acid. The genes that are found to be affected

mostly play a role in cell proliferation, growth and tumour suppression. This review aims to

compare and contrast the effect of specific methyl donor availability on the methylation

patterns found in normal and cancerous cell models.

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3. Methyl Donor Depletion on Cancer Associated

Genes

3.1. P16

P16, also known as CDKN2A or p16INK4A, is a tumour suppressor gene that regulates the

progression of cells from the G1 phase to the S phase of the cell cycle. The promoter gene of

P16 has been noted to be highly hypermethylated in many cancers such as salivary gland

tumours, nasopharyngeal carcinomas, breast cancers, cervical cancers and colorectal cancers

(Nikolic et al, 2015; Nawaz et al, 2015; Delmonico et al, 2015; Blanco-Luquin et al, 2015; Lee

et al, 2015; Dumitrescu et al, 2015; Don et al, 2015) however, the association of the aberrant

methylation to the presence of methyl donors has not been explored in much detail.

The general presence of hypermethylation of p16 promoter region has been discovered in

head and neck squamous cell carcinomas for a long time (Nakahara et al, 2006, Lee et al,

2004 and Cody et al, 1999) however this has only been linked to the deficiency of methyl

donors very recently and only one paper has been identified to have specific links to oral

cancer. A human study conducted in 2006 contributes supporting evidence that p16INK4a

promoter region is methylated as a result of low folate intake which in turn causes genetic

silencing of the pRB pathway, leading to tumour progression (Kraunz et al, 2006). Kraunz and

his colleagues set up an experiment whereby head and neck tumours were removed from

carcinoma patients and PCR was performed to quantify the amount of P16INK4A expression

in each of the 242 head and neck squamous cell carcinoma patients. This study provides

evidence for the hypermethylation of p16 tumour suppressor gene promoter in cancers

through alteration of the concentration of dietary methyl donors however, for this to be

considered a reliable mechanism contributing specifically to the change in methylation

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patterns, the subject required a lot more attention as there seems to be only one paper

published about the correlation.

The lack of research into p16 promoter hypermethylation linked to methyl donor deficiency

is consistent throughout other site-specific tumours but it has been mentioned somewhat.

In 2002, extensive site-specific p16 methylation was identified in 100% of rat tumours in

animals fed a folate deficient diet (Pogribny and James, 2002). The results showed that the

increase in p16 promoter methylation was positively associated with the increase in tumour

progression.

3.2. P53

One possible underlying mechanism of cancer development as a result of low dietary folate

is thought to be through mediation of p53 tumour suppressor gene expression, as evidenced

in cases of colorectal carcinomas (Schernhammer et al, 2008). P53 is an example of another

tumour suppressor gene and is involved in the stabilisation of DNA to prevent genetic

mutations upon genome replication. It has been noted that there is significant global

hypomethylation and also p53 specific hypomethylation in human colon carcinoma cells

under folate deficient conditions and that this methylation pattern is reversible when the

cells are supplemented with folate afterwards (Wasson et al, 2006). P53 promoter

hypermethylation was also observed as a result of dietary folate deficiency in and was linked

to tumour progression within a rat colon model (Kim et al, 2000).

In terms of oral carcinogenesis, overexpression of p53 has been reported in two cancerous

oral cell lines although the expression of p53 was altered by the supplementation of folic

acid in only one cell line. This suggests that folate may have a significant role in growth of

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established oral cancers through the p53-dependent and p53-independent pathways

(McCabe et al, 2010). Conversely, a recent study by Balbuena and Casson, published a few

months prior to the McCabe paper, describes how dietary folate is not associated with p53

expression and thus is not associated with oesophageal cancer (Balbuena and Casson, 2010).

Since the McCabe paper only shows alterations in p53 expression in one out of two already

established cancer cell lines supplemented with folic acid, this provides uncertain evidence

as to whether aberrant p53 methylation is linked specifically to folate intake in oral cancers.

Also, as all evidence and variable factors accounted for in the Balbuena and Casson paper

show no statistical significant relationship between p53 expression and folate intake, the

association between p53 methylation and folate in relation to oral cancer development and

progression is very unclear.

The evidence supporting the link between p53 mutation and folate depletion and

supplementation on colon carcinogenesis may suggest the mechanism of action of folate

depletion on p53 methylation is the same in oral cancers however the literature seems to

suggest otherwise and the conflicting evidence on folate-mediated p53 methylation in oral

cancer studies makes it difficult to assume this mechanism is in use in oral cancer cases and

also that it is consistent throughout all cancers.

3.3. DAPK1

Death associated protein kinase 1 is a mediator molecule for apoptosis and is one of the

most commonly upregulated genes associated with cancer throughout various tissues in the

human body, through promoter hypermethylation (Peng et al, 2010; Almeida et al, 2015,

Hafez et al, 2015; Laskar et al, 2014) however, a recent paper looking into papillary thyroid

cancer found an overall average decrease in DAPK promoter methylation compared to their

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controls. This research does not agree with other recent literature and is the only research to

find this decrease in DAPK promoter hypermethylation in cancer (Wang et al, 2014). All other

literature reviewed is relatively recent and confirms the upregulation of DAPK promoter

methylation so it can be considered that the Wang paper provides anomalous results within

this study area.

