University of Southern California Dissertations and Theses

Hepatitis B virus X protein regulation of β-catenin and NANOG and co-regulatory role with YAP1 in HCC malignancy

(USC Thesis Other)

Hepatitis B virus X protein regulation of β-catenin and NANOG and co-regulatory role with YAP1 in HCC malignancy

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content Hepatitis B Virus X protein regulation of β-Catenin and Nanog
and co-regulatory role with Yap1 in HCC malignancy

By

Chad Isamu Nakagawa

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

AUGUST 2015

1

Acknowledgements

I would like to thank Dr. Keigo Machida for accepting me into his lab and providing me
the opportunity to work and grow as a graduate student. I would also like to thank Dr. Hidekazu
Tsukamoto and Dr. James Ou for accepting to be on my committee and taking the time from
their busy schedules to help shape me into a better researcher. I would also like to thank Dr. Ou
and his lab for providing the mouse HBV liver tissue samples and plasmids for use in this study.
From the Machida lab I would like to extend my gratitude to Ahmed, Padmini, Joe, and Chia-lin
for helping with experiments and providing stimulating conversation. Also Dr. Hifzur Siddique,
thank you for helping with the overwhelming work load we received, for giving me the
confidence to move forward in my career, and for being a good friend. For that I give all my
gratitude.
I would also like to thank my family, whose endless support got me through even the
hardest of times. To my girlfriend Laura, who pushed me through when I felt like giving up. And
to all of my friends who always believed in me. Thank you.

2

Table of Content

II. Abbreviations 3
II. Abstract 4
1. Introduction 5 - 18
1.1. Hepatocellular Carcinoma 5
1.2. HCC Detection and Treatment 8
1.3. Hepatitis B Virus and HBx Role in Carcinogenesis 9
1.4. The WNT/β-catenin Pathway and its Relationship to
HCC
12
1.5. The YAP Pathway and its Interaction with β-catenin 14
1.6. NANOG Regulation and Role in Stemness 16
1.7. Rationale of the Study 18
1.8. Hypothesis 18
2. Materials and Methods 19 - 27
2.1. Western blot and immunohistochemistry 19
2.2. Quantification of immunohistochemistry staining 21
2.3. Mouse strains 21
2.4. Cell lines and passaging 21
2.5. Plasmids, lentivirus, and retrovirus 22
2.6. Luciferase assay 23
2.7. Colony formation assay 24
2.8. Hepatoblast groups and intrasplenic injection into wild-
type mice
25
2.9. Statistical analysis 27
3. Results 28 - 30
3.1. HBx plays a role in the regulation of β-catenin and
Nanog levels
28
3.2. Liver tissue shows more hepatocarcinogenesis in HBx
positive group
28
3.3. β-catenin and Nanog localize in the nuclei in HBx
positive tissue samples
29
3.4. BIRC5 plays an important role in late stage
tumorigenesis
29
3.5. β-catenin + Nanog show the highest transformative
ability in p53 +/+ hepatoblasts
30
4. Figures 31 - 38
5. Discussion 39 - 41
6. References 42 - 50

3

I. Abbreviations
AASLD American Association for the Study of Liver Disease
ACS American Cancer Society
BCLC Barcelona Clinic Liver Cancer
CBP CREB Binding Protein
cccDNA covalently closed circular DNA
CCRK Cell Cycle Related Kinase
CDK Cyclin-dependent Kinase
CLIP Cancer of the Liver Italian Program
CTL Cytotoxic T Lymphocyte
CTP Child-Turcotte-Pugh
CUPI Chinese University Prognostic Index
DAPI 4', 6-diamidino-2-phenylindole
DEN Diethylnitrosamine
DMEM Dulbecco's Modified Eagle's Medium
DNA Deoxyribonucleic Acid
DNMT DNA methyltransferase
DPBS Dulbecco's Phosphate Buffered Saline
EASL
EORTC
European Association for the Study of the Liver
European Organisation for Research and Treatment of Cancer
ECM Extracellular Matrix
EGF Embryonic Growth Factor
EtOH Ethanol
FBS Fetal Bovine Syrum
HAT Histone Acetyl Transferase
HBcAg Hepatitis B Core Antigen
HBeAg Hepatitis B e Antigen
HBsAg Hepatitis B Surface Antigen
HBV Hepatitis B Virus
HBx Hepatitis B Virus X Protein
HCA Hepatocellular Adenoma
HCC Hepatocellular Carcinoma
HRP Horseradish Peroxidase
IGF Insulin-like Growth Factor
JIS Japanese Integrated Staging
KIX Kinase-inducible Domain Interacting
MMP Matrix Metallopeptidase
NFE2L2 Nuclear factor (erythroid-derived 2)-like 2
PBS Phosphate Buffered Saline
PBST Phosphate Buffered Saline Tween 20
PVT Portal Vein Thrombosis
TBST Tris Buffered Saline Tween 20
YAP Yes Associated Protein
4

