Role of protein misfolding pathway in HBX induced hepatocellular carcinoma

ROLE OF PROTEIN MISFOLDING PATHWAY
IN HBX-INDUCED
HEPATOCELLULAR CARCINOMA (HCC)

TAN SU YIN

NATIONAL UNIVERSITY OF SINGAPORE
2011


ROLE OF PROTEIN MISFOLDING PATHWAY
IN HBX-INDUCED
HEPATOCELLULAR CARCINOMA (HCC)

TAN SU YIN
(B.Sc.(Hons.), NTU)

A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF MEDICINE
YONG LOO LIN SCHOOL OF MEDICINE

NATIONAL UNIVERSITY OF SINGAPORE
2011


I
ACKNOWLEDGEMENTS
It would not have been possible to complete my journey in this masters thesis without
the help and support from many kind people around me.

I would like to express my deepest appreciation to my supervisor, Dr Matiullah Khan,
for his invaluable guidance, advice and insightful discussions throughout my
university years and in this course of research.
My thanks also extend to Cancer Science Institute as well as National University of
Singapore, where the project was carried out, for the opportunity to pursue this degree
through their sponsorship and support.
My heartfelt thanks to Dr Azhar Ali and Dr Angela Ng for their valuable discussion
and technical advices whenever needed. I have benefited greatly from their wisdom
and sharing of expertise in scientific knowledge as well as in life experiences.
Special thanks to Dr Azhar Ali, Dawn and Sarawut for taking their precious time to
proof-read this thesis.
I would like to thank all the past and present members of this lab for the wonderful
working experience, especially Angie, Jess, Lizan, Dawn, Haris, Lifeng and Wanqiu
whom all have contributed to the success of this thesis in one way or another. It has
been a real pleasure working with all of you throughout these years.
My deepest gratitude to my dearest friends, Meg, Li Ren, Pei Li, John, Ben, Mei Ling
and Sarawut for their care and concern towards my well-being. Thanks for being such
wonderful colleagues and friends and I really treasure all the joys and laughter that we
have shared.
Lastly, I would like to express my most sincere thanks to my family. I could not have
completed without the immense love and unequivocal support from them. Heartfelt
thanks to my dad and my little sister for cheering me up and ensuring that I achieve
the goals in my life.
Thank You.
Tan Su Yin
August 2011


II
TABLE OF CONTENTS

ACKNOWLEDGEMENTS
TABLE OF CONTENTS

I
II
VII

SUMMARY
LIST OF TABLES

VIII

LIST OF FIGURES

IX

ABBREVIATIONS

XI

Chapter 1. Introduction

1

1.1

Liver Cancer

1

1.2

Hepatocellular Carcinoma (HCC)

1

1.2.1

Epidemiology

1

1.2.2

Aetiological factors of HCC

2

1.2.2.1 Hepatitis B virus (HBV)

2

1.2.2.2 Hepatitis C virus (HCV)

3

1.2.2.3 Aflatoxin B1 (AFB1)

4

1.2.2.4 Alcohol

5

Diagnosis and treatment

6

1.2.3.1 Screening tests

6

1.2.3.2 Treatment

7

Challenges in understanding HCC

8

1.2.3

1.2.4
1.3

Acute promyelocytic leukemia (APL)

9

1.3.1

PML-RARα

9

1.3.2

Nuclear hormone co repressor (N-CoR)

10


III
1.3.3

1.4

Current knowledge of PML-RARα,  N-CoR and its perceived
mechanism in APL

10

ER stress and unfolded protein response (UPR)

12

1.4.1

Protein folding

12

1.4.2

Unfolded protein response

13

1.4.3

UPR-induced apoptosis

15

1.5

Ubiquitin Proteasome Pathway (UPP)

19

1.6

Autophagy

19

1.6.1

Macroautophagy

20

1.6.2

Chaperone-mediated autophagy (CMA)