In terms of methyl donors, Flatley and colleagues did a variety of work looking precisely at

the role of folate on cervical carcinomas. The research group’s initial focus was on 3 tumour

suppressor genes within the cervical cancer environment, including CDH1 (see Section 3.5)

and DAPK, and showed a greater concentration of folate in cervical cells causes increased

promoter methylation of DAPK (Flatley et al, 2009). Later, they also linked this increase in

folate and DAPK methylation to an individual’s risk of high-resistance HPV persistence (Flatley

et al, 2014). Continued work from this group of researchers suggests that the results are

reliable and consistent and can be considered as strong evidence for the positive association

between folate and DAPK methylation.

While there is a lot of literature confirming the methylation of DAPK in many oral cancers

(Choudhury and Ghosh, 2015; Don et al, 2014; Arantes et al, 2015), no work has been

published on the role of any methyl donors in relation to the upregulation of DAPK in oral

cancers despite the vast amount of literature proving the presence of DAPK promoter

methylation in other epithelial cancers. Investigation into this particular study of DAPK

methylation would be beneficial towards oral cancer research.

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3.4. LINE-1

Long Interspersed Nuclear Element-1 is a transposable element of the human genome. This

means that the DNA sequence can change its position within the genome which can create

mutations within the cells. LINE-1 has been noted to be a biomarker for cancer risk (Barchitta

et al, 2015 and Kawakimi et al, 2011) and is hypomethylated in many cancers already such as

colon cancer (Zhou et al, 2015; Sunami et al, 2011; Ogino et al, 2008, Antelo et al, 2012),

breast cancer (Llanos et al, 2015; van Hoesel et al, 2012) non-small cell lung cancer (Liu et al,

2014) and oral squamous cell carcinomas (Baba et al, 2014 and Subbalekha et al, 2009).

Some studies describe the link between LINE-1 hypomethylation and methyl donors.

Schernhammer and colleagues later discovered that LINE-1 hypomethylation as a result of

low dietary folate intake is associated with an increased risk of colon cancer in humans

(Schernhammer et al 2010) and these findings are supported by Asada and colleagues’ work

which involved looking at the effect of a choline deficient diet on the methylation patterns of

LINE-1 in rat livers (Asada et al, 2006). In contrast to this, some opposing views were stated

in 2009 which claim no association between folate status, LINE-1 methylation, and risk of

carcinomas of the colorectal mucosa in humans (Figueiredo et al, 2009). Taking into account

that this study is based on normal colorectal samples and not carcinoma samples, the article

provides a possible explanation for the difference in their findings compared to the work of

Figueiredo, stating that folate concentration may not be relevant to the methylation of LINE-

1 in normal colon tissue but there relationship between folate and LINE-1 promoter

methylation could be revealed once the neoplasm develops (Schernhammer et al, 2010). A

positive association between folate concentration and LINE-1 methylation has also been

discovered in non-small cell lung cancer (Jin et al, 2009) which supports the argument

displayed by both Schernhammer and Asada.

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The connection between methyl donors and LINE-1 hypomethylation in oral cancers has not

yet been discussed but the previous literature demonstrates a possible link between LINE-1

methylation and folate concentration and outlines a greater need for scientific exploration in

this area and gives a possibility that folate may also lead to LINE-1 methylation in oral

cancers.

3.5. CDH1

Cadherin-1, also known as epithelial cadherin (E-cadherin) is the most studied protein within

the cadherin superfamily, encoded by the CDH1 gene, which is involved in mediating of cell-

cell interactions within tissues. The gene itself is is another example of a tumour suppressor

gene as the downregulation of CDH1 has been shown to affect cellular molarity within

tissues, allowing cancerous cells to invade the tissue. CDH1 is most commonly known for its

inactivation during breast carcinoma and metastasis (De Leeuw et al, 1997) but recently,

promoter hypermethylation of CDH1 has been noted in oral cancer (Pannone et al, 2014;

Kordi-Tamandani et al, 2010; de Moraes et al, 2008).

The relationship between CDH1 promoter hypermethylation and the availability of methyl

donors has only been discussed in relation to lung, cervical and breast carcinomas and, such

as with the LINE-1 gene, no literature is currently available on the role of methyl donors on

CDH1 methylation patterns in oral cancer. In 2009, research showed that folate

concentration is a factor contributing to the promoter hypermethylation of CDH1 in cervical

cancers but the authors acknowledge that this is not a simple correlation and other

contributing factors also enhance the hypermethylation process (Flatley et al, 2009).

Research from 2012 demonstrates how no association was found between folate intake and

CDH1 promoter methylation in breast tumours however, folate could contribute to the

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methylation in normal or premalignant tissues (Tao et al, 2011). Following this trend,

Vaissiere and colleagues also found no association between dietary folate intake and

methylation of CDH1 in lung cancer (Vaissiere et al, 2009). These articles are all relatively

new to date and provide strong evidence that there is no statistical correlation between

folate concentration and CDH1 promoter hypermethylation, suggesting that methyl donors

have little or no effect on this particular gene in these tissues. It may be assumed that it is

unlikely that a correlation is to be found between folate alone and CDH1 methylation in oral

cancers and other factors may be necessary to initiate significant methylation pattern

changes and carcinogenesis.