II. Abstract

Hepatocellular carcinoma (HCC) is the fifth most common type of cancer. The mortality
rate continues to rise every year. Hepatitis B virus (HBV) is a major risk factor associated with
the development of HCC, due to its ability to regulate cell stemness and proliferation factors, and
cause liver cirrhosis and constant inflammation. As sorafenib treatment is not curative, tumor-
initiating stem-like cells (TICs) contributes to the low survival rates of HCC patients. Gaining a
better understanding of the mechanisms that are involved in the activation of TIC regulators will
provide rationale for targeted treatments for those HCC patients. Here we show that HBV X
Protein (HBx) can upregulate both β-catenin and NANOG pluripotency transcription factor
expression, where they then translocate into the nucleus upon activation. Coexpression of
constitutively-active β-catenin or YAP1 with NANOG transactivate Survivin (Birc5) promoter in
a liver progenitor cell line. Coexpression of constitutively active β-catenin with NANOG
promotes self-renewal ability in hepatoblasts. Expression of HBx in the presence or absence of
NANOG expression in hepatoblasts promotes tumor development in immune-competent mice
transplanted with hepatoblasts. Non-invasive live animal imaging using ultrasound and microCT
method confirmed tumor development. Taken together, coexpression of constitutively active β-
catenin with NANOG promotes self-renewal ability and HBx protein plays a key role for
oncogenic transformation of hepatic progenitor cells.

5

1. Introduction
1.1 Hepatocellular Carcinoma
Amongst the different types of cancer, hepatocellular carcinoma (HCC) is the fifth most
common (Caldwell and Park, 2009), and accounts for more than 80% of liver cancer cases
(Farazi and DePinho, 2006). Common risk factors associated with the development of HCC are
obesity, chronic hepatitis infection, cirrhosis, alcoholism, diabetes, and intake of aflatoxin B1
(ACS, 2015). However, despite the prevalence of HCC it is still the third most common cause of
cancer related deaths, with an 18% five year survival rate from time of diagnosis, an overall 15%
increase over the last 35 years (Table 1) (ACS, 2015). The amount of new cases per year has
doubled over the last 20 years and risen to about 35,660, with men being three times more at risk
than women of developing HCC (ACS, 2015). The amount of expected deaths has also increased
to about 24,550 per year, giving HCC one of the highest increases in mortality over the past 25
years (40 – 60% increase) (ACS, 2015; Llovet et al., 2015). One of the more common treatment
methods for early stage HCC is liver resection or liver transplant (EASL-EORTC, 2012).
However in most cases, HCC isn’t diagnosed until it has progressed into late stages, and in such
Table 1. Trends in 5 year survival rates for HCC between the years 2003 - 2009. Data
adapted from the American Cancer Society (2015).
6

case that there is not a readily available liver to be transplanted, drug treatment with sorafenib,
transcatheter arterial chemoemobiolization, or other treatments are used (Colagrande et al. 2015).
The downside of these treatments, however, is that they are not curative but rather prolong the
life expectancy of patients in this circumstance (Llovet et al., 2008).
In a clinical setting, an important piece of information is the HCC stage. However, there
are 7 different methods of tumor staging, each taking into account different factors to stage a
tumor. These 7 methods are TNM, Okuda, BCLC, CLIP, JIS, CUPI, and French staging systems
(Subramaniam et al. 2013). The two most common of those are TNM and BCLC staging. TNM
is not a liver cancer specific grading system, and employs the use of 3 main factors to stage a
tumor (Subramaniam et al., 2013). Those three factors are the primary tumor features (T), the
presence of nodal involvement (N), and the presence of distant metastasis (M) (AJCC, 2010).
However recent research has proposed that the T portion of TNM staging should be changed to
focus on vascular invasion, as well as tumor number and size (Poon and Fan, 2003; Vauthey et
al., 2002). The main issue with the TNM scoring system is that it offers little insight into
potential treatment options available to patients with late stage HCC, as TNM is most applicable
for use on samples from patients who have undergone curative surgery and prediction of survival
rate is needed (Subramanim et al, 2013). A more recent form of HCC staging, accepted by both
the AASLD and EASL, is the Barcelona Clinic Liver Cancer (BCLC) staging (Bruix et al, 2011;
EASL-EORTC, 2012). The important factors taken into consideration by BCLC staging is size
of primary tumor, presence of metastasis, PVT, CTP, and bilirubin levels (Llovet et al., 1999).
Stages for BCLC are stage 0, A, B, C, and D, with the added benefit of suggested treatment
options depending on the stage, ranging from curative resection at stage A to supportive
treatment at end stage D (Subramanim et al., 2013). One of the downfalls of using BCLC staging
7