22

1.7

Current perspective in APL and study hypothesis and objectives

23

1.7.1

Current perspective in APL

23

1.7.2

Study hypotheses and objectives

24

Chapter 2. Materials and Methods

27

2.1

Materials

27

2.1.1

General Reagents

27

2.1.2

Antisera

28

2.1.2.1 Western Blotting

28

2.1.2.2 Immunofluorescence Staining

29

Primer Sequences

29

2.1.3.1 Semi-Quantitative RT-PCR primers

29

2.1.3.2 siRNA sequences

30

2.1.4

Drugs and Inhibitors

30

2.1.5

Hepatocellular Carcinoma Tissues

31

2.1.5.1 Human Samples

31

2.1.3


IV

2.1.6

2.1.7

2.2

2.1.5.2 Tissue Microarray

32

Cell lines

32

2.1.6.1 Liver cancer cell lines

32

2.1.6.2 HEK 293T and HeLa cell lines

32

Plasmids

32

2.1.7.1 pACT vector

32

Methods

33

2.2.1

Cell Culture Treatment

33

2.2.1.1 Cell culture

33

2.2.1.2 Treatment with drugs and inhibitors

33

2.2.1.3 Transfection

33

2.2.2

2.2.1.3.1 DNA transfection of 293T cells and HeLa cells

33

2.2.1.3.2 N-CoR siRNA knockdown in HCC cells

34

Protein extraction

34

2.2.2.1 Mammalian cells

34

2.2.3

Immunoblotting/Western Blotting

35

2.2.4

Immunohistochemistry

36

2.2.5

Immunofluorescence and confocal microscopy

36

2.2.6

RNA extraction

37

2.2.6.1 Mammalian cells

37

Reverse transcription polymerase chain reaction (RT-PCR)
amplification

38

2.2.7.1 DNA agarose gel electrophoresis

39

Biological Assays

39

2.2.8.1 Luciferase assay

40

2.2.7

2.2.8

2.2.8.2 ATP assay

40


V
2.2.8.3 Ubiquitination assay/ Immunoprecipitation

40

2.2.8.4 Solubility assay

41

Chapter 3. Results

42

3.1

Analysis of N-CoR status in human liver cancer cells and tumours

42

3.1.1

Loss of N-CoR protein in multiple human liver cancer cell lines

42

3.1.2

Loss of N-CoR protein in human liver cancers

42

3.2

3.3

Elucidating the mechanism of N-CoR loss

44

3.2.1

N-CoR loss in HCC cells is linked to misfolding

44

3.2.2

Inverse relationship between N-CoR and HBX expression levels

45

3.2.3

Co-expression with HBX preferentially localizes N-CoR to the
cytosol

46

3.2.4

N-CoR in HBX positive HCC cells is found to be co-localized
with HBX in the cytosol

48

3.2.5

N-CoR protein in liver tumour sections is also found to be
localized in the cytosol

52

3.2.6

HBX induces N-CoR insolubility

52

3.2.7

HBX promotes degradation of ectopic N-CoR in transfected
293T cells

53

3.2.8

HBX promotes ubiquitin-proteasome mediated degradation of
N-CoR

54

3.2.9

N-CoR loss in HBX positive HCC cells is mediated by the
ubiquitin-proteasome pathway

55

The role of misfolded N-CoR in HBX-induced UPR

58

3.3.1

HBX positive HCC cells exhibit amplification of ER stress

58

3.3.2

HBX promotes ATF6 activation

62

VI
3.3.3

3.4

HBX abrogates N-CoR mediated repression of the ATF6
promoter

63

Investigating the molecular mechanism underlying the misfolded NCoR induced transformation of HCC cells