3.6. RARβ

Retinoic acid receptor beta (RARβ) is a nuclear receptor which mediates cell signaling, cell

growth and differentiation. Cancer types such as breast cancer, non-small cell lung cancer,

prostate cancer and oral cancer have all been found to contain hypermethylated promoter

regions of the RARβ gene (Siegel et al, 2015, Huang et al, 2015, Hoshimoto et al, 2015,

Serenaite et al, 2015 and Lai et al, 2014). Dietary folate concentration has been associated

with the promoter methylation of RARβ in breast cancer (Pirouzpanah et al, 2015 and

Lubecka-Pietruszewska et al, 2013) and non-small cell lung cancer (Liu et al, 2009) but no

other evidence contributes to the role of folate on RARβ methylation in any other cancerous

tissues, including oral cancer. The limited amount of data on RARβ promoter methylation in

association with methyl donors and oral cancers means that there is a requirement for

additional research in this area.

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4. Conclusion

All genes discussed in this literature review undergo specific promoter hypermethylation

leading to gene hypomethylation in the carcinoma samples compared to normal human

tissue. To summarise, Table 1 shows the current literature regarding genes names, functions

and any evidence of changes in methylation patterns in relation to cancer and methyl donor

depletion thus far.

Gene name Function Evidence of link of methyl donor

availability to oral carcinogenesis

P16 regulates progression of cell cycle from

G1 to S phase

Yes - Kraunz et al, 2006

P53 Stabilises DNA to prevent genetic

mutations during genome replication

Possibly – contradicting views from

McCabe et al, 2010 and Balbuena and

Casson, 2010)

DAPK1 Mediates apoptosis – also involved in

inflammation

No publications to date but evidence of

association in other carcinomas

LINE-1 Transposable element of human genome

which can create mutations if transposed

at certain loci

No publications to date but evidence of

association in other carcinomas

CDH1 Mediates cell-cell interactions within

tissues and during embryonic

development

No publications to date and opposing

views on association in other carcinomas

RARβ Mediates cell signalling, growth and

differentiation

No publications to date but evidence of

association in other carcinomas

Table 1: Information on all genes reviewed in this paper from the current literature including the gene

of interest name, function within the human body and evidence of promoter methylation pattern

changes in association with methyl donor availability within oral cancers.

13

There are many other accounts of aberrant DNA methylation during the establishment,

development and progression of oral cancer. Several recent literature reviews and research

articles published in the past 5 years have confirmed specific methylation changes in the

following genes within oral carcinomas: LATS-1 (Reddy et al, 2015); PROX1 (Rodrigues et al,

2014; Yokobori et al, 2015; Sasahira et al, 2014); PTEN (Zheng et al, 2015; Ren et al, 2014;

Alyasiri et al, 2013); HSPB1 (Wang et al, 2013); hMLH1 (Gonzalez-Ramirez et al, 2011; de

Oliveira et al, 2014; Caldeira at el, 2011); MGMT (Kordi-Tamandani et al, 2010; Diez-Perez et

al, 2011; Melchers et al, 2015; Don et al, 2014). Despite this, no work has been published to

describe the role of methyl donor depletion on the methylation of these genes.

For the purpose of this review, genes detailed in each section have been selected due to

their involvement in already published literature which show promising results in terms of

their association with methyl donors and methylation of the gene promoter region in cancer.

It may be valuable to conduct more research on these genes in more detail with respect to

methyl donors and oral carcinogenesis to further understand the relationship between

methyl donors and cancer, specifically oral carcinogenesis.

In conclusion, there are many different genes which show aberrant DNA methylation oral

carcinogenesis however only recent publications have begun to explore the role of methyl

donors with respect to the methylation. There is a small pool of literature which mainly

focusses on the role of folate on methylation and only one or two individuals have looked at

the role of other methyl donors such as betaine, choline and methionine in relation to the

levels of DNA methylation in oral cancers and other human carcinomas found in different

anatomical locations. It seems the research focus is gradually changing to incorporate the

effect of methyl donor deficiency on the methylation of specific proliferative and tumour-

14

suppressor genes and thus progression towards dysplasia and carcinoma development.

Further work is needed to fully understand firstly, if methyl donors promote carcinogenesis

and if so, by which mechanisms these molecules target specific genes.

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5. Acknowledgements

The author would like to acknowledge Dr Vanessa Hearnden and Dr Craig Murdoch for their

assistance with the understanding of the topic and for their constant support throughout the

development of this review. The author would also like to acknowledge Professor Hilary J

Powers for her extensive knowledge in the subject area and continual encouragement on

this research. This work was supported by the University of Sheffield’s Department of

Human Nutrition and Department of Oncology and by Sheffield Hallam University’s

Biosciences and Chemistry Department within the Faculty of Health and Wellbeing.

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