Human Susceptability to HPC - Dissertation - Completed

SCHOOL OF BIOLOGICAL SCIENCES – RHUL

BS3020: Special Study Dissertation

FULL DISSERTATION TITLE:

Human genetic susceptibility to the development of

Hepatitis B induced hepatocellular carcinoma

STUDENT NAME: Sohail Jaweed Akhtar

STUDENT NUMBER: 100757715

EMAIL ADDRESS: [email protected]

DISSERTATION SUPERVISOR: Dr. Shobana Dissanayeke

TURNITIN RECEIPT NUMBER: _____________________

ACKNOWLEDGEMENTS (optional, keep text within the box):

I would like to express my sincere appreciation towards my supervisor and

tutor, Dr. S. Dissanayeke. I would like to thank her for her guidance, and aid in

developing my dissertation topic. Especially for the motivation she provided.

Thank you.

The deadline for submitting your work to the school office is

12 noon Thursday 5th March 2015 (Week 8, Term 2).

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Human genetic susceptibility to the development of Hepatitis B induced hepatocellular carcinoma

TABLE OF CONTENTS

1.0 SUMMARY............................................................................................3

2.0 INTRODUCTION ....................................................................................4

2.1 BACKGROUND ................................................................................... 4

2.2 INFERENCES IN RELATION TO GENETIC SUSCEPTIBILITY ................................... 5

2.3 RATIONALE OF THE STUDY..................................................................... 5

2.4 OBJECTIVES ...................................................................................... 6

3.0 DISCUSSION......................................................................................... 7

3.1 PATHOBIOLOGY OF HBV ..................................................................... 7

3.1.1 OVERVIEW OF HBV STRUCTURE, GENOME AND GENOTYPES ........................................... 7

3.1.2 CLINICAL DIAGNOSIS OF HBV INFECTION..................................................................... 8

3.1.3 THE HBV LIFE CYCLE ............................................................................................... 9

3.2 DISTINCTIVE MOLECULAR PATHWAYS OF HCC .......................................... 12 3.2.1 HCC MOLECULAR PATHWAYS ................................................................................. 12

3.2.2 DISTINCT ASSOCIATIONS IN HBV-RELATED HCC PATHWAYS ......................................... 12

3.3 HBV ONCOLOGICAL MECHANISMS IN RELATION TO HCC ............................. 16 3.3.1 OUTLINE OF HBV ONCOLOGICAL MECHANISMS .......................................................... 16

3.3.2 HBV DNA AMALGAMATION .................................................................................. 16

3.3.3 HBV VIRAL ONCOLOGICAL PROTEINS ........................................................................ 18

3.3.4 ONCOLOGICAL MECHANISMS ASSOCIATED WITH HBV IMMUNE RESPONSE ...................... 21

3.4 GENE SUSCEPTIBILITY IN RELATION TO HBV AND HCC ................................ 23 3.4.4 INITIAL FINDINGS OF GENE SUSCEPTIBILITY ................................................................. 23

3.4.4 ANALYSIS OF HOST GENETIC SUSCEPTIBILITY TOWARDS HBV-INDUCED HCC .................... 24

4.0 CONCLUSION ..................................................................................... 26

5.0 REFERENCES ...................................................................................... 29

6.0 FIGURES AND TABLES ........................................................................ 35

[I] ATTATCHMENTS .....................................................................................

I.I. TURNITIN RECEIPT .................................................................................

I.II CD: DISSERTATION & FIGURES ................................................. BACK COVER

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1.0 SUMMARY

Past studies have conveyed that individuals may differ significantly in their

susceptibility to infectious diseases. However thorough investigations between

symptoms of disease and human genetic susceptibility are lacking. The literature will

aim to illustrate associations of human genetic susceptibility on the progression of

hepatocellular carcinoma (HCC) with Hepatitis B (HB) infection. It is known that HB

virus (HBV) infected individuals have an increased risk of HCC, although this is

understood to be an indirect symptom of cirrhosis. This paper will ascertain HBV and

host liver mechanisms in relation to HCC, in collaboration with immunological,

epidemiological and genetic evidence to correlate host genetic susceptibilities.

By describing the mechanisms of HBV and HCC in relation to the host, it is clear that

the process of HCC can be both direct and indirect. Identification of genes that have

been found to contain polymorphisms that encourage the development of HBV-

induced HCC, suggests gene predisposition. It is concluded that host susceptibility is

significant enough to induce and develop HCC for those that are infected by HBV.

The recognition of HBV-HCC genetic susceptibility, may lead to better understanding

of the roles genetics have on viral cancer and pathogen-causing symptoms, possibly

leading to the development of pharmaceutical therapies.

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2.0 INTRODUCTION

2.1 BACKGROUND

Individual hosts within the same population have been well-known to differ

significantly in susceptibility of identical infectious diseases. Reports have stated

associations of single-nucleotide polymorphisms (SNPs) and rare mutations which

enable susceptibility to many diseases in humans; infectious or otherwise (Lin, Chen

et al. 1989).

The HBV is a member of the Hepadnavirus family and is classified into ten genotypes,

A-J (Hernandez, Venegas et al. 2014). As a mostly double-stranded DNA virus it is not

classified as a retrovirus, yet encompasses similar mechanics by using reverse

transcription of a pre-genomic RNA (pgRNA) intermediate (Summers, Mason 1982),

therefore defined as a pararetrovirus. Related viruses are found in several animals

including woodchucks, tree squirrels and Peking ducks (Breiner, Schaller et al. 2001).

Primarily infecting hepatocytes causing both acute and chronic liver disease, the

association of HBV chronic infection (CHB) with the development of HCC was first

designated by Blumberg, Gerstley et al. (1967). HB is transmitted via body fluids

either vertically through childbirth or horizontally by person-person contact (WHO

2014). Indirect infection can occur if bodily fluids are present on objects within

several days of environmental exposure entering intravenously, such as IV drug

needles (WHO 2014). HBV is present at high concentrations in the blood stream of

an infected individual, between 2000-20,000 IU/mL (Barcena 2009). Due to these

aspects; HBV is 100 times more infectious than HIV (WHO 2014).