66

A. N-CoR misfolding and Chronic inflammation

66

3.4.1

Upregulation of APR gene transcript in HCC cells

66

3.4.2

Pro-inflammatory cytokine abrogates N-CoR function

66

3.4.3

APR genes are not repressed by N-CoR

68

B. N-CoR misfolding and Epithelial Mesechymal Transition (EMT)

69

3.4.4

The levels of EMT gene transcripts in HCC cells are high

69

3.4.5

EMT genes are not repressed by N-CoR

70

C. N-CoR misfolding and Autophagy

71

3.4.6

Autophagy is activated in HCC cells

71

3.4.7

Formation of autophagosomes in HBX positive cells

72

3.4.8

Loss of N-CoR may be linked to autophagy

76

3.4.9

HBX activates autophagy

77

3.4.10 Autophagy supports the growth of HCC cells

77

Chapter 4. Discussion

81

4.1

Role of misfolded N-CoR in HCC pathogenesis

81

4.2

HBX induces a conformational change in N-CoR

81

4.3

HBX-induced conformational change leads to instability and
N-CoR protein degradation

82

4.4

Role of autophagy in the survival and growth of HCC cells

83

4.5

Role of HBX-induced misfolded N-CoR in the activation of UPR and
autophagy

84

VII
4.6

Future areas of research

86

4.6.1

Mechanism of autophagy mediated N-CoR loss in liver cancer
pathogenesis

86

4.6.2

Identification of misfolded N-CoR as a molecular target in HCC

86

4.6.3

Appropriate experimental controls

88

REFERENCES

90

VII

SUMMARY
Transcriptional control imparted by nuclear receptor co-repressor (N-CoR)
plays an important role in the growth suppressive function of several tumour
suppressor proteins. Abrogation of this transcriptional control due to a misfolded
conformation dependent loss (MCDL) of N-CoR has been implicated in acute
promyelocytic leukemia (APL). It was hypothesized that an APL like MCDL of NCoR might be involved in other malignancy. Indeed, the initial screening of N-CoR
status in various liver cancer (HCC) cell lines revealed an APL like MCDL of N-CoR
protein. The N-CoR protein presented in these HCC cells was misfolded and was
linked to the amplification of endoplasmic reticulum (ER) stress and cytoprotective
unfolded protein response (UPR). siRNA-induced N-CoR ablation led to selective
reduction of intracellular ATP level and significantly compromised the growth of
HCC cells, suggesting an important role of energy, possibly derived from N-CoR
catabolism, in HCC cell growth. These findings identify an important role of
autophagy-induced recycling of misfolded N-CoR protein in the selective activation
of autonomous survival and growth in HCC cells.


VIII
LIST OF TABLES

Table 2.1.

List of Chemicals, Reagents and Kits

27

Table 2.2.

List of Primary Antibodies

28

Table 2.3.

List of Secondary Antibodies

28

Table 2.4.

List of Primary Antibodies

29

Table 2.5.

List of Secondary Antibodies

29

Table 2.6.

List of semi-quantitative RT-PCR primers

29

Table 2.7.

List of siRNA sequences used in siRNA mediated gene
knockdown

29

Table 2.8.

List of drugs and inhibitors

30

Table 2.9.

Clinical characteristics of patient samples used in this study

31


IX
LIST OF FIGURES

Figure 1.1.

Model of bifunctional role of nuclear receptor corepressor in
acute promyelocytic leukemia pathogenesis

11

Figure 1.2.

UPR signalling pathways in mammalian cells

16

Figure 1.3.

ER stress pathways implicated in mediating cell apoptosis

18

Figure 1.4.

Kinetics of N-CoR misfolding in APL

25

Figure 3.1.A-B

Selective loss of N-CoR protein in HCC cells

43

Figure 3.2.

Evidence of N-CoR loss in primary human liver cancer
samples

44

Figure 3.3.

Genistein promotes stabilization of N-CoR protein in SK
Hep1 cells

45

Figure 3.4.

N-CoR protein level in HCC cells is inversely related to HBX
transcript level

46

Figure 3.5.

HBX promotes cytosolic retention of N-CoR protein

47

Figure 3.6.

N-CoR of HBX positive HCC cells is found in the cytosol

51

Figure 3.7.

