School Of Applied Sciences

Implications of promoter hypermethylation of the GSTP1 gene in prostate carcinogenesis and comparing its effectiveness as a clinical biomarker for prostate

cancer with PSA testing

A dissertation submitted as part of the requirement for the BSc Biological Sciences

James Pereira

12/5/14

Abstract

Prostate cancer is the second leading cause of cancer-related deaths in the US

and UK. Research has focused on determining the causes of prostate

carcinogenesis and recently has highlighted that epigenetics may play a pivotal

role in the development of prostate cancer. The aim of this paper was to identify

any novel implications for the promoter hypermethylation of the glutathione S-

transferase Pi 1 (GSTP1) gene in prostate carcinogenesis and to compare its

effectiveness as a clinical biomarker to prostate specific antigen (PSA) testing. The

GSTP1 gene is responsible for the conjugation of carcinogenic compounds with

glutathione to render them inactive during Phase II drug metabolism. Silencing of

the gene through hypermethylation exposes the cell to carcinogenic insult. This

paper has highlighted two carcinogenic compounds that may be involved in

prostate carcinogenesis, benzo[a]pyrene and PhIP. There is evidence that both

compounds are able to form adducts with DNA and evidence that PhIP may also

be responsible for abnormal cell proliferation and as such presents a pathway to

prostate cancer. Additionally, evidence was found that other cellular pathways act

to upregulate DNMTs and as such promote hypermethylation of the GSTP1 gene.

Interestingly, silencing of the GSTP1 gene in hepatocellular carcinoma results in

over activation of STAT3, a process that is known to be carcinogenic. Links to a

similar process in prostate cancer were found. This paper highlighted that the use

of PSA testing as a widespread screening biomarker for prostate cancer has

several issues including a lack of specificity, false positives and promoting

unnecessary needle biopsies. GSTP1 hypermethylation was shown to have high

specificity for prostate cancer, especially when combined with other DNA

methylation profiles. Additionally, it had the advantage of being able to distinguish

between latent and clinical disease and showed potential as a prognostic and

treatment efficacy biomarker.

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Acknowledgments

I would like to thank my supervisor Dr. Kevin McGhee for his support and guidance

throughout the duration of this project. I would also like to thank my housemate Joe

Allen for proofreading my dissertation.

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Contents

Chapter 1: Introduction1.0 Introduction 61.1.1 Prostate Structure 61.1.2 Androgens and Androgen Receptors 71.1.3 Prostate tumour development 81.1.4 Factors Affecting Prostate Carcinogenesis 101.1.4.1 Age 101.1.4.2 Senescence 101.1.4.3 Chronic Inflammation 111.1.4.4 Oxidative Stress 111.2 DNA methylation 111.2.1 Disruption of Transcription Factors 131.2.2 Hypermethylation 151.2.3 Histone Modifications 151.2.4 Factors Influencing DNA methylation 161.2.4.1 Age 161.2.4.2 Diet 171.3 GSTP1 181.4 Biomarkers 211.4.1 Prostate Specific Antigen 211.4.2 GSTP1 as a biomarker 211.5 Aims 241.6 Objectives 24

Chapter 2: Method2.0 Methodology 25

Chapter 3: Results3.0 Carcinogenic compounds 293.0.1 Benzo[a]pyrene 293.0.2 Heterocyclic Amines 313.1 Interaction of GSTP1 with other cellular systems 343.1.1 Transforming growth factor-B 343.1.2 STAT3 353.1.3 The Retinoblastoma protein 373.2 Clinical Biomarkers 383.2.1 PSA testing 383.2.1.1 PSA testing normal limits 383.2.1.2 PSA testing and prostate cancer mortality 383.2.1.3 Risks versus benefits of PSA testing 393.2.1.4 PSA false positives 413.2.1.5 PSA specificity 423.2.2 GSTP1 hypermethylation as a biomarker 423.2.2.1 GSTP1 hypermethylation specificity 433.2.2.2 Discriminatory power of GSTP1 hypermethylation 443.2.2.3 GSTP1 hypermethylation as a prognostic biomarker 453.2.2.4 GSTP1 hypermethylation as a treatment efficacy biomarker 46

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Chapter 4: Discussion and Conclusion4.1 Discussion 474.2 Conclusion 52

References 55

AppendicesEvaluative Supplement 67Interim Interview Comments 70

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Chapter 1: Introduction

On a global scale prostate cancer accounts for 14% of total new cancer cases and

6% of total cancer deaths in males in 2008. It presents a growing health problem

as longevity increases. It is now the most commonly diagnosed cancer in the US

and the UK, and the second leading cause of cancer-related deaths in men in both

countries (Dale et al 2004). The incidence rates of prostate cancer vary by more

than 25 fold world wide and this is thought to reflect the utilization of prostate-

specific antigen (PSA) testing that is able to detect clinically important tumors and

tumours with slow growth rates that might otherwise evade diagnosis (Jemal et al

2011).

1.1.1 Prostate structure

In men, the prostate gland is a tissue surrounding the urethra at the base of the

bladder. Despite the adult prostate lacking in discernible lobular structure (Shen

and Abate-Shen 2010) it can be defined as having a zonal architecture and

includes the central, periurethral transition and peripheral zones (Timms 2008)

(Figure 1). The outermost peripheral zone occupies the most volume and it is this

area that harbours the majority of prostate carcinomas. Mice are the most

frequently used organism as models for the study of the initiation and progression

of prostate cancer because that the dorsolateral lobe in mice is the most analogous

to the human peripheral zone (Berquin et al 2005). At the histological level both the

human and mouse prostate contain a pseudostratified epithelium with three

differentiated epithelial cell types: neouroendocrine, luminal and basal (Peehl

2005). The suggestion that the dorsolateral lobe is analogous to the human

peripheral zone is supported by gene expression profiling data (Berquin et al

6

2005), however, Shappell et al (2004) notes that there are anatomical and natural

history issues that impact on the ability to make straightforward analogies between

genetically engineered mouse models of prostate cancer and the human disease

being modeled.

1.1.2 Androgens and

Androgen Receptors

The development and maintenance of the prostate is dependent on androgens and

androgen receptors (AR) with their action governing both prenatal development of

the prostate and the continued survival of the secretory epithelia; the most

common cell type transformed in prostate adenocarcinoma (Heinlein and Chang

2004). Androgen action and the functional status of AR are important mediators of

prostate cancer progression. Numerous clinical studies have implicated increased

expression of AR with reduced recurrence free survival and disease progression

(Lee 2003). Additionally, low serum testosterone levels in patients with newly

Figure 1: Zonal structure of the human prostate

(Best Health 2014)

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diagnosed prostate cancer have been found to correlate with higher AR expression

and increased blood vessel density within tumours. Heinlein and Chang (2004)

summarise that, despite evidence obtained from animal models that elevated AR

expression can initiate prostate cancer development or is associated with recurrent

growth in the presence of low androgen, the persistent heterogeneity of human

prostate cancer suggests that increased AR expression is not associated with

prostate cancer initiation.

1.1.3 Prostate tumour development

Primary prostate tumours often contain multiple independent foci of cancer that are

often genetically distinct (Clark et al 2008) and therefore prostate cancer is

regarded as a multifocal disease. Shen and Abate-Shen (2010) suggest that the

heterogeneity of prostate cancer is potentially relevant for understanding the

distinction between latent and clinical disease as well as the strong correlation

between prostate cancer progression and aging. Despite this notion that prostate

cancer is a disease of older men, a study by Wolf et al (2010) suggests that cancer

initiation may take place at a relatively early age due to the frequent presence of

histological foci of prostate cancer in prostate specimens from healthy men in their

twenties to forties. Evidence supports the view that the prostate gland can be the

site of multiple neoplastic transformation events, many of which do not develop into

clinically detectable disease but simply give rise to latent prostate cancer

(Montironi et al 2007). There is debate as to whether clinical prostate cancer

initiates from a different pathogenic program than latent prostate cancer however, it

is also conceivable that most latent prostate cancer foci may not undergo activating

events that lead to their development into clinical disease (Bratt and Schumacher

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2011). Prostatic intraepithelial neoplasia (PIN) is widely considered to represent a

precursor for prostate cancer. There are two grades of PIN (low grade and high

grade) but it is the high grade that is suggested to be the most significant risk factor

for prostate cancer (Botswick 2000). PIN is characterized at a histological level by

a reduction in basal cells, enlargement of nuclei and nucleoli, cytoplasmic

hyperchromasia – darker staining of the cells due to increased DNA content and

nuclear atypia which includes chromatin clearing (Iwata et al 2010).

Despite the phenotypic heterogeneity exhibited by human prostate cancer, more

than 95% of prostate cancers are classified as adenocarcinoma, which presents

with a luminal phenotype. Interestingly, Ma et al (2005) were able to show that

many prostate cancers in mouse models also presented with a relatively luminal

phenotype supporting their use as a model for the study of prostate cancer.