is that the intermediate HCC, stage B, is not well defined and covers a wide array of factors (Di
Costanzo and Tortora, 2015). A large portion of HCC patients will fall under this category,
however there is much gray area between stage B and C, and some patients who may be staged
at B will not be eligible for stage B treatment (Subramanim et al., 2013). This can become
problematic for physicians trying to outline a treatment schedule for patients with BCLC stage B
HCC. However, despite the heterogeneity of BCLC stage B, the BCLC staging system is still
currently one of the most widely used staging systems (Colagrande et al., 2015). With the recent
influx of genomic analysis on HCC, an up and coming method of staging via gene signatures
seems likely to be a better prognostic tool than those currently available (Hoshida et al., 2010; Li
and Mao, 2013; Nault et al., 2013). While the effectiveness of scoring via the use of gene
signatures has yet to be seen, insights into important driver genes for HCC development are
becoming more prevalent.

Figure 1. Key genes of each pathway that plays a roles in the development of hepatocellular carcinoma. Adapted from
Shibata and Aburatani (2014).
HCC
WNT
Signaling
AXIN1
CTNNB1
Genomic
Instability
TP53
Viral Factors HBV/HBx
Chromatin
Remodeling
ARID1A
Oxidative
Stress
NFE2L2
8

The recent use of genomic analysis has revealed that HCC generally consists of 30 – 40
gene mutations (Llovet et al., 2015). However, among those mutated genes only about 15 – 20%
of those are considered to be driver gene mutations. Some of the more commonly mutated genes
in HCC are TP53, β-catenin, ARID1A, and AXIN1 (Guichard et al., 2012; Kan et al., 2013;
Scholer-Dahirel and Schlabach, 2011; Villanueva and Llovet, 2011; Villanueva and Llovet,
2014). TP53, β-catenin, and AXIN1 are important mutations that can affect cell cycle regulation
and differentiation (Figure 1). Mutations in these genes give cells a more dysplastic and de-
diffferentiated phenotype. ARID1A is an important regulator of chromatin remodeling, and
mutations in this gene prevent the DNA from correctly organizing into chromatin structures,
allowing for uncontrolled expression of genes (Llovet et al., 2015). Some of the affected genes,
like NFE2L2, are important in the control of oxidative stress, and when they become
dysregulated it allows for the accumulation of DNA damage and potentially more mutations
(Ahn et al., 2014). While these mutations are common, they do not always occur simultaneously,
as β-catenin and TP53 mutations are generally seen to be mutually exclusive, with a low
probability of them occurring in the same HCC (Tornesello et al., 2013).
1.2 HCC Detection and Treatment
While there are screening methods for HCC, ultrasound and blood tests, generally these
tests do not provide adequate results in the early stages of HCC development (ACS, 2015).
However, regular screenings are often seen in patients who have a high risk for the development
of the disease (e.g. those patients infected with HBV and simultaneous cirrhosis) (ACS, 2015).
Often times, at the point of HCC diagnosis the patients are at the intermediate to late stage, and
few approved treatment options exist (Colagrande et al., 2015). Patients who are diagnosed at
early stages of HCC generally have more options for curative treatment available like liver
9

resection, transplant, or ablation (Ye et al., 2010). Even amongst patients who are eligible to
receive a liver transplant, HBV positivity can increase the risk for HCC development in the new
liver, as circulating tumor stem cells in the blood eventually lodge in the new liver and form new
tumor masses (Hemming et al., 2001). Late stage HCC patients are left with a very limited
selection of treatment methods, usually chemotherapy, radiation, or clinical trials. However,
chemotherapy treatments like sorafenib are not urually a curative treatment option and only
meant to prolong the lifespan of late stage HCC patients < 1 year (Colagrande et al., 2015). The
lack of an effective HCC detection method and treatment options translate into the low 18% 5-
year survival rate observed.
1.3 Hepatitis B Virus and HBx Role in Carcinogenesis
Hepatitis B virus (HBV) is a hepadnaviridae that uses reverse transcription during
replication, despite not being a retrovirus (Ng and Lee, 2011). During infection, HBV binds to
hepatocytes via NTCP and is then endocytosed (Yan et al., 2012). The virus is uncoated,
releasing the viral core into the cytoplasm, followed by the transport of the viral genome into the
nucleus (Beck and Nassal, 2007). Using its viral polymerase, the 3.2-kb circular, partially
double-stranded DNA becomes a covalently closed circular DNA (cccDNA) (Ng and Lee, 2011).
Once this occurs the virus is free to produce the necessary components required to produce more
virion particles, as well as recirculate components back into the nucleus to further the production
of HBV (Beck and Nassal, 2007). The four open reading frames within the genome are the viral
envelope (S), core proteins (C), viral polymerase (P), and the X protein (X) (Figure 2) (Seeger
and Mason, 2000).
10