N-CoR protein is mainly localized in the cytosol in tumour

sections

51

Figure 3.8.

HBX can induce N-CoR insolubility

53

Figure 3.9.

HBX promotes N-CoR degradation

54

Figure 3.10.

HBX promotes ubiquitination of N-CoR protein

56


X
Figure 3.11.A-B Ubiquitin-proteasome mediated degradation of N-CoR protein
in HBX positive HCC cells

57

Figure 3.12.A-D HBX positive cells exhibit high level of ER stress

59

Figure 3.13.A-C Regulation of UPR by HBX and N-CoR

64

Figure 3.14.

Ectopic expression of HBX in HeLa cells repressed ATF6
promoter activity

65

Figure 3.15.

N-CoR loss in HCC cells might be associated with an upregulation of APR genes

67

Figure 3.16.

Pro-inflammatory cytokine promotes degradation of N-CoR

68

Figure 3.17.

APR genes were up-regulated in a dose dependent manner
after genistein treatment

69

Figure 3.18.

Expression level of EMT genes

70

Figure 3.19.

N-CoR did not repress EMT gene expression after genistein
treatment

71

Figure 3.20.

Level of LC3-II in HBX positive cells is elevated

73

Figure 3.21.

Autophagosomes are seen in HBX positive cells

73

Figure 3.22.

Bafilomycin promotes stabilization of N-CoR protein in SK
Hep1 cells

76

Figure 3.23.A-B HBX induced activation of autophagy is mediated by
misfolded N-CoR

78

Figure 3.24.

N-CoR loss is linked to ATP mediated growth of SK Hep1
cells

80

Figure 4.1.

Schematic representation of the regulation of UPR and
autophagy in normal and HCC cells.