Infection of chronic HBV is recognised to significantly increase the risk of

Hepatocellular carcinomas (HCC); attributed to cirrhosis of the liver (Parkin 2006).

World-wide, HCC is known to be the third leading cause of cancer death, being the

fifth most common form of cancer (Parkin 2006, Parkin 2001).

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Currently, research acknowledges that HBV infections account for the majority of

cirrhosis and primary liver cancer cases. 54-78% of HCC is determined by HBV

infection internationally; the remaining proportion is supplemented by HCV (25%)

and numerous risk factors including family history (Parkin 2006).

2.2 INFERENCES IN RELATION TO GENETIC SUSCEPTIBILITY

Analysis of molecular mechanisms and epidemiological studies, in relation to liver

carcinomas caused without cirrhosis (Yuen, Tanaka et al. 2009) may infer associations

of genetic susceptibility (Chen, Chen 2003). A significant amount of HCC is caused

without anatomical damage, although most cases are caused due to cirrhosis

(Fattovich, Stroffolini et al. 2004). Literature suggests that these instances of HBV

related liver carcinogenesis is caused by activating cellular cancer-related genes

(Honda, Yamashita et al. 2006, Chen, Chen 2003). In consequence, it is possible that

host and viral genetic factors are involved in risk of HCC. Leading to the following

hypothesis and aim: to provide evidential associations to indicate that the host is

genetically susceptible to progression of HCC induced by HBV related mechanisms.

2.3 RATIONALE OF THE STUDY

From a clinical perspective, the rationales of this study are highlighting the

importance of genetic influences on diseases. To provide information in relation to

possible polymorphisms, which may support advancements in HCC identification

programs in patients with HBV infection. To identify critical mechanisms in HB, HBV,

or cancer processes to aid development of specific therapeutic interventions, be it

immunological or otherwise.

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2.4 OBJECTIVES

By ascertaining and demonstrating probable HBV and HCC molecular pathways to

lay a foundation on how oncological mechanisms of HBV induce hepatocellular

carcinogenesis. Associations of host genetic interactions can be made, which are

conceivably involved with the viral to host molecular interactions and pathways.

Considering there is little research completed on detailed analysis of host gene

susceptibility for HBV induced HCC. Any genome-based analysis study requires

support for a successful conclusion. This can be achieved by critically analysing

literature based on supported molecular mechanisms in relation to viral oncology. To

then relate the mechanisms with host genetics, and therefore identify any genetic

susceptible polymorphisms that may directly or indirectly encourage or favour HBV-

induced HCC. This strategy will explain resolutions for the extensively unknown

predictive mechanisms of HBV and HBV-induced HCC, to either disapprove or accept

the hypothesis.

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3.0 DISCUSSION

3.1 PATHOBIOLOGY OF HBV

3.1.1 Overview of HBV structure, genome and genotypes

Belonging to Hepadnaviruses, HBV contains a partially relaxed double-stranded

circular DNA (rcDNA) covalently linked to DNA polymerase (Pol) at the 5′ end of the

minus strand, is lipid enveloped, and contains unknown preferential hepatocellular

properties. The lipid envelope consists of a hepatitis B surface antigen (HBsAg)

encircling an inner nucleocapsid composed of hepatitis B core antigen (HBcAg). This

double-shelled structure is 42nm in diameter and functions as the infectious HBV

virion and contains the 3.2kb rcDNA genome (Sayers, Barrett et al. 2009)

The genome encodes four overlapping open reading frames: (ORFs) S, C, P, and X

(Fig1) (Sayers, Barrett et al. 2009). The pre-S/S ORF encodes the three surface

envelope proteins; large, middle, and small HBsAg. The pre-C & C ORF encodes the

‘e’ antigen (HBeAg) and the core antigen (HBcAg) respectively. Both the S and C

ORFs are categorised by the initiation in-frame codons, which enable the translation

of various proteins from the RNA sequence. The P ORF expresses the terminal

protein (TP) and the Hepadnaviral Pol. The relatively small overlapping ‘X’ gene

encodes a 16.5kd protein, named as the X protein (HBx) or X antigen (HBxAg)

interchangeably, with multiple functions essential for viral replication and inducing

HCC. Although, certain X gene linked mechanisms and biological functions are

presently unknown. (Grimm, Thimme et al. 2011).

As identified previously, HBV is classified into ten genotypes, A through H, with two

new additions, I & J, in 2014 (Hernandez, Venegas et al. 2014). The genotypes differ

by at least 8% from one and other, individually containing several subtypes and have

distinct geographical distributions (Hernandez, Venegas et al. 2014). Comprehensive

analysis in relation to distinct oncogenecity of HBV genotypes has been made.

Particular studies for example, have determined that HBV genotype C is found to be

an independent risk factor for HCC development (Chan, Hui et al. 2004). Concerns

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must be made when discussing carcinogenesis by HBV as whole rather than

individual genotypes. However, considering that there is only a small difference (at

least 8%) between classes, it is more essential to understand the products

synthesised by the shared genome rather than detailing the differences unless

significant.

3.1.2 Clinical Diagnosis of HBV infection

The initial phase of CHBV infection is characterised by HBeAg-positivity, high viral

load and alanine transaminase (ALT) (Saikia, Talukdar et al. 2015). HBeAg appears

upon HBV infection in the bloodstream, as it is secreted from the cells and

accumulates in serum, serving as a marker of HBV replication. This indicates that the

host is both a carrier and infectious. The magnitude of viral load, detected by HBV

DNA levels, is proportionate to how active HB infection is. The clinical significance of

high levels of ALT is that it is aetiologically associated to hepatocellular injury (Saikia,

Talukdar et al. 2015). Together, the results deduce that a patient has CHB. Whilst

these levels specifically show that the subject is in the immune active phase, one of

five classified phases of CHB infection. It is when HB infection becomes chronic that

associated HCC development occurs (Guidotti, Rochford et al. 1999).