Prostate cancer differs from other epithelial tumours in that it lacks distinguishable

subtypes which may differ in both prognosis and/or treatment approach. The

majority of prostate carcinomas are described as acinar adenocarcinomas whilst

other classifications of the cancer include ductal adenocarcinoma, mucinous

carcinoma and signet ring carcinoma; however these are extremely rare Gringon

(2004). The most significant variant of the cancer is neuroendocrine prostate

cancer, which is classified as either a carcinoid tumour or a small cell carcinoma;

this variation is said to represent less than 2% of prostate cancer incidences.

Common sites of secondary metastasis for prostate cancer include lung, liver and

pleura. However if a prostate cancer does mestastasize it consistently moves to

bone where it forms characteristic osteoblastic lesions (Logothetis and Lin 2005).

1.1.4 Factors Affecting Prostate Carcinogenesis

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1.1.4.1 Age

There are several factors that are thought to affect prostate carcinogenesis,

however, the single most important risk factor is advanced age. The chances of

developing prostate cancer increase from 1 in 10,000 for men under 40 to 1 in 7 by

the age of 60 (Thompson 2006). Studies have examined the molecular

consequence of aging, centering on the gene expression changes associated with

cellular senescence, inflammation and oxidative stress. (Bethel et al 2009).

1.1.4.2 Senescence

Cell senescence is a process of cell cycle arrest in which cells become

nonproliferative but remain fully viable (Courtois-Cox et al 2008). Recent work has

identified cellular senescence as a potent mechanism of tumour suppression that

prevents the cell containing the malignant phenotype from proliferating after

oncogenic insult (Shen and Abate-Shen 2010). Cellular senescence has been

shown to occur during prostate enlargement, particularly enlargement as a result of

aging. A study by Chen et al (2005) on genetically engineered mice found that

conditional inactivation of the Pten tumour suppressor gene results in PIN lesions

that display a senescence phenotype providing evidence that senescence is a

protective mechanism suppressing the progression from latent to clinical disease

therefore additional oncogenic events are required for the progression from cancer

precursor lesions to adenocarcinoma (Zemskova et al 2010).

1.1.4.3 Chronic Inflammation

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Studies have also been able to provide a casual link between chronic inflammation

and prostate cancer. Van Leenders (2003) notes that in aging men regions of

prostate atrophy are often associated with increased epithelial proliferation and

have been termed proliferative inflammatory atrophy (PIA). Further work by De

Marzo et al (2003) showed that regions of PIA are often found in close proximity to

PIN and adenocarcinoma and suggested that these lesions may also act as a

precursor to prostate cancer.

1.1.4.4 Oxidative Stress

Another major influence on prostate carcinogenesis is though to be the oxidative

stress. (Minelli et al 2009). Oxidative stress is a result of the imbalance of

detoxifying enzymes and reactive oxygen species, leading to cumulative damage

to lipids, proteins and DNA (Gupta-Elera 2012). Increases in the oxidized DNA

adduct 8-oxy-7,8,dihydro-2’-deoxyguanosine (8-oxy-dG) are correlated with a

reduction in major antioxidant enzymes in human PIN and prostate cancer

(Botswick et al 2000). Additionally, Ouyang et al (2005) was able to show

increased levels of 8-oxy-dG in mouse prostate, correlated with the onset of PIN

following the loss of function of the Nkx3.1 homeobox gene in the mouse prostate.

1.2 DNA methylation

Epigenetics is described as a stable alteration in gene expression potential that

takes place during development and cell proliferation, without any change in gene

sequence (Das and Singal 2004). DNA methylation is a common form of epigenetic

signaling, used to lock genes in the off position and has important function in a

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variety of cellular processes including embryonic development, genomic imprinting,

X-chromosome inactivation and preservation of chromosome stability.

Methylation occurs at the cytosine bases of Eukaryotic DNA, which are covalently

modified through the addition of a methyl group at the 5’ carbon of the cytosine

ring. In mammals this predominantly occurs at the dinucleotide CpG. CpG rich

areas, known as CpG islands, are positioned in the regulatory regions of genes

and are somewhat protected from methylation but despite this, approximately 60-

90% of the dinucleotides across the genome are modified (Zilberman and Henikoff

2007). DNA methylation helps to maintain transcriptional silence in the

nonexpressed and the noncoding regions of mammalian genome; exemplified by

the heavy methylation of pericentromeric heterochromatin: regions of heavily

condensed and transcriptionally inactive DNA (Jones 2012). Methylation of these

areas ensures that this DNA is late-replicating and suppresses the expression of

any potentially harmful viral sequences or transposons that may have integrated

into sites containing such highly repetitive sequences (Baylin 2005)

Figure 2: The mechanism of DNA methylation. Adapted from: (Fukushige and Horii 2013)

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The conversion of cytosine bases to 5-methylcytosine is undertaken by DNA

methyltransferase (DNMT) enzymes. These enzymes are responsible for the

transfer of a methyl group from the universal methyl donor S-adenosyl-L-

methionine (SAM) to the 5-position of a cytosine base (Figure 2). There are four

members of the DNMT family: DNMT1, DNMT3A, DNMT3B and DNMT3L.

DNMT3A and DNMT3B both encode for de novo methyltransferases (Sigalotti et al

2010) whilst DNMT1 encodes the maintenance methyltransferase. Unlike the other

DNMTs, DNMT3L has not been shown to have any inherent enzymatic activity,

however studies have suggested that it enhances the methylation activity of

DNMT3A and DNMT3B (Suetake et al 2004). Numerous studies involving gene

knockout analysis in mice have proven that the DNMT1 and DNMT3A/DNMT3B

genes are all essential for viability (Jin and Robertson 2013).

1.2.1 Disruption of Transcription Factors

The addition of methyl groups does not affect base pairing but can influence

protein-DNA interactions by protruding into the major groove (Lazarovici et al

2013). This can translate into transcriptional inhibition by interfering with the

initiation of transcription. The molecular consequence of CpG methylation is

generally believed to disrupt transcription factor (TF) – DNA interactions either

directly (Nan et al 1998) or through the recruitment of proteins that compete for the

TF binding sites (Boyes and Bird 1991). Direct disruption occurs through the

inhibition of binding of sequence specific factors whose binding sites contain the

CpG dinucleotide. Alternatively, the repressive potential of methylated DNA can be

mediated by a group of proteins known as methyl-CpG binding proteins (MBPs).

MBPs can be categorized into two families: The first family are referred to as

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methyl-CpG binding domain (MBD) protiens and include MeCP2, MBD1, MBD2,

MBD3 and MBD4. All the proteins share an 80 amino acid MBD and prevent

transcription through a transcriptional repression domain (Lan et al 2010). The

second family of MBPs are known as Kaiso-like proteins and include ZBTB4 and

ZBTB38. These proteins lack the MBD but instead recognize DNA sequences

containing methyl-CpG sequences through a zinc finger domain and are able to

repress transcription through a POZ/BTB domain (Filion et al 2006). MBPs are able

to recognize methylated DNA, modify surrounding chromatin and recruit

transcriptional corepressor molecules to silence gene expression. Robertson and

Jones (2000) suggest that the exact mode of transcriptional repression in vivo most

likely results from a combination of both the recruitment of corepressor molecules

and the modification of the surrounding chromatin and states that these processes

may be dependent on the CpG density.

The body of evidence for the loss of transcription factors resulting in the spread of

DNA methylation is growing. Brandeis et al (1994) used transgenic mice to show

that a small number of transcription binding sites at a promoter region, in particular

those for Sp1, are important in protecting the Aprt gene from de novo methylation.

More recently Gebhard et al (2010) examined methylation resistant CpG islands in

the human genome in acute leukemia cell line and normal blood monocytes and

found that transcription factor binding is correlated with resistance to de novo

methylation. Additionally, the concept of a methylation-determining region (MDR)

has been introduced by Lienert et al (2011). This study showed that promoter

sequences of 1kb referred to as MDRs are usually sufficient to recapitulate DNA

methylation patterns in mouse stem cells. It has been considered that the loss

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MDR protective activity caused by decreased expression of TFs and mutations in

TF binding sites may define sites for de novo methylation in cancer.

1.2.2 Hypermethylation

DNA methylation of CpG islands in the promoter regions of genes can result in the

inactivation and silencing of these genes; in tumour cells this process is often

incorrectly regulated. This process, known as hypermethylation, occurs in virtually

every type of cancer whereby the inactivation of tumour suppressor genes through

hypermethylation inhibits cell homeostasis (Esteller 2002). Mechanisms for the

establishment and maintenance of DNA methylation patterns during both

tumourigenesis and normal development remain poorly understood however

Fukushige and Horii (2013) describe one theory: First, there must be an initial

random methylation event that provides an selective advantage to the cell resulting

in clonal selection and proliferation (Jones and Bayling 2007). Following this cis-

acting factors recruit DNMTs to methylation target site. Finally loss of certain

transcription factors results in the spreading of DNA methylation into affected CpG

islands (Turker 2002).

1.2.3 Histone Modifications

Histone modifications also play critical roles in the epigenetic silencing of genes.