86


XI

ABBREVIATIONS

AFB1

aflatoxin B1

AFP

alpha-fetoprotein

AML

acute myeloid leukemia

APL

acute promyelocytic leukemia

ASK1

apoptosis-signal-regulating kinase 1

Atg

Autophagy-related genes

ATF

activating transcription factor

BA-1

Bafilomycin A1

BiP

binding Ig protein

BSA

bovine serum albumin

CHOP

C/EBP homologous protein

CMA

chaperone-mediated autophagy

CT

computed tomography

DCP

des-gamma-carboxy prothrombin

DAPI

4,6-diamidino-2-phenylindole

DMSO

dimethyl sulfoxide

DNA

deoxyribonucleic acid

EDEM

ER-degradation-enhancing  α-mannosidase-like protein

EDTA

ethylenediaminetetraacetic acid

ERAD

ER associated degradation

eIF2α

elongation factor 2α

EM

electron microscopy

EMT

epithelial-mesenchymal transition

ER

endoplasmic reticulum

ERSE

endoplasmic reticulum stress element

ERAD

ER-associated degradation

FAB

French American and British

FBS

fetal bovine serum


XII

GFP

green fluorescent protein

GRP

glucose-regulated protein

HBV

hepatitis B virus

HBX

hepatitis B virus X protein

HCC

hepatocellular carcinoma

HCV

hepatitis C virus

HDACs

histone deacetylase complexes

HMW

high molecular weight

HRAS

Harvey-ras proto-oncogene

Hsp

heat shock protein

IF

immunofluorescence

IHC

immunohistochemistry

IRE1

inositol requiring kinase 1

JNK

c-Jun N-terminal kinase

kb

kilo base

kDa

kilo Dalton

LAMP2A

lysosomal-associated membrane protein 2A

MCDL

misfolded conformation dependent loss

mins

minutes

mRNA

messenger RNA

MRI

magnetic resonance imaging

mTOR

rapamycin

N-CoR

nuclear receptor co-repressor

OSGEP

O-Sialoglycoprotein endopeptidase

PBS

phosphate buffered saline

PDI

protein disulpide isomerase

PERK

RNA-activated protein kinase like endoplasmic reticulum kinase

PI3K

phosphatidyl-inositol 3 kinase

PI3KC3

class III phosphatidyl inositol 3-kinase

PLB

passive lysis buffer


XIII

PML

promyelocytic leukemia

POD

PML oncogenic domains

PtdIns

phosphatidyl inositol

PtdIns(3,4,5) P3 phosphatidylinositol-3,4,5- triphosphate
PTM

post translational modification

PVDF

polyvinylidene difluoride

RA

retinoic acid

RARα  

retinoic  acid  receptor  α

RFA

radiofrequency ablation

ROS

reactive oxygen species

RT-PCR

reverse transcription-polymerase chain reaction

S1P

site-1 protease

SDS

sodium dodecyl sulphate

SDS-PAGE

SDS-Polyacrylamide gel electrophoresis

siRNA

small interfering RNA

SMRT

silence mediator of retinoic and thyroid receptors

TRAF2

TNF receptor-associated factor 2

Ub

Ubiquitin

UGGT

UDP-glucose:glycoprotein glucosyltransferase

UPP

ubiquitin proteasome pathway

UPR

unfolded protein response

UPRE

unfolded protein response element

US

ultrasound

Vps34

vesicular protein sorting 34

XBP1

X-box DNA-binding protein 1


1

Chapter 1. Introduction
1.1

Liver Cancer
Liver cancer is one of the most lethal malignancies worldwide. Globally it

ranks the second most frequent cause of cancer death in men and the sixth in women
(Ahmedin et al., 2011). Liver cancer consists of several histologically different
primary hepatic malignancies, such as cholangiocarcinoma (bile duct cancer),
hepatoblastoma (liver cancer affecting young children) and haemangiosarcoma
(cancer arising from the blood vessels of the liver). Hepatocellular carcinoma (HCC),
also called malignant hepatoma, is by far the most common type.
1.2

Hepatocellular Carcinoma (HCC)

1.2.1

Epidemiology
Hepatocellular carcinoma (HCC) is a major cause of morbidity and mortality

in human. It is the fifth most common cancer worldwide, and the third leading cause
of cancer-related deaths (Ferlay et al., 2008) with an estimate of one million deaths

annually (Bosch et al., 2004; Cha and Dematteo, 2005).
The incidence of liver cancer varies around the world and is highest in
Mongolia with 116.6 cases per 100,000 person-years for men; 74.8 cases per 100,000
person-years for women (Ferlay et al., 2008). Over 80% of HCC cases occur in
developing countries such as sub-Saharan Africa, Southeast Asia, and East Asia
(including Mongolia). In contrast, the incidence of HCC is much lower in developed
countries such as North America with 6.8 cases per 100,000 person-years for men; 2.2
cases per 100,000 person-years for women.


2

In Singapore, HCC is the fourth most frequently occurring cancer in men with
an overall incidence of 18.9 per 100 000 person-year (Singapore Cancer Society). The
incidence is highest in the Chinese population as compared to the other ethnic groups
(Yuen et al., 2009).
1.2.2

Aetiological factors of HCC
Significant differences in HCC incidence across different countries reflect

regional differences in the prevalence of specific aetiological factors. The most
prominent factors associated with an increased risk of HCC include chronic hepatitis
B and C viral infection, aflatoxin B1 contamination and severe alcohol abuse. Other
aetiological factors which occurred at a lower frequency include demographic
characteristics (gender and age), lifestyle (smoking) and clinical factors (metabolic
abnormalities, diabetes, and obesity).
Overall, viral infection has been indexed as the major risk factor because more
than 80% of HCC in humans are attributable to infection with either hepatitis B or C
virus or both (Parkin et al., 2006). Nevertheless, it should be highlighted that the risk

of HCC increases in the event of multiple risk factors.
1.2.2.1 Hepatitis B virus (HBV)
Hepatitis B virus (HBV) is a 3.2kb partially double stranded DNA virus that
can elicit an acute and chronic inflammatory response in the liver. It infects
approximately 2 billion individuals globally and causes an estimated 320 000 deaths
annually (Lavanchy, 2004). As such, HBV-associated HCC is a disease of global
importance and is ranked the top 5 most frequent cancers worldwide (Marrero, 2006).
In Singapore, one third of HCC patients are HBV positive (Yuen et al., 1999).