Although most hosts develop anti-HBe antibodies soon after infection in addition to

levels of HBeAg become undetectable indicating immunity - the observation being

immunologically labelled as seroconversion. One third goes on to rapidly develop

HBeAg‐negative CHB, characterised by fluctuating levels of ALT, HBV DNA, and

diagnosis of active hepatitis (Saikia, Talukdar et al. 2015). This tier of CHB infection

can swiftly progress to cirrhosis and HCC. HBeAg as a potential predictive marker can

also identify the risk of HBV-related HCC, as a higher prevalence of HBeAg relates to

an increased risk of HCC diagnosis. This is most probable, as HBeAg reflects active

HBV DNA replication. Patients that are positive for both HBeAg and HBsAg have a

stated six times relative risk of HCC, compared with those positive for HBsAg alone.

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3.1.3 The HBV life cycle

HBV extracellular route, Attachment and Specificity

From the infection site, HBV is transported through the blood stream to converge at

the liver. Whilst the host is a carrier and before entering the hepatocytes, mutations

accumulate in the viral genome; whether this process increases the risk of HCC

development is unknown. It is found to be an important variable in antiviral

treatment and development of chronic liver disease (Sumi, Yokosuka et al. 2003). The

virus accesses the hepatocytes for replication (Fig2) by traversing the sinusoidal

epithelium by fenestrations, consequently entering the space of Disse; the space

between a sinusoid and a hepatocyte.

HBVs capability to attach, fuse, and penetrate hepatocytes are poorly understood; as

a cell-surface receptor is yet to be wholly recognised, therefore being hypothetical.

This is due to limitations of HBV investigations in in vitro systems (Breiner, Schaller et

al. 2001). Enveloped viruses typically enter host cells by interacting with a specific

receptor on the cell membrane by a viral surface protein. Viral entry occurs by either

membrane fusion or receptor-mediated endocytosis.

The absence of a confirmed receptor has led to an unconventional hypothesis

(Breiner, Schaller et al. 2001) stating that roaming liver sinusoidal endothelial cells

(LSECs) mediate the uptake of HBV into the liver, rather than hepatocytes. The novel

hypothesis was presented to overcome observations of LSEC inducing both Duck

HBV-Host barrier mechanisms as well as a pronounced scavenging process in test

animals (Breiner, Schaller et al. 2001). Although recent studies overshadow this by

suggesting hepatocyte multistep routes by initial attachment. Using infection

competitive experiments and inhibiting mechanisms used in duck HBV experiments,

Schulze et al. (2007) established that HBV initial attachment occurs on hepatocyte-

associated heparan sulphate proteoglycans receptors. As these receptors are found

on numerous variable cells, and that HBV are highly specific to hepatocytes. The

literature hypothesises that HBV follows additional hepatocellular-associated steps,

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suggesting a multistep process (Schulze, Gripon et al. 2007). This multi-step

hypothesis is recognised by S. Urban (2008). Describing HBV specificity towards

hepatocytes; S. Urban (cited by Grimm, Thimme et al. 2011) concludes that HBV

binds to an unknown hepatocyte-specific preS1-receptor which requires activation of

the virus. This results in exposure of the myristoylated N-terminus of the envelope L-

protein, inducing specificity (cited by Grimm, Thimme et al. 2011).

HBV Genome release and Conversion

Upon viral penetration the nucleocapsid is released into the cytoplasm advancing

towards the nucleus by way of microtubule connection, the core disintegrates

possibly by α and β importin mediation (Palmeri, Malim 1999), releasing the (rcDNA)

with its covalently linked polymerase in to the nucleus, HB viral mechanisms of

release and uncoating are presently unknown.

HBV infection requires covalently closed circular (cccDNA) formation and

amplification. Therefore within the nucleus, the rcDNA is converted into multiple

copies of cccDNA, a multistep process which includes the completion of the positive

strand of the DNA as well as the detachment from the bound Pol (Guo, Jiang et al.

2007). This process results in the cccDNA structure becoming resistant to anti-viral

agents; as it forms a chromatin-like assembly, acting as a small chromosome (Guo,

Jiang et al. 2007).

HBV Transcription, Translation and Reverse transcription

The cccDNA serves as the viral transcriptional template of all viral RNA synthesis by

the hosts RNA Pol II (Guo, Jiang et al. 2007). Viral DNA integration to host genome is

not required for HBV replication (Jones, Hu 2013). Transcription regulation is

undergone by numerous promoters and enhancers, including the identified enhancer

I (EnhI) and enhancer II (EnhII) located in HBV. Both enhancers’ functions are

connected with host promoters exhibiting increased activity in cell lines of hepatic

origin (Doitsh, Shaul 2003). EnhI regulates the X promoter and is involved in cell cycle

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control as well as apoptosis (Doitsh, Shaul 2003). Therefore EnhI assists the

production of the regulatory protein HBx.

HBx is involved in HBV transcription and replication; it is labelled as a trans-activator.

By being implicated with viral promoters plus enhancers, HBx up-regulates gene

expression and protein synthesis (Su, Schneider 1996). A 2009 study established this

by restoring HBx-deficient HBV replication intensities up to wild type (WT) levels,

upon introduction of the HBx protein in mouse liver (Keasler, Hodgson et al. 2009).

As the viral RNA strands enter the cytoplasm, the mRNA strands are translated by

host ribosomes to synthesise proteins. The HBx as well as three envelope proteins are

translated from the sub-genomic RNAs. The pgRNA is translated to synthesise the

core protein as well as HBV viral polymerase. (Doitsh, Shaul 2003, Grimm, Thimme et

al. 2011). The principle function of HBV Pol is to mediate HBV replication through

reverse transcription with pgRNA. This is undertaken in three sequential stages. A

priming step initiates the process of negative strand DNA synthesis. Then the

negative strand of DNA is generated by reverse transcription with degradation of

pgRNA (Jones, Hu 2013). Thirdly, the complementary strand of DNA is synthesised,

attaching on to the end of the negative strand to form a loop encompassing the HBV

Pol, circularising the partially double-stranded DNA structure (Jones, Hu 2013).