The assemblage of histone proteins into nucleosomes enables them to function as

DNA packaging units and transcriptional regulators (Kondo et al 2004). It is the

amino-terminal tails of histones that protrude from the nucleosome that are subject

to chemical modifications such as methylation (Jenuwein and Allis 2001).

Commonly it is the core histones H3, H4 and the linker histone H1 that are subject

15

to posttranslational modifications. (Cohen et al 2011). Chemical modifications of

the histone proteins can disrupt the access of regulatory factors and complexes to

chromatin and therefore may directly affect gene expression. The evidence linking

histone modification to DNA methylation and MBPs is accumulating, DNMTs are

thought to play a role in the direct repression of transcription through cooperation

with histone deacetylases; it is well known that the acetylation of histones acts as

an activating modification. Tumour suppressor gene silencing associated with DNA

methylation in cancer has been shown by Nuguyen et al (2001) to be associated

with loss of histone acetylation. This is further supported in a study by Okino et al

(2007), who found that GTSP1 was not associated with histones with activating

modifications in cancerous human prostate cells.

1.2.4 Factors Influencing DNA Methylation

1.2.4.1 Age

Age has long been considered one of the most important risk factors for the

development of cancer, generally this has been attributed to the cumulative

exposure to carcinogens over time, as well as the time required to receive the

multiple oncogenic insults required for the onset of neoplasia (Ahuja and Issa

2000). Naturally physiological aging is accompanied by functional changes such as

a gradual reduction in immune function and it is noted by Ahuja and Issa (2000)

that these cannot be solely attributed to genetic mutations. Epigenetic changes

such as methylation can take effect over several cell generations, resulting in the

gradual changes in function characteristic of older cells. Li et al (2004) evaluated

the age dependent methylation status of estrogen receptor alpha (ESR1) gene in

prostate cancer and found that the methylation rate of ESR1 increased

16

dramatically with age from 50% in patients aged 60 years and under to 89.7% for

patients aged 70 years and over. A positive correlation was also found between

age and methylation, thus proposing a mechanism linking aging and prostate

cancer.

1.2.4.2 Diet

Dietary factors are also believed to contribute to differences in cancer incidence

among populations with DNA methylation mediating some of the lifestyle factors on

disease risk. Rates of clinical prostate cancer have been shown to be 15-fold higher

in men from the United States than in men from Asian countries (Li 2007). It is

thought that the dramatically increased intake of soy isoflavone in Asian diets could

be a determining factor in this instance. A study by Day et al (2002) investigated the

effect of an isoflavone compound on DNA methylation in male mice fed a diet of

genistein. Consumption of genistein was found to correlate with changes in prostate

DNA methylation at CpG islands. There is considerable interest given to dietary

factors that contribute to abberant methylation patterns. Factors include methyl

donors that directly contribute to the methyl pool and are substrates involved in

DNA methylation. This is highlighted within the red square on Figure 2.

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Dietary methionine is transferred to the universal methyl donor SAM. This donor

releases a methyl group to the 5-position of a cytosine base during methylation.

1.3 GSTP1

Glutathione-S-transferases (GSTs) are a family of enzymes that assist in the

metabolism of harmful chemicals, leading to their detoxification and subsequent

elimination from the body (Okino et al 2007). They are described as a superfamily

Figure 3: How dietary methionine contributes the aberrant methylation patterns. Adapted from

Ho et al (2011)

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of dimeric phase II enzymes and exhibit broad catalytic diversity due to the

existence of eight cytosolic classes. The major role of the GSTs is to catalyse the

conjugation of electrophilic compounds to reduced glutathione and the reduction of

organic hyperoxides in order to prevent cytotoxicity (Dragovic et al 2014) and

protect against carcinogenic agents. In this role, GST action follows phase I drug

metabolism which is often catalyzed by members of the cytochrome P450 (CYP)

supergene family.

The GSTP1 gene is located at 11q13.2 (Figure 3) and contains 7 exons. It encodes

for the pi-class GST enzyme. Whilst the literature on the individual action of the pi-

class GST is limited it is well known that GST enzymes play a key role in the

metabolism of xenobiotics. Initially, CYP enzymes introduce a functional group to a

chemically inactive xenobiotic compound. In doing so they provide an electrophilic

centre that is attack by reduced glutathione in a reaction catalyzed by GSTs.

Following this reaction, the xenobiotic is removed from the cell during phase III of

drug metabolism which requires the action of drug transporters such as multi drug

resistance associated protein (Hayes and McLellan 1999).

Figure 4 Location of the GSTP1 gene. (Genecards 2013)

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Not all xenobiotic compounds require activation by CYP enzymes and may instead

become activated by interaction with free radicals or through cyclooxygenases.

Examples of compounds that undergo conjugation with reduced glutathione include

alkyl and aryl halides, unsaturated carbonyls and isothiocyanates (Sherratt and

Hayes 2001) (Figure 4).

Figure 5 Examples of GST catalysed reactions. The GST substrates shown are as follows:1,

a 2atoxin B1-8,9-epoxide; 2, benzylisothiocyanate; 3, dibromoethane; 4, maleylacetoacetate;

5, a modelo-quinone. (Sherratt and Hayes 2001)

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1.4 Biomarkers

The National Institutes of health define biomarker as a trait that is objectively

measured and evaluated as an indicator of normal biological processes,

pathogenic processes or pharmaceutical response to a therapeutic intervention

(Ilyin et al 2004). Recently medical literature has shown a rapid increase in interest

in biomarkers and this is reflected in an increase in the number of biomarkers that

have been discovered and studied. Despite this the only biomarker routinely used

in prostate cancer diagnosis is prostate-specific antigen (PSA).

1.4.1 Prostate Specific Antigen

PSA is a kallikrein-like serine protease produced almost exclusively by the

epithelial cells of the prostate. Circulating levels correlate with the disruption of the

prostate basal membrane epithelial cells and this may be a result of benign

prostate hyperplasia, prostatitis, trauma to the prostate or adenocarcinoma and for

this reason it is described as organ specific but not cancer specific (Strope and

Andriole 2010). PSA is synthesized by all prostate epithelial cells and this weakens

the specificity of PSA as a cancer biomarker. Furthermore, additional variation in

PSA levels can be introduced by different analytical methodologies and therefore

PSA levels must be interpreted carefully dependent on individual clinical scenarios.

Otero et al (2014) suggest that despite its clinical value, PSA is not the ideal

biomarker for prostate cancer detection and management.

1.4.2 GSTP1 as a biomarker

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More recently it has been suggested that GSTP1 CpG island hypermethylation

could be used as a molecular biomarker for prostate cancer. Several strategies for

the detection of CpG hypermethylation have been developed and include Southern

blot analysis, polymerase chain reaction amplification of DNA treated with 5-

methyl-cytosine sensitive restriction endonucleases and bisulfite genomic

sequencing. Nakayama et al (2004) state that in order to be useful for prostate

cancer screening an early detection assays for GSTP1 CpG island

hypermethylation must target readily available clinical specimens such as

peripheral blood, urine, ejaculate or expressed prostatic secretions. Additionally, it

is a requirement that as a biomarker it must be highly sensitive (referring to the

proportion of correctly identified positive results) and specific (referring to the

number of correctly identified negative results) to prostate cancer when testing

clinical specimens. It has already been already been established that only prostate

cancers or prostate cancer precursor lesions contain hypermethylated DNA which

is indicative of high specificity for prostate carcinogenesis (Nakayama et al 2003).

The ability of assay techniques to detect prostate cancer or precursor lesions is

likely to be determined by whether or not DNA sequences containing GSTP1

hypermethylation are present in the available clinical specimen.

The basis for most current prostate cancer screening and early detection is

peripheral blood specimens as they are easily obtained. DNA with evidence of

hypermethylation changes may appear in peripheral blood as a result of three

processes. Firstly, it may be a result of circulating cancer cells that contribute to

prostate cancer metastases. Secondly, it may arise from intravascular death of

prostate cancer cells resulting in the release of DNA or chromatin fragments from

22

the cells. Finally, it may be detected in circulating phagocytic cells that have

previously ingested prostate cancer cells (Maxwell et al 2009)

Urine, ejaculate or expressed prostate fluids are generally obtainable from men at

risk of prostate cancer development. In order to detect DNA with hypermethylation

changes, shedding of prostate cancer cells or cell fragments into prostatic ducts is

required. The two precursor lesions PIA and PIN are entirely encompassed within

prostatic ducts and can be expected to shed cells, which may then be detected.

Despite the general tendency of prostate cancers to invade out of the prostatic

ducts, a study by Gonzalgo et al (2003) showed that hypermethylated sequences

could be detected in secretions from 86% of prostatectomy specimens from men

with prostate cancer. They note that the presence of hypermethylated DNA in the

specimens may have come from the shedding of either prostate cancer or PIN

cells into prostate ducts.