3

An array of processes is involved in HBV-induced hepatocarcinogenesis.
These include host-viral interactions (Block et al., 2003; Nowak et al., 1996; Wieland
et al., 2004; Rehermann et al., 2004), sustained cycles of necrosis-inflammationregeneration (Block et al., 2003; Lok et al., 2001), viral-endoplasmic reticulum
interactions (Kojima, 1982), viral integration into the host genome (Brechot, 2004)
and targeted activation of oncogenic pathways by viral proteins (Matsubara et al.,
1990).
The 16.5 kDa HBV X protein (HBX) is suspected to be an important
oncogenic factor in hepatocarcinogenesis (David et al., 2006). In vivo studies have
shown that it can trans-activate a large number of promoters related to inflammation
and cell proliferation through protein-protein binding. This mechanism allows HBX
to undergo favourable alteration in the cellular environment for further viral
replication (Tang et al., 2006). In the infected liver host cells, HBX appears to induce
variety of responses such as genotoxic stress response, transcription modulation,
protein degradation, cellular signalling pathways, cell cycle checkpoints and apoptosis
(Murakami, 2001). HBX has since been proposed to be strongly related to the
progression of HCC however its exact role in malignant transformation has yet to be
fully elucidated.
1.2.2.2 Hepatitis C virus (HCV)

Hepatitis C virus (HCV) is a 9.6kb non-cytopathic virus of the flaviviridae
family. It possesses a positive single-stranded RNA genome encoding non structural
proteins (NS2, NS3, NS4A, NS5A and NS5B) and structural proteins (core, E1, E2).
HCV has been implicated to be responsible for an estimate of 170 million chronic
infections worldwide. Further, it is a major risk factor for the development of HCC


4

(McGlynn and London, 2005), predominantly in developed nations such as Europe,
United States and Japan (Bosch et al., 1999)
Analogous to HBV infection, HCV induces liver inflammation followed by a
continuous cycle of hepatocyte cell death and regeneration. However, unlike HBV, its
genome is RNA-based, thus it is unable to integrate to host genome to induce
hepatocarcinogenesis (Rehermann et al., 2005). Instead, HCV has been shown to
possess the   propensity   to   evade   the   host’s   immune response to promote
tumourigenesis (Gale and Foy, 2005). The HCV’s  RNA and core proteins are found
to impair dendritic functions which are important for T-cell activation (Pachiadakis et
al., 2005) and evade immune-mediated cell death by interacting with interferon-α  and  
tumour necrosis factor-α  (Melén et al., 2004; Gale and Foy, 2005). Additional roles of
the HCV core proteins in the pathogenesis of HCC include inhibition of apoptosis
(Kamegaya et al., 2005), interference with cell signalling pathways to modulate cell
proliferation (Hino O. et al., 2002; Macdonalds et al., 2003) and modulate p53
transcription thereby affecting the p53-regulated pathways (Majumder et al., 2001). In
mouse models, HCV core proteins have been shown to induce hepatic steatosis as
well as reactive oxygen species (ROS) and oxidative stress (Moriya et al., 1998;
2001). These data collectively suggest that viral proteins play a direct role in inducing
hepatocarcinogenesis.
1.2.2.3 Aflatoxin B1 (AFB1)
Aflatoxin B1 (AFB1) is a mycotoxin that is produced by the Aspergillus fungi.