Maturation, Re-importation or Exocytosis

While the new HBV DNA is being synthesised, core proteins self-assemble around

the viral Pol-DNA complex to form new viral cores. DNA-containing nucleocapsids

can be either re-imported into the nucleus for cccDNA amplification, or can be

enveloped for secretion (Jones, Hu 2013). Meanwhile viral surface proteins begin to

build up in the membrane of the endoplasmic reticulum. Where they bud into the ER

lumen, and are secreted by the cell. Enveloping the DNA-containing nucleocapsids

forms the infectious virions (fig2), finally the virions exocytose through the Golgi

body via the normal secretory pathway.

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3.2 DISTINCTIVE MOLECULAR PATHWAYS OF HCC

3.2.1 HCC molecular pathways

HCC development is not understood clearly. Global reports into genetic alterations

and molecular profiles with large-scale analysis of HCC, have demonstrated multiple

heterogeneous alterations within gene expression profiles, suggesting complexity

(Fattovich, Stroffolini et al. 2004, Shin, Park et al. 2003). The HCC process is associated

with accumulation of genetic and environmental changes that occur during initiation,

promotion, progression and malignant conversion of the disease (WHO 2014). Such

variability is associated with the numerous HCC contributing factors. Deregulation of

various pathways to influence the survival of cancerous cells by suppressing

pathways involved in apoptosis and regulating cell cycle, such as the alterations of

the p53 pathway, is well documented. Additionally transformed expression of growth

factors and genes involved in angiogenesis, the process of new blood capillary

growth, may participate in HCC development.

3.2.2 Distinct associations in HBV-related HCC pathways

By listing the vital pathways which are responsible for HBV-related HCC, this will aid

in the establishment for how HBV mechanisms influences the host pathways, to

enable the identification of the genes responsible. Gene expression profiles from

microarray technologies and other genetic approaches have established associations.

An investigation has highlighted predominant genetic instability of specific molecular

pathways (Laurent-Puig, Legoix et al. 2001) in relation to HBV infection in cases of

HCC compared to lower instability in HBV-unrelated HCC. Furthermore, gene

expression profiling (Laurent-Puig, Legoix et al. 2001) has revealed that HBV

associated HCC are inclined to affect the mitotic cell cycle, cause p53 mutations, and

cause unfavourable prognosis (Boyault, Rickman et al. 2007). Although associations

have been made, there is a lack of comprehensive studies on human signalling

networks in relation to HBV-induced HCC. Therefore, assumptions are made in

relation to gaps of pathways. With association studies, animal studies, related HBV

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viruses, and HBV molecular understandings supporting the prediction of where and

how HBV-induced HCC occurs in relation to molecular pathways.

Wnt/β-Catenin pathway

Abnormal activation of the Wnt/β-Catenin pathway through mutations in either β-

Catenin or Axin genes has been associated approximately 20% of HCC cases (Legoix,

Bluteau et al. 1999) depending on which or if both genes are defective. The Wnt

signalling pathway is highly integrated in homeostasis, cell proliferation,

differentiation, and apoptosis mechanisms. Consequently, the pathway is a prime

target for cancer generation. In most cases deregulation of the pathway by HBV is

caused by either the inactivation of the tumour suppressor gene adenomatous

polyposis coli or mutation of the proto-oncogene β-catenin. The tumours caused by

β-catenin, display distinct physiological patterns in HCCs and activates expression of

liver metabolic enzymes involved in detoxification and the urea cycle as well as being

associated with host genetic instability (Legoix, Bluteau et al. 1999).

Transforming Growth Factor-β (TGF-β) Pathways

HCC can be influenced by proteins and cellular factors of many signalling pathways.

TGF-β is a human secreted cytokine protein that is involved in numerous pathways

and is important for controlling the immune system (Pardali, Moustakas 2007).

Majority of human tissues have high expression rates towards genes that induce

TGF-β production. In contrast, other anti-inflammatory cytokines such as Interleukin-

10 (IL-10), whose expression is minimal in non-stimulated tissues, apparently

requiring activation by commensal or pathogenic flora (Li, Mai et al. 2012). TGF-β

becomes persistently induced during hepatitis and promotes cirrhosis progression.

Taking up numerous roles, TGF-β in the early stages of liver damage acts as a tumour

suppressor (Pardali, Moustakas 2007). Whilst at stages of abnormal tissue growth,

TGF-β is up-regulated and functions as a tumour promoter that develops neoplastic

growths (Yen, Lin et al. 2012), inducing metastasis activities. It was shown that the use

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of inhibitors of TGF-β prevented the development of HCC (Coulouarn, Factor et al.

2008), illustrating the destructiveness of these proteins.

Ras Proteins in the MAPK/ERK pathway

Human ras proteins are small Guanosine-5'-triphosphate (GTP) binding proteins. The

primary role of GTP is to act as a substrate for the synthesis of RNA or DNA during

the transcription process or during DNA replication respectively (Santos, Nebreda

1989, Filchtinski, Sharabi et al. 2010). Ras proteins function as molecular switches to

influence cell growth, differentiation and apoptosis (Santos, Nebreda 1989). Whilst

oncogenic mutation of ras is rare in HCC, certain kinases has been implicated in

tumourigenesis, such as mitogen-activated protein kinases (MAPK), which are part of

the shared Ras pathway; MAPK/ERK (Pearson, Robinson et al. 2001), which further

resulted in disruption of cell proliferation and apoptosis mechanisms.

The p53 Pathway

Tumour protein p53 (p53) is encoded by the anti-oncogene TP53 gene in humans.

The p53 protein is crucial for prevention of cancer as it functions as a tumour

suppressor (Isobe, Emanuel et al. 1986). This is due to the proteins involvement in

several cell and genome regulation mechanisms involved in conserving stability by

preventing genome mutation. In response to intracellular and extracellular stress

signals caused by DNA damage, cellular stress and immune response p53 expression

is up-regulated (Toledo, Wahl 2006). Binding to DNA, p53 activates expression of

several genes directly and hundreds down-stream for cell mediation and regulation

(Toledo, Wahl 2006). This allows P35 to activate DNA repair proteins upon genome

damage. It can arrest growth by holding the cell cycle at the G1/S regulation point

on DNA damage recognition, to allow DNA repair to take place (Marion, Strati et al.