GSTP1 CpG island hypermethylation may also be used as a biomarker to aid in

prostate cancer diagnosis. Where hypermethylation changes are only present in

prostate cancers, PIN lesions and PIA lesions, they may provide strong support to

classic diagnostic techniques such as needle biopsy specimens. Needle biopsy

can be a challenging technique as there are many conditions that mimic the

histological appearance of prostate cancer (Nakayama et al 2004). Additionally, as

many as 30% of men are diagnosed with prostate cancer by a repeat biopsy

procedure, after the initial biopsy failed to detect the presence of cancer (Chon et

al 2002). It is thought that combining hypermethylation assays and biopsy

techniques may result in better diagnosis of prostate cancer.

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1.5 Aim

The first aim of this paper is to seek out novel implications of hypermethylation of

the GSTP1 promoter region in prostate carcinogenesis and to compare GSTP1

promoter hypermethylation to PSA testing in order to evaluate their roles as clinical

biomarkers

1.6 Objectives

Objectives:

- Identifying how hypermethylation of GSTP1 removes the cells protection

from carcinogens

- Identifying any interactions between GSTP1 and other cellular pathways,

that may promote carcinogenesis.

- Highlight the issues surrounding PSA testing

- Determine the effectiveness of GSTP1 hypermethylation as a biomarker

24

Chapter 2: Methodology

Table 1 Table of search terms

Search Term Search Engine Search Engine Date Searched

Prostate Cancer 1,420,000 Google Scholar 24/10/13Incidence Rates of Prostate Cancer

646,000 Google Scholar 24/10/13

Prostate Structure 1,530,00036,800,000

Google ScholarGoogle

24/10/1324/10/13

Androgen Receptors

344,000 Google Scholar 28/10/13

Development of prostate tumours

410,000 Google Scholar 29/10/13

Prostate cancer precursor lesions

61.700 Google Scholar 29/10/13

Classification of prostate cancer

772,000 Google Scholar 4/11/13

Advanced age in prostate cancer

1,050,000 Google Scholar 5/11/13

Cell senescence 332,000 Google Scholar 5/11/13Chronic inflammation of the prostate

203,000 Google Scholar 9/11/13

Oxidative stress in prostate cancer

104,000 Google Scholar 9/11/13

DNA methylation 962,000 Google Scholar 11/11/13CpG methylation 113,000 Google Scholar 11/11/13DNMT enzymes 18,900 Google Scholar 13/11/13Methylation and transcription factors

412,000 Google Scholar 14/11/13

Methyl binding proteins

1,840,000 Google Scholar 16/11/13

Hypermethylation 75,900 Google Scholar 17/11/13Histones in gene silencing

57,300 Google Scholar 19/11/13

Age as a risk factor in methylation

110,000 Google Scholar 19/11/13

Influences of dietary factors on methylation

36,500 Google Scholar 20/11/13

Glutathione S- 42,600 Google Scholar 23/11/13

25

transferasesGSTP1 23,200

232,000Google ScholarGoogle

23/11/1323/11/13

Biomarkers 1,140,000 Google Scholar 27/11/13PSA testing 115,000 Google Scholar 29/11/13GSTP1 as a biomarker

12,900 Google Scholar 30/11/13

GSTP1 hypermethylation in sample fluids

2,630 Google Scholar 3/12/13

Needle biopsy in prostate cancer

73,600 Google Scholar 4/12/13

Benzo[a]pyrene 221,000 Google Scholar 4/1/14Benzo[a]pyrene conjugation

25,700201

Google ScholarPubmed

9/1/149/1/14

Benzo[a]pyrene and GSTP1

2,330 Google Scholar 12/1/14

Heterocyclic amines in prostate cancer

26,500 Google Scholar 15/1/14

PhIP in prostate cancer

3,020 Google Scholar 19/1/14

PhIP and GSTP1 hypermethylation

662 Google Scholar 19/1/14

PhIP interaction with estrogen receptor a

18,300 Google Scholar 24/1/14

PhIP interaction with DNMTs

950 Google Scholar 1/2/14

Transforming growth factor B and DNMT expression

13,600 Google Scholar 5/2/14

STAT3 139,000 Google Scholar 7/2/14STAT3 and GSTP1 820 Google Scholar 9/2/14Retinoblastoma protein

110,00 Google Scholar 11/2/14

pRb as a transcriptional repressor of E2F

8,010 Google Scholar 14/2/14

E2F influence over DNMT expression

2,280 Google Scholar 14/2/14

Normal limits of PSA testing

37,300 Google Scholar 17/2/14

PSA testing and mortality rates

36,900 Google Scholar 19/2/14

Benefits of PSA 42,100 Google Scholar 21/2/14

26

testingPSA false positives 21,900 Google Scholar 21/2/14PSA testing and unnecessary needle biopsies

4,380 Google Scholar 23/2/14

PSA testing and overtreatment

5,370 Google Scholar 26/2/14

Specificity of PSA testing

39,200 Google Scholar 28/2/14

GSTP1 hypermethylation detection

8,030 Google Scholar 3/3/14

Specificity of GSTP1 hypermethylation

4,330 Google scholar 5/3/14

DNA methylation panels for prostate cancer

23,100 Google Scholar 7/3/14

Discriminatory power of GSTP1 hypermethylation

1,020 Google Scholar 9/3/14

GSTP1 as a prostate cancer prognostic biomarker

4,050 Google Scholar 15/3/14

GSTP1 a prostate cancer treatment efficacy biomarker

2,470 Google Scholar 21/3/14

The research for this paper was undertaken by searching terms in Google scholar

for relevant papers. In some instances some search terms were also placed into

Pubmed or Google to find additional papers and diagrams for explanation. Google

scholar allows you to filter papers by date published, a useful tool for finding the

most current research. It was found in this instance that Google scholar provided

access to a greater number of published papers than Pubmed, which is why it was

used throughout the duration of the project.

27

The research generally consisted of finding key papers surrounding a topic and

then entering key terms and themes found in these fundamental papers into the

search engine. Searching for papers in this way meant that the links between

topics were better understood and were subsequently supported by data from

further studies. In all cases the best effort was made to find the most recent studies

on a topic but wherever possible the original paper on a topic was cited.

28

Chapter 3: Results

3.0 Carcinogenic Compounds

It is well established that glutathione S-transferase Pi-1 (GSTP1) is responsible for

the conjugation of a variety of genotoxic compounds with glutathione rendering

them inactive. Therefore hypermethylation of the GSTP1 promoter region resulting

in reduced or non-expression of the pi class glutathione-s-transferase protein will

leave the cell exposed to attack by carcinogenic compounds.

3.0.1 Benzo[a]pyrene

An example of one such compound is Benzo[a]pyrene (B[a]P). B[a]P is a polycyclic

aromatic hydrocarbon (PAH) which is ubiquitously found in urban atmospheres as

a result of burning carbon based fuels (Larson and Baker 2003). B[a]P is

activated by phase I cytochrome P450 isozymes resulting in a subset of reactive

metabolites that have been implicated in prostate carcinogenesis (Grover and

Martin 2002). This is necessary for phase II detoxification where the reactive

metabolites are conjugated with reduced glutathione rendering them more water

soluble and less reactive, reducing their toxicity. The genotoxicity of PAHs is

thought to be associated with the formation of reactive oxygen species (ROS) and

the redox cycling of metabolites such as B[a]P quinones which result in the

formation of DNA adducts – a piece of DNA covalently bonded to a carcinogen

(Tarantini et al 2011) If these DNA adducts are not repaired, they can lead to base

substitution mutations, small insertions or deletions or more complex gene

29

rearrangements (De Marzo et al 2007). Studies on rats have detected increased

production of DNA adducts, as well as the oxidative stress indicator 8-Oxo-2-

deoxyguanosine (8-oxo-dG) following the formation of ROS (Briede et al 2004) and

it is widely known that 8-oxo-dG is a detrimental oxidative lesion because of its

mutagenic effect (Marnett 2000). Additionally, Park et al (2006) were able to show

that reactive PAH o-quinones have the potential to cause DNA adducts and 8-oxo-

dG lesions.

A study by Kabler et al (2009) investigated the relative toxicity of activated B[a]P

against controls and the protection afforded to cells by GSTP1 by studying the

number of surviving cells from cultures of 250 cells, across a 4 day period. It was

established that cytochrome p450 isozyme CYP1A1 was able to efficiently activate

B[a]P to a more reactive metabolite evident by the 27 fold enhancement of

cytotoxicity in cells expressing CYP1A1 when compared to controls with non-

expression of the gene. The protection afforded to cells by GSTP1 was highlighted

by the deliberate depletion of the substrate glutathione to 20% of control levels by

a glutathione biosynthesis inhibitor. This resulted in a reduction in cytotoxic

protection from 16 fold to 5 fold. The reduction of the substrate glutathione is

representative of a reduction in enzyme activity and as such similar conclusions

can be drawn about the reduction, or total prevention of enzyme activity caused by

hypermethylation of the promoter region of the GSTP1 gene. Therefore evidence

indicates that hypermethylation results in reduced protection against the

carcinogenic potential of B[a]P and provides a pathway for prostate

carcinogenesis.

