This document is a dissertation submitted for a BSc in Biological Sciences. It examines the implications of promoter hypermethylation of the GSTP1 gene in prostate carcinogenesis. It first provides background on prostate cancer development and factors influencing carcinogenesis like age, senescence, inflammation and oxidative stress. It then discusses DNA methylation and the GSTP1 gene. The dissertation aims to identify implications of GSTP1 hypermethylation in prostate cancer development and compare its effectiveness as a biomarker to PSA testing. It finds evidence that carcinogenic compounds may contribute to carcinogenesis and GSTP1 interacts with other cellular pathways. It also highlights issues with PSA testing and shows GSTP1 hypermethylation has

1. 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

2. 2
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.

3. 3
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.

4. 4
Contents
Chapter 1: Introduction
1.0 Introduction 6
1.1.1 Prostate Structure 6
1.1.2 Androgens and Androgen Receptors 7
1.1.3 Prostate tumour development 8
1.1.4 Factors Affecting Prostate Carcinogenesis 10
1.1.4.1 Age 10
1.1.4.2 Senescence 10
1.1.4.3 Chronic Inflammation 11
1.1.4.4 Oxidative Stress 11
1.2 DNA methylation 11
1.2.1 Disruption of Transcription Factors 13
1.2.2 Hypermethylation 15
1.2.3 Histone Modifications 15
1.2.4 Factors Influencing DNA methylation 16
1.2.4.1 Age 16
1.2.4.2 Diet 17
1.3 GSTP1 18
1.4 Biomarkers 21
1.4.1 Prostate Specific Antigen 21
1.4.2 GSTP1 as a biomarker 21
1.5 Aims 24
1.6 Objectives 24
Chapter 2: Method
2.0 Methodology 25
Chapter 3: Results
3.0 Carcinogenic compounds 29
3.0.1 Benzo[a]pyrene 29
3.0.2 Heterocyclic Amines 31
3.1 Interaction of GSTP1 with other cellular systems 34
3.1.1 Transforming growth factor-B 34
3.1.2 STAT3 35
3.1.3 The Retinoblastoma protein 37
3.2 Clinical Biomarkers 38
3.2.1 PSA testing 38
3.2.1.1 PSA testing normal limits 38
3.2.1.2 PSA testing and prostate cancer mortality 38
3.2.1.3 Risks versus benefits of PSA testing 39
3.2.1.4 PSA false positives 41
3.2.1.5 PSA specificity 42
3.2.2 GSTP1 hypermethylation as a biomarker 42
3.2.2.1 GSTP1 hypermethylation specificity 43
3.2.2.2 Discriminatory power of GSTP1 hypermethylation 44
3.2.2.3 GSTP1 hypermethylation as a prognostic biomarker 45
3.2.2.4 GSTP1 hypermethylation as a treatment efficacy biomarker 46

5. 5
Chapter 4: Discussion and Conclusion
4.1 Discussion 47
4.2 Conclusion 52
References 55
Appendices
Evaluative Supplement 67
Interim Interview Comments 70

6. 6
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

7. 7
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 AndrogenReceptors
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
Figure 1: Zonal structure of the human prostate
(Best Health 2014)

8. 8
(Lee 2003). Additionally, low serum testosterone levels in patients with newly
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

9. 9
events that lead to their development into clinical disease (Bratt and Schumacher
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).

10. 10
1.1.4 Factors Affecting Prostate Carcinogenesis
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).

11. 11
1.1.4.3 Chronic Inflammation
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

12. 12
signaling, used to lock genes in the off position and has important function in a
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)

13. 13
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

14. 14
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

15. 15
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

16. 16
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

17. 17
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.

18. 18
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
of dimeric phase II enzymes and exhibit broad catalytic diversity due to the
Figure 3: How dietary methionine contributes the aberrant methylation patterns. Adapted from
Ho et al (2011)

19. 19
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)

20. 20
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 ̄atoxin B1-8,9-epoxide; 2, benzylisothiocyanate; 3, dibromoethane; 4, maleylacetoacetate;
5, a modelo-quinone. (Sherratt and Hayes 2001)

21. 21
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
More recently it has been suggested that GSTP1 CpG island hypermethylation
could be used as a molecular biomarker for prostate cancer. Several strategies for

22. 22
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
the cells. Finally, it may be detected in circulating phagocytic cells that have
previously ingested prostate cancer cells (Maxwell et al 2009)

23. 23
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.

24. 24
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

25. 25
Chapter 2: Methodology
Table 1 Table of search terms
Search Term Search Engine Search Engine Date Searched
Prostate Cancer 1,420,000 Google Scholar 24/10/13
Incidence Rates of
Prostate Cancer
646,000 Google Scholar 24/10/13
Prostate Structure 1,530,000
36,800,000
Google Scholar
Google
24/10/13
24/10/13
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 5/11/13
Chronic
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 11/11/13
CpG methylation 113,000 Google Scholar 11/11/13
DNMT enzymes 18,900 Google Scholar 13/11/13
Methylation 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 17/11/13
Histones 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-
transferases
42,600 Google Scholar 23/11/13
GSTP1 23,200
232,000
Google Scholar
Google
23/11/13
23/11/13

26. 26
Biomarkers 1,140,000 Google Scholar 27/11/13
PSA testing 115,000 Google Scholar 29/11/13
GSTP1 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 4/1/14
Benzo[a]pyrene
conjugation
25,700
201
Google Scholar
Pubmed
9/1/14
9/1/14
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 7/2/14
STAT3 and GSTP1 820 Google Scholar 9/2/14
Retinoblastoma
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
testing
42,100 Google Scholar 21/2/14
PSA false positives 21,900 Google Scholar 21/2/14
PSA testing and 4,380 Google Scholar 23/2/14

27. 27
unnecessary
needle biopsies
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.
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

28. 28
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.

29. 29
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
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

30. 30
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.

31. 31
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
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

32. 32
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
carcinogenesis and it has been shown that estrogens can induce prostate
inflammation in rats (Coffey 2001). This shows a link between PhIP induced

33. 33
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
interacts with the cell to increase expression of DNMT1 is an important focus for

34. 34
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
DNA methylation through an increase in DNMTs expression, particularly in cancer

35. 35
(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).
Specifically, STAT3 regulates the expression of genes controlling cell proliferation,

36. 36
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
of STAT3 leading to the inability of the cell to induce apoptosis. If this is true then

37. 37
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 Retinoblastomaprotein
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).

38. 38
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
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

39. 39
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
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

40. 40
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.

41. 41
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.
Figure 5 PSA testing from Moyer and US Preventive
Services Task force (2012)

42. 42
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
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

43. 43
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
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

44. 44
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
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

45. 45
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
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

46. 46
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 efficacybiomarker
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.

47. 47
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
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

48. 48
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
developing our understanding of TGF-B’s role in prostate carcinogenesis.
Establishing how pRb functionality may be lost in prostate cancer may reveal a

49. 49
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
resulting from PSA testing being highlighted as another problem. Significantly PSA

50. 50
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.

51. 51
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

52. 52
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
specificity and the problems associated with false positives and follow up

53. 53
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.
In summary, the basis for prostate carcinogenesis is complex and requires the
failure or abnormality of various cellular systems. Understanding how GSTP1

54. 54
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.

55. 55
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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