2009, Kastan, Kuerbitz 1993). If DNA repairs prove unsuccessful, p53 can initiate

apoptosis of the cell. It has been reported that almost half of all human tumours is

due to a single point mutation of the TP53 gene. Of the remaining cancers and in

25% of primary HCCs (Ueda, Ullrich et al. 1995), the normally expressed protein P53

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signalling is defective leading to deregulation of cell cycle arrest and defective

apoptosis by either inducing abnormal pro-apoptotic or anti-apoptotic activities

(Knoll, Furst et al. 2011).

HBx and HBx interactive protein (HBxIP)

Initially identified by Melegari et al. (1998), HBxIP interacts with HBx, which eliminates

the transactivation properties of HBV enhancers and promoters. HBxIP in

differentiated HCC cells reduced WT HBV replication to the same levels observed

following infection with an HBX-minus infection. The investigation hypothesised that

HBxIP negatively regulates HBx activity, altering the life cycle of HBV and clearing the

infection. It was found that a specific mutation on HBxIP phosphorylation site

prevented HBx interaction. Although currently there are unknown SNPs.

Mutations or/and interactions with antiapoptoic proteins (Marusawa, Matsuzawa et

al. 2003) can inhibit HBxIP, and a more detailed genome associated study should

take place, to understand the benefits of HBxIP as a pharmaceutical therapy against

HBx.

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3.3 HBV ONCOLOGICAL MECHANISMS IN RELATION TO HCC

3.3.1 Outline of HBV oncological mechanisms

HBV infection can promote carcinogenesis by at least two direct mechanisms (fig3).

First, integration of the viral DNA in the host genome can induce chromosome

instability and cause direct carcinogenesis by disrupting cell proliferation, viability

and differentiation (i.e. cis-activation) (Yaginuma, Kobayashi et al. 1985). The second

mechanism of carcinogenesis linked to HBV infection is based on the expression of

viral proteins. In particular HBx, to modulate cell sustainability, proliferation and

linked to disrupting vital p53-dependent activities, an imperative step in initiating

HCC. Indirectly (fig3), the host induced by the stress of infection can promote cancer,

including cellular stress primarily oxidative and ER stress, and an overactive immune

attack. By detailing the following mechanisms, any host genetic or epigenetic

associations can be easily followed up for analysis.

3.3.2 HBV DNA amalgamation

Integration of HBV DNA into the human genome is one of the most important steps

in HBV-related carcinogenesis (Matsubara, Tokino 1990). It has been suggested that

HBV DNA integrates into human host chromosomes in hepatocytes in the early

stages of natural acute infections (Matsubara, Tokino 1990). Both HBV DNA insertion

and host cellular DNA replication occurs during liver cell proliferation, secondary to

the apoptosis of neighbouring hepatocytes. Considering that HBV replication does

not require viral DNA integration into the host’s genome, it is stated by various

studies that DNA amalgamation occurs randomly and that HBV insertion

mutagenesis occurs rarely. This hypothesis is challenged and rejected by

supplementary studies that use improved methods of analysis, and in this literature

judges the hypothesis to be a null hypothesis to be disproved.

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Disapproving the null hypothesis

Random distribution of HBV integration sites was initially proposed due to

observations of multiple integrations being established in numerous human

chromosomes in human HCC (Yaginuma, Kobayashi et al. 1987). Additionally, studies

that identified the insertion of HBV DNA into cancer susceptible host genes

(Paterlini-Brechot, Saigo et al. 2003, Murakami, Saigo et al. 2005), such as telomerase

reverse transcriptase (TERT) which is involved in regulating telomeres which in turn is

a critical nucleotide sequence for maintaining genomic activity. Were scrutinised as

the observations of gene amplification stated by the reports important for

carcinogenesis were not been commonly observed in all subjects HCC diagnosis in

multiple studies (Tamori, Yamanishi et al. 2005).

The rare mutagenesis hypothesis can be explained. As in these cases, the integrations

may be acting directly on chromosomes or else as non-coding DNA which are

regulating the transcription of neighbouring genes (Wittkopp, Kalay 2011), known as

cis-acting interactions.

The use of Alu-PCR

The issue with all of the stated investigations is that they are limited to low accurate

methods and are unable to provide a high output analysis which is both effective and

consistent, such as the use of Southern blotting. This was answered by the use of

modified polymerase chain reaction (PCR), primarily Alu-PCR studies especially those

that implemented large-scale analysis. The seemingly random distribution was

challenged by the PCR technique and is stated in literature (Murakami, Saigo et al.

2005, Paterlini-Brechot, Saigo et al. 2003) as a highly effective tool for the study of

HBV integration sites.

Investigations using Alu-PCR that aids in amplifying sequences adjacent to added

primers from a commonly repeated segment, produces a unique DNA fingerprint of

amplitude lengths. The sequences are then inputted through numerous databases

and bioinformatics tools, for identification of HBV location on human genomes.

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The Alu-PCR studies analysed HBV DNA insertion sites revealing high rates of gene

targeting by HBV DNA insertion. Numerous studies including the ones stated identify

a high ratio of cancer causing genes (Murakami, Saigo et al. 2005, Paterlini-Brechot,

Saigo et al. 2003) including genes that are involved in tumour suppression, apoptosis

control and specific pathways.

Furthermore, HBV insertions have been associated with major genetic alterations

(Chen, Chen 2003) and not just by means of cis mechanisms (Murakami, Saigo et al

2005), such as chromosomal translocation and large duplication in TERT and other

control mechanisms. It can therefore be predicted that viral integration takes an

important step towards liver cancer and specifically HCC. From ample evidence DNA

amalgamation is not distributed randomly and that HBV insertion mutagenesis

occurs frequently in HCC patients (Murakami, Saigo et al. 2005, Paterlini-Brechot,

Saigo et al. 2003).