30

3.0.2 Heterocyclic Amines

Epidemiological studies have also indicated a link between prostate cancer

incidence and mortality and the consumption of charred meat. One mechanism

proposed for way in which charred meat can stimulate cancer is the formation of

heterocyclic amines (HCAs) (Sugimura et al 2004). HCAs are formed during the

cooking of meats at high temperatures and these can be metabolized to

biologically active compounds that adduct to DNA. 2-amino-1methyl-

6phenlimidazo[4,-5b]pyridine (PhIP) is the most common HCA found in charred

meats and has been shown to have the highest carcinogenic potential of these

compounds. Bioactivation of PhIP first involves the activation to a DNA binding

species by N-hydroxylation, catalysed by cytochrome P-450s. Subsequently, the

compound undergoes esterification to form a major adduct at the 8’ carbon of

guanine bases (Arlt et al 2011). Several studies exposing rats to PhIP have been

able to show the development of several tumour types including carcinomas of the

prostate in males. Importantly, both the alpha and pi class isoforms of GSTs have

been shown to inhibit adduction of activated PhIP metabolites to DNA (Nelson et al

2001).

Nakai et al (2007) studied the mutation inducing effects of PhIP in rats in several

tissues. They found that PhIP at a dose of 70mg/kg, three times a week for four

weeks was enough to induce mutations. The most frequent mutations included G:C

to A:T transitions, G:C to T:A transversions, G:C to C:G transversions and -1 base

31

pair deletions in the colon, spleen and all lobes of the prostate. They failed to find

PhIP induced mutations in the liver and kidney. This was attributed to the cell

turnover rate whereby liver and kidney epithelial cells turnover extremely slowly

compared to the PhIP target tissues.

Despite several studies noting the association of PhIP with inducing mutation and

subsequently initiating cancer, a study by Nagao et al (2001) investigating DNA

adducts, mutation and cancer incidence failed to find a strong correlation between

the frequency of mutations and cancer incidence. Additionally, despite Nakai et als’

(2007) findings that PhIP induced mutations in all lobes of the rats prostate,

carcinoma was only found in the ventral lobe. Furthermore, not all target tissues of

PhIP-induced mutations have been associated with PhIP induced cancer

(Sugimura et al 2004). Therefore, it has been suggested that whilst mutations

resulting from PhIP consumption may be involved in the initiation of prostate

cancer, there must be a mechanism for the proliferation of the tumour in response

to PhIP in order to explain the tissue specific differences.

One proposed mechanism is the estrogenic effect of PhIP. Lauber et al (2004)

showed that PhIP is able to stimulate cell proliferation through estrogen receptor-a.

It has been shown that estrogen receptor-a is expressed in the rat ventral prostate

and that estrogen together with testosterone upregulates the expression of

estrogen receptor-a mRNA. This form of positive feedback could explain the

selective nature of tumour growth on the ventral lobe of the rat prostate.

Interestingly, human prostate cancer is also zonal and is largely confined to the

peripheral zone. Inflammation is a known to be a key risk factor in prostate

32

carcinogenesis and it has been shown that estrogens can induce prostate

inflammation in rats (Coffey 2001). This shows a link between PhIP induced

mutations, and subsequent proliferation of tumour cells resulting from PhIP –

estrogen receptor-a interaction in human prostate carcinogenesis. Furthermore,

abnormal upregulation of estrogen receptor-a in humans, combined with a diet rich

in charred meats could result in inflammation of the prostate and therefore places

the individual at increased risk of developing prostate cancer.

A study by Li et al (2012) has shown a relationship between the consumption of

PhIP with the hypermethylation of the GSTP1 promoter region and the loss of

expression of E-cadherin, a molecule involved in the maintenance of the normal

tissue architecture. There is evidence that dysregulation of E-cadherin is strongly

associated with human prostate cancer progression (Fan et al 2012). The study by

Li et al (2012) showed that in untreated mice high levels of E-cadherin was found

uniformly on the plasma membrane of mice luminal epithelial cells. In mice treated

with PhIP partial loss of expression of E-cadherin was seen in low grade PIN

lesions at week 30 of the study. At week 40 the expression was further reduced in

high grade PIN epithelial cells. Following quantification of the promoter region

methylation status in DNA samples obtained from mice, it was concluded that that

promoter hypermethylation of GSTP1 is associated with loss of expression of E-

cadherin from luminal epithelial cells showing known cancer precursor lesions. The

study also tracked the levels of expression of DNMT1, the maintenance

methyltransferase. Interestingly, whilst DNTM1 was expressed at low levels in

prostate epithelial cells in untreated mice, DNMT1 expression was significantly

increased in cells at weeks 30 and 40 of the study. Understanding how PhIP

33

interacts with the cell to increase expression of DNMT1 is an important focus for

future research as it may be involved in the initiation and maintenance of GSTP1

promoter hypermethylation.

PhIP appears to be a significant risk factor for the silencing of GSTP1 through

promoter hypermethylation. Silencing of GSTP1 exposes the cell to carcinogenic

attack through the inability to metabolise carcinogenic molecules or prevent adduct

formation. However, it is important to understand that whilst the cell is exposed to

carcinogenic insult if the GSTP1 gene is silenced, there are still a variety of cellular

defense mechanisms in place to prevent the replication of cells with irreparable

genomic damage. Understanding links between these defense mechanisms and

GSTP1 promoter methylation is important to the mechanism of prostate

carcinogenesis.

3.1 Interaction of GSTP1 methylation with other cellular systems

Prostate carcinogenesis is a complex, multifactorial event that may require both

genetic insult and abnormalities in cellular mechanisms (Nwosu et al 2001).

Understanding how hypermethylation of the GSTP1 promoter region is linked to

other cellular processes is essential in understanding its role in the genesis of

prostate cancer.

3.1.1 Transforming growth factor-B

Transforming growth factor-Β (TGF-B) is a cytokine that regulates mammalian

development, differentiation and homeostasis and is ubiquitously expressed in all

virtually all cell types and tissues (Lee et al 2012). It acts as a key regulator for

34

DNA methylation through an increase in DNMTs expression, particularly in cancer

(Zhang et al 2011). Benign and malignant cells show differential effect of TGF-B

mediated activities: in benign cells TGF-B inhibits DNMT expression whilst in

cancerous cells TGF-B stimulates DNMT expression. Recently, research into the

links between TGF-B and DNMT expression has implicated the ERK pathway as a

major mediator of TGF-B induced expression of DNMTs in prostate cancer (Zhang

et al 2011). The study showed that there was a positive correlation between

treatment of prostate cancer cells lines with TGF-B and the expression of

phosphorylated-ERK (p-ERK) – used to detect ERK activation. On the other hand,

in benign cell lines, p-ERK expression was rapidly inhibited after the addition of

TGF-B. Further study was able to show that the exposure of prostate cancer cell

lines to TGF-B for 24 hours increased the expression of DNMT1, DNMT3A and

DNMT3B by around 15%. Interestingly, treatment with an antibody responsible for

the ERK inhibitor led to a downregulation of the DNMTs mRNA expression of

between 41.5-57.6%. This evidence shows link between TGF-B and the

upregulation of DNMTs that may contribute to promoter hypermethylation of

GSTP1 in prostate cancer cells. Further work to explain the differential expression

of p-ERK in benign and cancerous cell lines is needed and may prove to be

significant in explaining the role of hypermethylation of the GSTP1 promoter region

in prostate cancer.

3.1.2 STAT3

STAT3 is a member of the signal transduction and activator of transcription family

that transduces signals from cytokines and growth factor receptors on the cell

surface and regulates gene expression responses in the nucleus (Kou et al 2013).

35

Specifically, STAT3 regulates the expression of genes controlling cell proliferation,

survival and immune responses. Persistent activation of STAT3 signaling is

oncogenic, contributing to processes such as cell proliferation, preventing

apoptosis and suppressing anti-tumour immune responses (Schroeder et al 2014).

A study on the effects of differing levels of GSTP1 on STAT3 activat ion in

hepatocellular carcinoma has shown that GSTP1 is important in the regulation of

the transcriptional activity of STAT3. Kou et al (2013) were able to show that

overexpression of GSTP1 inhibited epidermal growth factor’s (EGF) ability to

phosphorylate STAT3 – effectively preventing its activation. STAT3’s role in cell

cycle proliferation and cell cycle progression is naturally of interest in respect to

carcinogenesis and this study was able to show that liver carcinoma cells

transfected with 2μg of GSTP1 for 36 hours exhibited reduced proliferation when

compared to control cells. Additionally the study was able to show that

overexpression of GSTP1 was able to induce cell cycle arrest, most likely through

the inhibition of STAT3 signaling. The results of this study are of interest to

prostate carcinogenesis, particularly because whilst GSTP1 is over expressed in a

variety of human cancers such as lung, colon and bladder cancer reduced

expression of GSTP1 is characteristic of hepatocellular carcinoma and prostate

cancer. Barton et al (2004) show that STAT3 is present in cancerous areas of

prostate but not within the normal margins whilst Niu et al (2001) were able to

show that malignant cells expressing persistently activated STAT3 become

dependent on it for survival and as such disruption of activation or expression of

STAT3 resulted in apoptosis. Combining the findings of these studies shows that

GSTP1 promoter hypermethylation may result in persistent, unchecked activation

36

of STAT3 leading to the inability of the cell to induce apoptosis. If this is true then

promoter hypermethylation of GSTP1 may result in the inability of the cell to defend

itself against carcinogenic agents as well as the inability to induce apoptosis

following genomic damage; leading to prostate carcinogenesis.