Epidemiological studies have shown that AFB1 consumption increases HCC risk by
approximately four-fold. Further, it has been observed that areas of high HCC
incidence and high AFB1 intake correspond to areas where HBV is endemic


5

(Groopman et al., 1996; Qian et al., 1994). Previous studies have also shown that the
combination of AFB1 and HBV is likely to increase the risk of HCC by 60-fold (ElSerag and Ruldolph, 2007; McGlynn and London, 2005).
Once ingested, AFB1 is metabolized to form a pro-mutagenic DNA adduct
which results in a specific point mutation from guanine to thymine at codon 249
(AGG to AGT) in the tumour suppressor, p53 (Moradpour and Blum, 2005; Wild and
Montesano, 2009). To some extent, the loss of p53 function via mutation is believed
to induce loss of cell growth control and eventually promote hepatocarcinogenesis.
Further, this loss of p53 function has also been reported to assist the mutational
activation of oncogenes such as human Harvey-ras proto-oncogene (HRAS) (Riley et
al., 1997). Together, the mutational actions of AFB1 may contribute to the
development of HCC.
1.2.2.4 Alcohol
Chronic alcohol intake has long been recognized as an important HCC risk
factor. Epidemiology data suggests that alcohol has been found to be a more
prominent risk factor for HCC in low incidence areas than in high incidence areas.
This may be due to the lower mean alcohol consumption and/or the dominant effect of
HBV infection in the high-risk population (El-Serag and Ruldolph, 2007; Bosch et al.,
2005; McGlynn and London, 2005).
Alcohol-induced hepatocarcinogenesis

has

been

associated with

the

production of inflammatory cytokines through monocytic activation (McClain et al.,
2002). Consequently, Kupffer cells are activated with the eventual release of
chemokines and cytokines, leading to hepatocytes necrosis. Alcohol also damages the
liver through an oxidative-stress mechanism. First, it promotes the development of


6

fibrosis and cirrhosis and creates a conducive HCC microenvironment (Campbell et
al., 2005). Second, oxidative stress may decrease STAT1-directed  activation  of  IFNγ  
with consequent hepatocyte damage and constant tissue destruction ultimately leads to
hepatocarcinogenesis (Osna et al., 2005).
1.2.3 Diagnosis and treatment
HCC patients are commonly diagnosed at very late stages due to the
asymptomatic features during the course of neoplastic development and the lack of
reliable biomarkers. Thus, this disease has a very poor prognosis with more than 50%
of the patients dying within 1 year and less than 6% with a 5 year survival rate
(Hoofnagle, 2004).
1.2.3.1 Screening tests
The most widely used marker today for HCC is alpha-fetoprotein (AFP). In
recent years, clinical research has discovered a fucosylated AFP (AFP-L3) to be a
new tumour marker with sensitivity of about 56% and specificity of >95% (Li et al.,
2001). More importantly, it has been suggested to be a specific indicator for poorlydifferentiated and unfavourable diagnosis (Tateishi et al., 2006).
Another established serum biomarker for HCC is an abnormal prothrombin,
des-gamma-carboxy prothrombin (DCP) (Liebman, 1989; Aoyagi et al., 1996). DCP

has been reported to be the most significant predisposing factor for the development
of portal vein invasion which is an indicator of end-stage liver disease (Koike et al.,
2001). Hence, it is more commonly used as a diagnostic marker rather than for
surveillance. Together, DCP may complement AFP or AFP-L3 for HCC diagnosis
purposes. Other promising biomarkers for HCC include Golgi-protein 73 (Kladney et