3.3.3 HBV Viral oncogenic proteins

The second direct mechanism of HCC induced by HBV infection is the involvement of

HBV viral proteins in deregulating and disrupting cell pathways to induce and

propagate cancer. The HBx protein encoded by the X gene is the viral function most

frequently implicated in oncogenesis. The protein is highly involved in hepatocyte

transformation as it influences multiple genes. Labelled as pleiotropic protein; it acts

on cell cycle regulation, hosts DNA repair and signalling pathway mechanisms (Lee,

Mok et al. 2014). The number of diverse functions HBx delivers makes it difficult to

build a specific hypothesis about its mode of action. What is clear is that HBx is a

multifunctional protein that is highly oncogenic.

HBx association with apoptosis and cell growth

Majority of human hepatocellular carcinomas are showed to be related to the

mutation of the TP53 gene and/or the anti-oncogene product p53. HBx is stated to

mediate HCC by inducing and inhibiting apoptosis. This is due to the HBx proteins

ability to form a complex or inactivate p53, to deregulate p53 activities, accounting

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for numerous cellular transformations. Although in relation to p53 inactivation, it has

been observed to occur rarely in HBV-induced HCCs, and remains controversial

(Feitelson 1999). Concerning HBx ability to form p53 complexes, a study revealed

that transgenic mice expressing HBx in the liver are more likely than WT mice to

develop hepatocellular carcinoma. The reason given is that HBx promotes cell cycle

progression whilst it forms a p53 complex, sequestering the anti-oncogene product

from its role in mediated apoptosis, causing uncontrolled growth (Kew 2011). The

association of cellular growth can be explained by HBx ability to both trans-activate

HBV promoters as well as cellular functions associated with cell growth, such as the

proto-oncogene gene expression promoter c-Fos (Wu, Forgues et al. 2002).

An additional interaction of HBx with p53, causes the inhibition of p53 ability to

initiate nucleotide excision repairs, by preventing DNA repair proteins which also

take part in inducing apoptosis. This allows accumulation of DNA mutations

contributing to carcinogenesis (Elmore, Hancock et al. 1997). Experimental

observations also suggest that the HBx protein increases TERT and telomerase

activity, prolonging the lifespan of hepatocytes and contributing to malignant

transformation (Kew 2011). It is deliberated that HBx antigens are protein kinases

which may undergo autophosphorylation, defined as the ability to phosphorylate

kinase by the aforementioned protein, HBx. Although the targets for phosphorylation

of HBxAg are unknown, phosphorylation of tumour suppressor gene components

and cell cycle proteins are recognized to alter their activities in controlling cell

growth (Hernandez, Venegas et al. 2012).

HBx role in transcriptional trans-activation and cell signalling

HBx can increase the rate of gene expression to cause oncogenesis. By acting as a

transcriptional trans-activator that can up-regulate a large number of proto-

oncogenes such as c-Fos (Wu, Forgues et al. 2002). HBX trans-activation can also

lead to activation of signalling pathways in relation to oncogenesis, proliferation,

inflammation and immune responses. Among these, HBx can directly or indirectly

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affect nuclear factor and activator proteins, which induce gene expression. The

proteins and factors respond to stimuli such as cytokines and stress, leading to

increased cycle progression (Lara-Pezzi, Armesilla et al. 1998). Which can induce

malignant transformation, as previously mentioned. HBx further functions as a trans-

activator by interacting with both nuclear transcription factors and signal

transduction pathways, such as Ras and MAPK. HBx role in inducing these specific

pathways is to increase the rate of cellular gene expression which improves the rate

and efficacy of viral replication.

Additional viral proteins involved in carcinogenesis

Other than HBx, there may be other viral proteins involved in liver cancer. The

envelope proteins, mainly L and possibly M are found to have associated oncological

properties. An investigation (Xu, Huang et al. 2001) that overproduced envelope

proteins showed that mainly L proteins and perhaps M proteins resulted in

intracellular accumulation and induced cellular stress, which led to the development

of cancer. In a study that uses HBV L envelope only producing transgenic mice, the

resulting mice liver consistently developed HCC. This suggests that the L- viral

envelope protein may be hepatotoxic (Chisari, Ferrari 1995). Although associations

have been made, they were formed in animal models and not respective of humans.

Viral mutations of the HBV proteins including core, pre-C and envelope genes may

be induced by liver disease or HCC, suggesting an association between the viral

proteins and damage to the liver (Feitelson 1999). These studies cannot be relied

upon as well. As the occurrence of liver diseases in these studies is not clear, as it

may either be due to the liver damage or the effect of the proteins. As HBV prepares

for exocytosis, the accumulation of viral glycoproteins may induce ER stress, which

over time can lead to cell responses inducing mutagenic reactants and an overactive

immune response. Envelope proteins may not be directly inducing mutations; it may

be due to the frequent and relentless occurrence of the viral proteins that causes the

hepatocyte to mutate. Until further comprehensive studies are made on HBV viral

proteins, associations can only be specified.

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3.3.4 HBV Immune system response in relation to cancerogenesis

T lymphocytes involved in cell-mediated immunity are a major hindrance to chronic

HBV development and interestingly involved in cancer development. The level of

active cytotoxic T-cells is vital for elimination of acute hepatitis. Subjects with CHB

have insufficient level of T-cell to viral antigens (Guidotti, Rochford et al. 1999). T

lymphocytes that use non-cytolytic mechanisms, namely CD8 T-cells are also

involved in the resolution of hepatitis, involving interferon and tumour necrosis

factors (Guidotti, Rochford et al. 1999). Typically, a reduced number of HBeAg in

addition with increased T-cell recognition of all HBV antigens is associated to

recovery from CHB.

Cytotoxic T-cells during HB infection have been stated to significantly contribute to

liver injury, by causing apoptosis of infected hepatocytes and by producing antiviral

cytokines capable of eliminating HBV from viable hepatocytes (Crispe, Dao et al.