3.1.3 The Retinoblastoma protein

The retinoblastoma protein (pRb) participates in a well-characterized cell cycle

regulatory pathway that functions to restrict cell cycle progression late in G1 in

response to growth inhibitory signals (Burke et al 2012). Inactivation of the pathway

can lead to the loss of homeostatic balance and therefore promotes abnormal

proliferation of cells, characteristic of tumours. It is well known that pRb acts as a

transcriptional repressor of the transcription factor E2F. A study by McCabe et al

(2005) sought to establish a relationship between the increased DNMT1 levels in

prostate epithelial cell lines lacking the pRb functionality. The study established a

conserved sequence in the promoter region of the DNMT1 gene in mice and

humans that showed considerable similarity to known E2F binding sites.

Subsequent introduction of one of the activating E2Fs, E2F1 resulted in a dose

dependent increase in levels of promoter activity showing that DNMT1 is

transcriptionally controlled by pRb and E2F. Kimura et al (2003) have shown that

DNMT1 is a growth related transcript and without proper cell cycle regulation of

DNMT1 by pRb, DNMT1 will be excessively transcribed and will contribute to

abnormal methylation patterns. This directly links to the promoter hypermethylation

of GSTP1 observed in 90% of prostatic tumours (Cairns et al 2001), and is

particularly relevant because the study was undertaken on prostatic epithelial cell

lines – the most transformed cell in prostate cancer (Rhim et al 2011).

37

3.2 Clinical Biomarkers

3.2.1 PSA testing

PSA testing is fiercely debated in regards to whether the harms inherent in PSA

screening for prostate cancer may outweigh the benefits

3.2.1.1 PSA testing normal limits

Currently, a PSA level of 4.0ng/ml is regarded as the upper limit of normal in

sample fluids and men exhibiting higher levels of PSA are recommended to

undergo a needle biopsy to produce a diagnosis (Heidenreich et al 2011). Studies

have challenged the use of “normal” PSA levels, arguing that a substantial

proportion of men within the determined normal range have been diagnosed with

cancer following a needle biopsy procedure. Thompson et al (2004) found that in a

study of 2950 men, 15.2% of participants with a normal PSA level (≤4.0ng/ml) were

diagnosed with prostate cancer following a needle biopsy procedure at the end of

the study. Worryingly, this percentage rose to 26.9% when examining men with a

PSA level between 3.1-4.0ng/ml. This provides evidence for the reduction of the

“normal“ PSA level upper limit and casts doubt over the use of PSA screening in

prostate cancer diagnosis. This conclusion is further supported by evidence from a

study by Holmstrom et al (2009), who found that to effectively rule out a diagnosis

of prostate cancer, during a follow up biopsy, PSA concentrations would be

required to be ≤1.0ng/ml.

3.2.1.2 PSA testing and prostate cancer mortality

38

PSA testing has been shown to increase the number of men diagnosed with

prostate cancer across screened and control groups. A meta-analysis of five

studies by Ilic et al (2011) found that screening is associated with a 35% increase

in the number of men diagnosed with prostate cancer. Whilst an increase in

diagnosis would appear to support the use of PSA screening in reducing the

mortality of prostate cancer, it was found that there was no significant decrease in

prostate cancer-specific mortality. Naturally, the role of a biomarker in diagnosis is

to accurately confirm the presence of disease for subsequent treatment and as

such would be reflected in a reduction in mortality rate (Madu and Lu 2010). This

provides further evidence against the use of PSA testing as a widespread

screening test for cancer.

A small exception to these findings was observed in the found in the European

Randomized Study of Screening for Prostate Cancer (ERSPC). In the subgroup of

men aged 55-69 years it was identified that 1410 men must be invited for

screening and 48 men must be subsequently diagnosed with cancer and receive

early intervention to prevent one additional prostate cancer death at 10 years (Ilic

et al 2011). However, this small reduction in mortality rate may be offset by the

harms associated with screening.

3.2.1.3 Risks versus benefits of PSA testing

Benefits from prostate cancer screening can take up to 10 years to accrue

(Johansson et al 2009) and the association between prostate cancer and

advanced age means PSA testing may not be advantageous for men with an

anticipated life expectancy of between 10-15 years, particularly because of the

39

known harms associated with screening: Immediate harms generally refer to

problems associated with prostatic biopsies and include modest harms such as

pain, fever and urinary tract infections (Croswell et al 2011). More serious

problems include the risk of sepsis of which there is a reported rate of 0.4% post-

procedure (Rietbergen et al 1997). Additionally, there are harms associated with

treatment of prostate cancer and these need to be factored into the risk and reward

concept of PSA screening. Esserman et al (2009) suggest that PSA screening

increases, by two-fold, the number of cancers detected that would not cause a

problem within a man’s lifetime if left untreated. This must be considered when

reviewing the complications and side effects of prostate cancer treatment.

Treatment for prostate cancer can cause erectile dysfunction, urinary incontinence,

bowel dysfunction and death (Wilt et al 2008). Sanda et al (2008) reports that one-

year after treatment with brachytherapy, external-beam radiotherapy or radical

prostatectomy, 54% to 75% of men couldn’t maintain erections and 6%-16%

suffered from urinary incontinence. The Prostate Cancer Outcomes Study finds

that these problems are frequently long term with 64%-79% reporting erectile

problems at five years post-treatment (Potosky et al 2004). In the context of the

ERSPC study, 48 men are at risk of such problems in order to prevent one death

from prostate cancer. Croswell (2011) argues that overdiagnosis of prostate cancer

from PSA screening is of great concern, with many men exhibiting histologically

evident but clinically silent cancer. Evidence suggests that the minimal reduction in

mortality obtained from widespread PSA screening does not outweigh the

reduction in quality of life in patients unnecessarily subject to biopsies and

overtreatment following screening.

40

3.2.1.4 PSA false positives

Additional issues arise from false positives found during PSA screening. Analyses

of both the ESRPC trial and the Prostate, Lung, Colorectal and Ovarian (PLCO)

cancer screening trial, by Kilpelainen et al (2010), and Croswell et al (2009) found

that between 10.4% and 12.5% of participants received at least one false-positive

test result after three to four screening rounds. Fowler et al (2006) found that false

positives can lead to increased stress related to perceived risk of the disease and

related sexual dysfunction when compared to negative screening results.

Furthermore, these problems could persist for up to a year following screening, and

the issues surrounding subsequent biopsies augmented such problems.

41

Further support for these findings is published in Moyer and US Preventive

Services Task Force (2012) who found that of 1000 men aged 55-69, screened

ever 1-4 years for 10 years with PSA testing, 100-120 receive false positive results

(Figure 5)

3.2.1.5 PSA specificity

Finally it is well known that PSA is not a prostate cancer specific marker. Increases

in PSA concentrations have also been associated with other prostatic diseases

such as benign prostatic hyperplasia, a common disease affecting 75-90% of men

by the age of 80 (Roehrborn et al 1999), and prostatitis – an inflammation of the

prostate sometimes caused by bacterial infection (Schatterman et al 2000). The

problems associated with PSA screening have led to increased research for more

specific and more reliable biomarkers of prostate cancer. Since the turn of the

century growing interest in the field of epigenetics has highlighted that

hypermethylation of the GSTP1 promoter region could become a useful tool in

prostate cancer early detection and diagnosis.

3.2.2 GSTP1 hypermethylation as a biomarker

Several studies have highlighted the significance of GSTP1 hypermethylation as

an epigenetic biomarker for prostate cancer. Firstly reviewed by Henrique and

Jeronimo (2004), it was found that GSTP1 promoter hypermethylation was the

most common epigenetic alteration in prostate cancer – detected in over 90% of

42

prostate cancers. They describe that methylation specific PCR (MPCR) methods

allow for the successful detection of GSTP1 methylation in body fluids including

serum, plasma, urine and ejaculates from prostate cancer patients. These

specimens are readily available and can be obtained from non-invasive procedures

such as needle biopsies and as such GSTP1 hypermethylation shows promise as

a biomarker.

3.2.2.1 GSTP1 hypermethylation specificity

To be truly effective as a biomarker it must show high specificity for prostate

cancer. A meta-analysis of 15 studies by Wu et al (2011) established a specificity

of 89%. This places the GSTP1 hypermethylation at a significant advantage in

comparison to PSA screening. However despite its high specificity, it is not 100%

prostate cancer specific (Chiam et al 2014); occurring in other cancers such as

hepatocellular carcinoma. This is problematic, particularly because whilst testing in

plasma, serum or urine methylated DNA will likely come from cancer cells, in blood

samples methylated DNA can also be released from white blood cells (Roupret et

al 2008) that may have ingested a cancer cell in a different area of the body.