7

al., 2000; Marrero et al., 2005), glypican-3 (Nakatsura et al., 2003; Capurro et al.,
2003), hepatocyte-growth factor (Yamagamim et al., 2002), insulin growth factor 1
(Mazziotti et al., 2002), vascular endothelial growth factor (Poon et al., 2004;
Schoenleber et al., 2009) and transforming growth  factor  β-1 (Song et al., 2002; Yao
et al., 2007).
Other than biomarkers, imaging studies play a crucial role in the diagnosis of
HCC. Imaging techniques most commonly used for the diagnosis of HCC include
ultrasound (US), computed tomography (CT), magnetic resonance imaging (MRI),
and angiography. While ultrasonography is widely accepted for HCC surveillance,
spiral computed tomography (CT) or dynamic magnetic resonance imaging are the
required imaging techniques for diagnostic confirmation and intrahepatic tumor
staging. Currently, with the development of imaging modalities, invasive biopsy is
infrequently required prior to treatment, and the diagnosis of HCC is strongly
dependent on hemodynamic features (arterial hypervascularity and washout in the
venous phase) on dynamic imaging.
1.2.3.2 Treatment
To date, the only proven cures for HCC are surgical resection or liver
transplantation. However, for liver transplantation, the scarcity of donor liver remains
a universal problem. In addition, a major concern for resected HCC patients is the
possibility of recurrence that can occur in about 75% of the resected patients (Llovet
and Bruix, 1999).

In cases when HCC lesions are unresectable, an alternative option is nonsurgical treatment. For example, radiofrequency ablation (RFA) uses a needle with an
electrode placed percutaneously into targeted tumour in the presence of ultrasound


8

scanning. The local heat generated then melts the tumour tissues (Gough-Palmer and
Gedroyc, 2008). Other treatments include conventional or molecular targeted
chemotherapy and radiotherapy.
1.2.4

Challenges in understanding HCC
Hepatocarcinogenesis is a multistep process involving diverse aetiologies,

ranging from metabolic disorders to hepatoxins to viruses. As documented by
numerous independent HCC studies, the genetic aberrations in HCCs are
heterogenous whereby distinct molecular players but related genetic pathways are
affected (Thorgeirsson et al., 2006). Nevertheless, the exact underlying mechanism
of hepatocarcinogenesis remains elusive. To aggravate the situation, early
hepatocellular carcinoma is characteristically silent and slow growing with few
symptoms until late in disease. The lack of clinically validated biomarkers and
clinical identification of hepatocellular carcinoma at advanced stages present
difficulties for diagnosis and treatment. While advances in computed tomography and
magnetic resonance imaging have markedly increased the sensitivity and specificity
of testing, they are still flawed with a relatively high false-positive rate. Several
surgical and nonsurgical therapies have been developed, but used with varying
degrees of success.
Hepatocellular carcinoma in general is a disease of multifactorial etiology and
confers many management challenges. It is therefore imperative to identify and
characterize new molecular mechanisms associated to HCC. This allows a better

understanding of the disease for the identification of effective diagnostic and
prognostic markers.


9

1.3

Acute promyelocytic leukemia (APL)
Acute promyelocytic leukemia (APL), designated as AML-M3 (acute myeloid

leukemia) in FAB (French American and British) classification (Bennett et al., 1976),
is a disease characterized by the chromosomal translocation involving retinoic acid
receptor-alpha   (RARα)   gene   on   chromosome   17   with   the   promyelocytic   leukemia  
gene (PML) on chromosome 15. It represents about 10% of all AML in adults with a
lower incidence reported in children (Stone et al., 1990; Chan et al., 1981).
1.3.1

PML-RARα
The ramification of chromosomal translocation mentioned earlier is the

generation of a fusion oncoprotein PML-RARα. Since its discovery in the 1990s, the
transformation role of PML-RARα  in  APL  has  been  well studied. One direct evidence
shown in two studies is the generation of an APL-like disease when this fusion protein
was targeted to the myelocytic compartment in transgenic mice (He et al., 1997;
Grisolano et al., 1997). Studies have elucidated the role of PML-RARα  as  a  dominant  
negative transcription repressor. PML-RARα  has  been  proposed  to  recruit  the  N-CoR
containing chromatin-remodeling complex to their promoters which eventually
repress the target genes for differentiation of granulocytes (Lin et al., 1998; Grignani
et al., 1998). It also recruits methylating enzymes and hypermethylates promoters of

retionic acid (RA) target genes resulting in transcriptional repression. The overall
outcome of this PML-RARα-induced transcriptional inhibition is differentiation arrest
of APL cells.