2000). This recorded response not only eliminates infected cells but infects viable

hepatocytes, if levels of apoptosis remains high, the overall liver can result in

increasing damage. Considering that a well-known risk factor in developing HCC is

liver damage, indirectly the immune system may aid in the development of HCC.

Controversially, selected studies suggest that T-cells preclude HBV clearance without

liver damage (Maini, Boni et al. 2000); in this study the application was based on

animal models concerning CD8 cells and supplementary components. In 2010 a

study on suppression of anti-tumour immune response, concluded that regulatory T-

cells in CHB causes suppression, aiding in tumour progression (Zhang, Mei et al.

2010). Comparing the studies, it is not reasonable to conclude whether the

suggestion of there being no damage is predominant, as there are numerous studies

suggesting the opposite. It is rational to suggest that the immune system is vital for

HBV clearance, and if working correctly under the right circumstances can clear the

virus without any significant damage. However the environment in which the

hepatocytes replicate will be consequently encircled by numerous mutagens such as

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oxidants, in response to the injury being inflicted. Therefore, initiating further

immune responses, such as the increased accumulation of cytotoxic natural killer

cells (NK cells), aggravating the liver further. Once the immune response is unable to

control virus replication, it may contribute to liver pathology.

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3.4 GENETIC ASSOCIATIONS TO THE DEVELOPMENT OF HBV-INDUCED HCC

As mentioned earlier, a significant proportion of HBV-associated HCC occurs in the

absence of liver cirrhosis (Fattovich, Stroffolini et al. 2004). The argument for HBV

having a direct influence in the process of cancer is derived from this fact, and is

illustrated by the direct HBV oncological mechanisms section, although indirect

mechanisms via liver damage remains a factor. The detection of gene expression

profiles implicated in inflammatory and DNA repair responses in non-carcinoma CHB

infected livers (Li, Zhao et al. 2011, Shin, Park et al. 2003), infers specific pathways

such as various signalling cascades. By suggesting HBV route to carcinoma is caused

by specific pathways and elucidating this in the literature. This forms a foundation for

the genetic association hypothesis. As all molecular mechanisms and cellular

pathways are initiated by the epigenetic regulation of gene expression and silencing.

If there are any SNPs or mutations that affect the regulatory genes to either induce

or impede HCC development. This proves that host genetic susceptibility is a factor

in HBV-induced HCC.

3.4.1 Initial findings of gene susceptibility

In 1991 a study presented evidence of inherited susceptibility connected to HBV

infected hosts with HCC in eastern china. Using complex segregation analysis, to

determine whether a major gene underlies the distribution of a phenotypic train and

whether it is dominant, recessive or co-dominant. The investigation analysed 490

extended families to support the existence of a recessive allele, resulting in an

increased life time risk of HCC whilst infected with HBV and genetic susceptibility.

The investigation results can be extrapolated to suggest that within the life times of

1000 HBV-infected individuals within the stated population, with equal numbers of

males and females. 21 cases of genetically susceptible males and 12 cases of

genetically susceptible females will develop HCC. Whereas 43 cases in non-

susceptible males and 5 in non-susceptible females will go on to develop HCC.

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Suggesting that approximately 40% of HCC cases occur among HBV infected and the

genetic susceptible. The data also suggests that there is a relationship between sex,

genetic susceptibility and HBV infection. Suggesting that the biological differences in

females by some means interferes with the other two variables, in that females

appear to develop HCC in the presence of both HCC and gene susceptibility.

Whereas, HBV infection is enough for some males to develop HCC. Due to

technological restrictions and the method used at the time the recessive allele was

not identified, but is proven to exist.

3.4.2 Analysis of host genetic susceptibility towards HBV-induced HCC

There are a limited number of genome association study investigations that have

been published, as gene susceptibility in relation to HBV-induced HCC is a novel

concept. However, considering that HBV causes up to 54-78% of HCC globally

(Parkin 2006, Parkin 2001). Gene profiling studies based on HCC in general may

contain sufficient data on HCC induced by HBV infection. By identifying studies that

represent a significant quantity of HBV-related HCC gene profiling data, it is possible

to complement findings in relation to direct studies of host genetic susceptibility.

In one such example, a study through exomic sequencing of several factors that are

associated with HCC identified ARID2 as a tumour suppressor gene (Li, Zhao et al.

2011). As a subunit of a PBAF chromatin-remodelling complex, facilitates ligand-

dependent transcriptional activation by nuclear receptors, and has been identified as

a tumour suppressor. There is no doubt that if a host had a defective gene in ARID2,

hepatocellular carcinomas are more likely to occur. The investigation cannot rule out

the possibility that individual ethnicities, viral subtypes or other environmental

factors defines the advantage by ARID2 mutations, although the investigation

suggest that the HBV infections are the major contributor to mutations (Li, Zhao et al.

2011). So a disadvantage mutation and therefore a genetic susceptibility in ARID2,

acting as part of the transcriptional pathway, can result in HCC.

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A more suitable genetic susceptibility study identified a susceptible mutation in IL-10

(Shin, Park et al. 2003). Part of the immune system, IL-10 is an anti-inflammatory

cytokine. In humans, IL-10 is primarily produced by monocytes and by certain

lymphocytes, such as regulatory T-cells. Upon viral infection IL-10 is found to be

released by cytotoxic T-cells to inhibit the action of NK cells. As the NK cells role is to

provide rapid responses to viral infected cells, a mutation in their development can

increase the risk of HCC. By means of genetic association analysis, a study showed

that a haplotype, a cluster of SNPs on a chromosome that are likely to be inherited

together, of the IL-10 protein titled as IL10-HT2 was strongly associated with

hepatocellular carcinoma (Shin, Park et al. 2003) in a large Korean population

characterised with HBV. The haplotype is stated to be common in Caucasians and

African Americans (Shin, Park et al. 2003). Highly significant acceleration to HCC

outcome was apparent among the IL10-ht2-bearing patients. The study suggest that

IL-10 polymorphisms play a critical role in immunity, inflammation progress and

cancer development. It can be concluded that an increased IL-10 production in IL10-

ht2 carrying individuals inhibits the innate immune system which may cause the

acceleration of the progression of CHB to HCC.