It has been suggested that to increase the specificity for prostate cancer that

GSTP1 should be included in a panel of hypermethylated genes, raising both

specificity and sensitivity to prostate cancer. One study demonstrated that the

combination of 9 DNA methylation profiles increased detection sensitivity from

94.3% to 98.3% and specificity increased from 83.3% to 100% (Jeronimo et al

2004). The strength of using a panel of DNA methylation markers is obvious when

the specificity of PSA screening has been shown to be around 20% (Chiam et al

43

2014). Wu et al (2011) has suggested the use serial testing in which PSA testing is

first used, followed by the use of GSTP1 hypermethylation to confirm any positive

results. Clearly the risks associated with needle biopsy procedures warrant strong

evidence to advocate their use, and it would seem that using a combination of

biomarkers prior to a needle biopsy would be a sensible decision. Better still,

building on Wu et al’ (2011) conclusions, would be to combine PSA testing with a

panel of DNA methylation markers in an attempt to eliminate any lack of specificity

for prostate cancer. The only issue with using PSA as a preliminary test is the

issues surrounding what can be considered the ‘normal’ range; risking missing the

15% of people diagnosed with prostate cancer with PSA levels below 4.0ng/ml.

3.2.2.2 Discriminatory power of GSTP1 hypermethylation

Another key issue of PSA screening is its inability to discriminate between prostatic

diseases such as BPH, precursor lesions such as high grade PIN and

adenocarcinoma. Again this comes the risk of false-positives, unnecessary needle

biopsies and overtreatment. Studies into varying levels of GSTP1 methylation in

PIA lesions, PIN lesions and cancer cells show that it may be possible to

distinguish these prostatic diseases if GSTP1 hypermethylation is used as a

biomarker. Nakayama (2003) found that GSTP1 hypermethylation was not present

in normal prostate epithelium or in hyperplastic epithelium but steadily increased in

percentage with the progression from PIA lesions (6.3%), high grade PIN lesions

(68.8% ) and adenocarcinoma lesions (90.9%). This power of discrimination

confers 2 advantages of GSTP1 hypermethylation over PSA screening. Firstly, it is

widely known that elevated PSA levels have been associated with benign prostate

hyperplasia, as they are elevated following any disturbance to the prostate

44

epithelia. The common occurrence of benign prostate hyperplasia in men over 40

means a significantly increased risk of false-positives from PSA testing. This study

provides evidence that this is not the case with detection of GSTP1 promoter

methylation. Secondly the ability to distinguish between and detect the advance of

cancer precursor lesions is of value to clinicians because whilst they do have the

capability to advance to adenocarcinoma, most remain clinically silent and do not

require treatment. Again this kind of specificity is not available with PSA screening

an as such using GSTP1 hypermethylation as a biomarker may prevent

overtreatment.

3.2.2.3 GSTP1 hypermethylation as a prognostic biomarker

The literature concerning GSTP1 as a potential prognostic biomarker for prostate

cancer is mixed. One study was able to show a 4.4 fold increased risk of PSA

relapse (referring to two successive increases in PSA level to a concentration

greater than 0.3ng/ml following a radical prostatectomy) with the detection of

hypermethylated GSTP1 in preoperative patient serum samples (Bastian et al

2005). However studies by both Woodson et al (2006) and Bastian et al (2007)

found no significant association between GSTP1 hypermethylation and PSA

relapse. The literature is further complicated by a study by Rosenbaum et al (2005)

that found that detection of hypermethylation of GSTP1 in human prostate tissues

was indicative of a decreased risk of PSA relapse. Importantly, PSA relapse is

thought of as a biochemical recurrence of prostate cancer but does not confirm the

presence of cancerous tissue. Freedland et al (2005) state that approximately 40%

of men will experience biochemical recurrence after initial treatment and that

detectable PSA levels may arise from benign, non-cancerous prostatic tissue left

45

after prostate removal that still produce PSA. Critically speaking, these variables

combined with the differences in methodologies are thought to account for

discrepancies in results between studies (Chiam et al 2014). Further research and

meta-analysis of current research into GSTP1 promoter hypermethylation as a

prognostic biomarker is required to ascertain its value in this role.

3.2.2.4 GSTP1 hypermethylation as a treatment efficacy biomarker

There is potential to use GSTP1 hypermethylation as a biomarker to predict the

response to and overall survival rate of patients undergoing chemotherapy.

Hovrath et al (2011) examined levels of hypermethylated GSTP1 in the plasma of

patients with prostate cancer undergoing chemotherapy through methylation

specific PCR. They found that patients with decreased methylated GSTP1 after the

first chemotherapy cycle were likely to present with a greater than 50% reduction in

PSA levels prior to the 4th chemotherapy cycle. The ability to track the progress of

treatment is important in any clinical setting and as such GSTP1 hypermethylation

may be a useful chemotherapy efficacy biomarker for prostate cancer.

46

Chapter 4: Discussion and Conclusion

4.1 Discussion

The role of GSTP1 in the detoxification of carcinogenic compounds is already well

established. However, what is less well understood is how silencing of this gene

exposes the genetic material of the cell to carcinogenic attack and subsequent

proliferation of cancerous cells. GSTP1 provides significant protection to prostate

cells compounds such as B[a]P and PhIP but hypermethylation of the promoter

region results in the silencing of GSTP1 and therefore allows the reactive

metabolites of B[a]P and PhIP to form DNA adducts. It is well established that

adducts are direct precursors to mutations and more complex gene

rearrangements and the results of this Investigation confirm the carcinogenic

potential of both molecules.

Research has indicated that prostate epithelial tissue, with a high cell turnover rate,

is at significantly greater risk of PhIP induced mutations when compared with

tissues with a slower turnover rate. Furthermore, links between PhIP and

subsequent cell proliferation following interaction with estrogen receptor-a have

been found. Therefore this study contributes the idea that GSTP1 hypermethylation

removes the cells protection from reactive HCA metabolites thus increasing the risk

of mutations whilst PhIP is able to induce abnormal cell proliferation. These

processes combined represent a significant pathway to prostate carcinogenesis

47

This paper was able highlight an area for future investigation concerning links

between PhIP, E-cadherin, GSTP1 hypermethylation and DNMT1 expression.

PhIP is able to upregulate DNMT1 and increases in DNMT1 expression can be

directly linked to increased levels of promoter methylation of the GSTP1 gene.

GSTP1 hypermethylation is also correlated with a loss of expression of E-cadherin,

a molecule whose dysregulation has been implicated in prostate cancer. The

finding that GSTP1 hypermethylation can influence E-cadherin expression requires

greater research to establish the nature of the relationship and potential

mechanisms by which GSTP1 is able to regulate the expression of the molecule. A

loss of function in E-cadherin’s may present another way in which GSTP1

hypermethylation is able to contribute to carcinogenesis. Additional study is also

necessary to understand how PhIP is able to influence DNMT1 expression

because it promotes hypermethylation of the GSTP1 gene and because it may

provide an explanation for the hypermethylation of 65 other genes that have also

been associated with prostate cancer.

Transforming growth factor-B (TGF-B) has been implicated as a regulator of DNMT

expression. It is known that DNMT1, DNMT3A and DNMT3B are upregulated in

prostate cancer cell lines as a result of TGF-B. The retinoblastoma protein (pRb) is

a protein that acts as an indirect transcriptional repressor of DNMT1 through

repression of the transcription factor E2F. Cells lacking pRb functionality are known

to excessively transcribe DNMT1. This paper presents that both of these cellular

pathways contribute to hypermethylation of GSTP1 and therefore contribute to the

carcinogenic process. Establishing the mechanisms behind the differential

activation of the ERK pathway in benign and cancerous cells is important in

48

developing our understanding of TGF-B’s role in prostate carcinogenesis.

Establishing how pRb functionality may be lost in prostate cancer may reveal a

potential therapy for restoring the function of pRb in cancerous prostate epithelial

cells

Studies of hepatocellular carcinoma have shown that GSTP1 is important in the

activity of STAT3: GSTP1 has been shown to inhibit over activation of STAT3 in

hepatocellular carcinoma. Persistent activation of STAT3 has been described as

oncogenic and research has shown that malignant cells became reliant on STAT3

expression. Whilst it is understood that these processes are confirmed in

hepatocellular carcinoma no current research has shown similar findings in

prostate cancer cell lines. GSTP1 is downregulated in both hepatocellular

carcinoma and prostate cancer and therefore GSTP1 hypermethylation could result

in persistent activation of STAT3 in prostate cells leading to the inability of the cells

to induce apoptosis. Further research is required to provide evidence for these

findings.

PSA testing, despite its widespread clinical use, has several problems that have

led researchers to question its use as an effective biomarker for prostate cancer.