By means of a GWAS study Zhang et al. (2010) (Cited by Chan, Wong et al. 2011)

investigation found an association between HBV-related HCC and a SNP in an intron

of the KIF1B gene355. From a cohort of Chinese ethnic individuals divided into two

groups; 355 chronic HBV carries with HCC and 360 chronic HBV carriers without HCC.

The association was confirmed in 5 additional independent Chinese samples,

consisting of 1,962 individuals with HCC, 1,430 control subjects. However the k1f1b

was found not to be associated with progression to CHB in a more recent study

(Zhong, Tian et al. 2012). By using Equivalence-based method analysis, the study

confirmed the absence of association, concluding that distinct genetic susceptibility

factor contributes to the progression from hepatitis B virus infection to HCC.

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4.0 CONCLUSION

The literatures aim is to provide evidential associations to indicate that the host is

genetically susceptible to progression of HCC induced by HBV related mechanisms.

Firstly, by providing evidence that HBV has direct HCC mechanisms, may infer

possible host susceptible genes. By using this as a foundation, analysis of literature

should be accomplished to obtain evidence for a genetic predisposition of HBV-

induced HCC.

This paper has determined HBV and host liver mechanisms in relation to HCC, and

found that HBV induces HCC separately from cirrhosis. HBV-induced HCC has both

direct and indirect mechanisms associated with HCC development. HBVs oncological

viral protein, HBx, was identified and explained in detail, due to its high efficacy in

propagating HCC. By illustrating HBV oncological mechanisms and pathways, every

gene in relation to these mechanisms could be potentially host susceptible. The

literature confirmed associations of human genetic susceptibility on the progression

of HCC with HB infection, by identifying polymorphisms, namely SNPs, and the HBxIP

protein. The literature on the basis of data analysis of gene associated studies,

identified that genetic predisposition has variable efficacy on the development of

HBV-related HCC. In general a significant quantity of the population can be

predisposed from genetic differences. Although depending on the type of

polymorphism the significance varies, additional factors, such as sex, can play a role

reducing the significance of population related polymorphisms.

It is clear from the explanations; that mechanisms of HBV are not entirely known, in

addition to contradicting hypothesis of the roles, and mechanism of HBV, as well as

the numerous ways HBV causes HCC. These factors hinder the identification of

susceptible genes due to the complexity of how the virus acts. Conversely, this allows

for a large number of susceptible genes, however from a clinical perspective this is

disadvantageous. Analysing the literature from the mechanism sections of the

27 of 37


dissertation, initiated the identification of the susceptible genes. Additionally, the

research that aided the explanation of mechanisms is also used as supporting

evidence towards the hypothesis.

Many of the studies, including the gene association studies, were accomplished in

Asia. As HBV is a predominate problem in the eastern hemisphere compared to other

regions. This therefore creates issues regards to HBV genotype and population

factors. Considering that identification of the polymorphisms was also found in other

ethnicities, this has mitigated one of the issues. Given that, predominately HBV is

more of a problem in this geographical location, it can be argued that the

dissertation is more appropriate.

An additional issue in the literature analysis was attempting to identify relative gene

association studies. The hypothesis of this paper is original, and obtaining published

papers that solely investigated the hypothesis was difficult. To sustain the evidence,

gene profiling studies based on HCC in general was used. Data was extracted in

relation to HBV-induced HCC and were factored for reliability and statistical

significance. Issues were raised when determining whether the gene mutation of

HCCs was cause or effect.

The genome wide investigations on HBV-related HCC studies are relatively new;

upon statistical analysis Zhang et al. (2010) is disregarded, upon further research the

paper stated HBV associations were pending confirmation. Considering this fact,

comprehensive investigations are required to sustain the conclusions stated by all

the genome studies.

In summary, there is enough evidence to suggest that the hypothesis is correct; that

there are associations of a genetic predisposition towards HBV-induced HCC.

Although there are many issues to consider, the dissertations circumstantially

provided suggestions that gene predisposition exists and by analysing the successful

genome studies there is a case for gene susceptibility. Although, depending on the

28 of 37


genetic alteration, variable predisposition exists. However, a combined influence of

several SNPs or other population independent mutations would have a dramatic

effect on clinical outcomes of HBV-induced HCC. And since the conclusion is that

host predisposition exist this becomes more likely.

The rationales stated at the beginning remains the same, however since HB viruses

are highly oncogenic, it is better to develop and use anti-viral therapies than to

consider genetic related therapies. Before these findings can be translated into

further clinical studies and possible practice, independent validations of the genome

studies should take place, and further comprehensive genome-related studies should

take place.

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6.0 FIGURES AND TABLES

Fig1. A linear genome schematic diagram. The figure illustrates the open reading frames relative sizes

and the synthesised proteins in relation to the genomic regions. The genome encodes four

overlapping open reading frames: (ORFs) S, C, P, and X. The pre-C & C ORF encodes the ‘e’ antigen

(HBeAg) and the core antigen (HBcAg) respectively. The largest genomic region, P encodes Viral DNA

polymerase. The pre-S/S ORF, depending on genomic region, encodes the three surface envelope

proteins; large, middle, and small HBsAg. Open reading Frame X encodes HBxAg. Data obtained and

manipulated from (Sayers, Barrett et al. 2009).

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Fig2

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Fig.3 Summarisation of the direct and indirect pathways HBV induces HCC. HBV DNA integrates into

host to alter the host genome, or to cause genome instability which eventually will cause genome

alteration. Genomic alteration if not repaired can lead to HCC. Not shown in figure: regulatory

mechanisms are also affected by HBV, inhibiting repairs. HBVs cancer inducing viral protein, HBx, can

affect several pathways and mechanisms including the not shown repair mechanisms to cause HCC.

Additionally HBV can indirectly cause HCC by the route of liver damage shown on the left of the red

vertical line.

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Human Susceptability to HPC - Dissertation - Completed