Several studies have highlighted problems with the use of a “normal” upper limit for

PSA concentration. Meta-analyses have shown that there is no significant

decrease in mortality from prostate cancer and PSA testing has also been found to

have significant implications on the quality of life of patients through problems such

as false positives and unnecessary needle biopsy procedures. Furthermore, the

benefits of PSA testing can take around 10 years to accrue with overtreatment

49

resulting from PSA testing being highlighted as another problem. Significantly PSA

testing is not prostate cancer specific and therefore cannot be relied upon to

produce an accurate diagnosis

This paper hasn’t generated any novel findings about PSA testing but it has

summarized a significant number of problems associated with its use as a

widespread biomarker for prostate cancer. These problems were helpful in

determining potential strengths of using GSTP1 hypermethylation as a biomarker.

Despite this, PSA testing has been shown to be effective in specific cases and

further research into factors that may determine when PSA testing is most effective

for an individual is required.

The literature concerning the use GSTP1 methylation as a biomarker for the

detection of prostate cancer has been growing since the turn of the century. Its

high specificity and the ability to detect it in a range of readily available body fluids

highlighted its potential. Subsequent studies have confirmed it to be highly

sensitive and highly specific to prostate cancer especially when combined with

other DNA methylation profiles. Combining the use of DNA methylation profiles and

PSA testing prior to the decision to undertake a needle biopsy procedure is a

promising avenue that may eliminate some of the problems, highlighted in the

current literature, with using PSA testing to advocate the use of a needle biopsy.

Further research should include studies using the serial testing method described

to determine the effectiveness of such a process in accurately diagnosing prostate

cancer as well as its effectiveness in eliminating the problems of false positives

and unnecessary needle biopsies arising from PSA testing.

50

GSTP1 hypermethylation also has the ability to distinguish between cancer

precursor lesions and adenocarcinoma, and is not affected by other prostatic

diseases such as BPH. GSTP1 hypermethylation has the ability to provide

clinicians with the ability to detect cancer precursor lesions and monitor them to

ensure they remain clinically silent and do not advance to adenocarcinoma. This

would result in a significant advantage over PSA testing by preventing

unnecessary needle biopsy procedures or overtreatment. Further work in this area

should focus on determining a scale that effectively allows clinicians to use

hypermethylation levels as a biomarker for disease progression.

The value of GSTP1 as a prognostic biomarker couldn’t be established. The

literature concerning GSTP1 hypermethylation and PSA relapse is inconclusive

and warrants further investigation. Studies require the alignment of methodologies

and more stringent guidelines to determine what constitutes PSA relapse.

Furthermore studies would benefit from some form of control for PSA levels that

might arise from benign tissue left over following treatment such as radical

prostatectomies.

GSTP1 hypermethylation has been shown to have potential as a chemotherapy

treatment efficacy biomarker but continued study to validate current research is

required. The ability to accurately track how effectively a therapy is working is of

significant value to clinicians and as such presents a promising role for GSTP1

hypermethylation

51

4.2 Conclusion

The aim of this paper was to seek out novel implications of hypermethylation of the

promoter region in prostate carcinogenesis. Firstly, this was undertaken by

identifying carcinogenic compounds that GSTP1 was directly responsible for

protecting the cell from. Two common carcinogenic compounds were evaluated

and research confirmed previous knowledge about direct carcinogenic properties

of both molecules.

The second objective was to identify any interactions that GSTP1 hypermethylation

may have with other cellular systems. Overall it appears that future research

should focus on pathways in the cell that promote the upregulation of DNMTs, as

found when investigating TGF-B and pRb. Promisingly, GSTP1 has been shown to

inhibit the activation of STAT3 in liver carcinoma cell lines and reduce proliferation

of these cells. Reduced expression of GSTP1 is characteristic of both

hepatocellular carcinoma and prostate cancer and therefore if parallels can be

drawn between the two diseases it suggests that the inability of hypermethylated

GSTP1 to regulate STAT3 activation could be a novel avenue for investigation into

in prostate carcinogenesis

Comparing GSTP1 hypermethylation to PSA testing was important to evaluate its

potential as a biomarker. The third objective was to highlight issues that have

surrounded PSA testing. The literature surrounding issues with PSA testing was

vast and no new problems could be found. However a review of the literature

highlighted that the greatest issues surrounding PSA testing was a lack of

52

specificity and the problems associated with false positives and follow up

procedures. Whilst pursuing the fourth objective it was established that a

combination of PSA testing and DNA methylation profiles could eliminate the

issues surrounding PSA testing as a widely used diagnostic marker. It was

highlighted that research in this area was required but serial testing looked

promising as a more effective diagnostic tool for prostate cancer.

Reviewing GSTP1 as a prognostic biomarker proved inconclusive due to largely

differing results obtained in different studies. This was attributed to differing

methodologies and problems with defining PSA relapse. More promisingly,

however, was the potential for GSTP1 hypermethylation to be used as a biomarker

for the efficacy of chemotherapy treatment. As a relatively new concept, there is

little data to support its use in this role but it may prove to be an encouraging

avenue for future research.

With further funding research into the role of GSTP1-STAT3 interaction in prostate

carcinogenesis looks promising especially when drawing parallels with

hepatocellular carcinoma. Additionally, research verifying the effectiveness of a

serial testing method for prostate cancer diagnosis may help to prevent the issues

that have been highlighted with using PSA testing as advocate for needle biopsy

procedures. Furthermore, establishing whether GSTP1 hypermethylation could

provide clinicians with a means to track chemotherapy efficacy is essential as it is

evident that it shows promise as a treatment efficacy biomarker.

53

In summary, the basis for prostate carcinogenesis is complex and requires the

failure or abnormality of various cellular systems. Understanding how GSTP1

hypermethylation contributes to this process can provide directions for future

investigation into the disease and potential areas for therapeutic intervention.

Furthermore, its specificity for prostate cancer has highlighted it as a strong

candidate as a diagnostic biomarker for prostate cancer particularly if used in

conjunction with other established biomarkers. Continued research into its use as a

biomarker may reveal that it can perform equally as well as a diagnostic tool, a

prognostic tool or as a biomarker for treatment efficacy.

54

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Appendices

Evaluative Supplement

The desk-based nature of this study meant that the project had several limitations.

The first and most obvious limitation was that there was no new experimental

evidence to contribute to the literature already surrounding the topic. With this in

mind, the project focused on reviewing current evidence and finding potential links

between the reported research that might highlight a novel pathway for

investigation. Whilst this proved to be difficult, some areas for future research were

found and may prove fruitful following investigation.

Access to certain papers was limited during the study due to restricted access.

This was more of a problem with the most recent papers and presents a strong

limitation of this study. Naturally a review of this kind relies on reporting links

between the most current knowledge of a topic in order to make it scientifically

strong and as such future investigations of this kind will need to review the most

current literature. However, this was only an issue with a small number of papers

and was unlikely to strongly impact on any of the findings of the project.

Another problem highlighted by this study was that ideally a greater amount of

evidence would be reviewed before reaching the conclusions that were discussed

in the paper. Whilst this study used data from reported meta-analyses, in the future

performing my own statistical calculations of the data would be preferable, however

due to a lack of resources and time constraints this was not possible during this

project.

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Future work into determining the processes behind prostate carcinogenesis is key

to developing therapy for the disease. With the advent of epigenetics in the 21st

century, research has focused on the role of epigenetic interactions in the

development of the disease. This work has highlighted a number of genes,

including the GSTP1 gene, actively involved in cellular defense, the cell cycle and

cellular maintenance that are the subject of abnormal epigenetic interactions.

Prostate cancer is a complex disease that more than likely has numerous

pathways of carcinogenesis but understanding these pathways is becoming easier

as research progresses. This project has highlighted the importance of

hypermethylation in cancer development and current research shows promise in

determining the exact role of hypermethylation in carcinogenesis. However, we still

do not understand the factors that lead to the initiation of hypermethylation and it is

likely that these factors may be the target for therapeutic intervention. Additionally,

the implications of hypermethylation of these genes on other cellular pathways are

not well understood but this project has shown that such these implications are

also sources of proliferation of cancerous cells and therefore presents another area

to focus future work concerning prostate carcinogenesis.

This project has given me a strong interest in the field of epigenetics. It is a field

that is growing and developing very quickly and harbors great potential for the

research into the causes of various genetic diseases and therefore the

development of new therapies for these diseases. This project has also been

educational in the sense of that it has taught me how to research and report on a

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topic effectively and in the appropriate manner. This is a transferable skill to any

form of employment.

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Student Research Project Interview – Agreed Comments Form

Student Name: James Pereira Programme: BSc Biological Science

Date: 28/11/13 IRP

Supervisor Name: Dr Kevin McGhee

Two copies of this form are needed – student to retain one copy the other is to be handed in to the student admin office C237.Student Signature: Supervisor Signature:

James was required to do a heavy amount of research at the start of term but has brought together a substantial amount and has written approximately 1,500 words

His introduction is going well however a little behind schedule for this year, but has assured me that this will be made up over the Christmas break

The introduction so far shows good understanding of the topic as well as what is needed to progress

Hopefully by the next time we meet after Christmas, the introduction will be complete and he will be ready to progress to the results section of the project.

I am very much looking forward to seeing the completed article

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