Figure 2. Diagram of the Hepatitis B Virus genome, with its four overlapping open reading frames HBx (X), surface
antigen (S), core protein (C), viral polymerase (P). Adapted from Ng and Lee (2011)
In endemic areas of Asia, the amount of people infected with HBV is over 350 million
(Lee et al., 2013). Of those infected, it is estimated that cirrhosis will occur in 30% of people
depending on the extent of HBV infection, and that HCC will develop in 10 – 60% depending on
extent of infection and cirrhosis (Lin and Kao, 2012). One of the methods for determining the
severity of HBV infection is to test the blood for hepatitis B virus surface antigen (HBsAg) (Lok
and McMahon, 2007). In highly endemic areas, transference of HBV often occurs via perinatal
transmission, with a 90% risk of infants having chronic HBV infections by their first year (Chen,
1993). Other methods of transference are, but not limited to, blood transfusions with
contaminated blood, sharing needles with an HBV infected individual, and having unprotected
sex with an infected individual (Buddeberg et al., 2008; Fairley and Read, 2012; Hughes, 2000).
X
P
S
C
Partially
dsDNA
5’
3’
5’ 3’
11

Generally the incubation time between first exposure and development of HBV is about 75 days
but can vary from as little as 30 days to as much as 180 days (WHO, 2015). In the first 20 years
of a chronic HBV infection, the replication levels are high, with patients showing high serum
HBV DNA, HBsAg, and HBeAg (Chen, 1993). Interestingly, the immune response during this
time is low. After this point however, HBV enters a second phase in which it slows replication
and the immune system starts to produce T cells that recognize HBV antigens (Chen, 1993). The
cytotoxic T cells recognize HBcAg and HBeAg and produce anti-viral cytokines in response,
which leads to hepatic necrosis and can eventually lead to hepatic cirrhosis (Iannacone et al.,
2007). The progression of cirrhosis usually occurs at about 2% per year, paralleling a slow
decline in liver function, until the HCC develops (Chen, 1993). Most of the patients who develop
HCC this way are HBsAg positive, due to the integration of the HBV genome during the period
of infection (Beasley et al., 1982). The hepatocytes that have had HBV genome integration occur
are usually not recognized by the CTL immune response as they only produce HBsAg (Chen,
1993). One reason for such prevalence of HBsAg positive cirrhotic patients developing HCC is
the integration of one of the four HBV genes into the genome, that gene being the HBx gene.
Of the four genes within the HBV genome, HBx has been suggested to play a key role in
the development of HCC through its involvement in cellular replication and stemness (Kekule et
al., 1993; Klein and Schneider, 1997; Murakami et al., 2005). HBx is a 17kDa, 154-amino acid
viral protein that does have a role in viral replication; however, this role is not well defined (Ng
and Lee, 2011). HBV positive patients with HCC have been shown to have higher levels of HBx
than HBV positive patients without HCC (Paterlini et al., 1995). HBx has been shown to not
bind directly to DNA, but instead interacts with transcription factors in the nucleus and kinases
in the cytoplasm to regulate β-catenin, GSK3β, Src-Kinase, p53, c-Jun, NF-kβ, BIRC5, and
12

others (Cha et al., 2004; Ding et al., 2005; Elmore et al., 1997; Kim et al., 2008; Park et al.,
2013; Sze et al., 2013). HBx has also been shown to increase expression of DNMTs, as well as
recruit DNMT1 and DNMT3A to regulate IGF and CDH1 (Tian et al., 2013). It has also been
shown to cause hypomethylation of tumor-suppressor genes; however, this mechanism is not
well understood (Tian et al., 2013). HBx is also one of the HBV genes to become integrated into
the host genome. Often times HBx integration leads to a C-terminus truncation mutant of HBx,
which has been shown to lead to a more aggressive HCC via the upregulation of c-Jun and
MMP10 expression (Sze et al., 2013). HBx has also been thought to be able to bind directly to
the YAP promoter region via a CREB element in order to induce YAP1 expression and promote
tumor growth (Zhang et al., 2012).
1.4 The WNT/β-catenin Pathway and its Relationship to HCC
The WNT/β-catenin pathway is an important component in the maintenance of stemness
in stem cells (Tarafdar et al., 2013). Under normal circumstances, β-catenin is held in a scaffold
of APC and AXIN, allowing GSK3β to phosphorylate 4 Ser/Thr sites (Ser33, Ser37, Thr41,
Ser45) on β-catenin (Cha et al., 2004; Ding et al., 2005), following a pre-phosphorylation event
by CK1 at a Ser/Thr site upstream of S33 (Metcalfe and Bienz, 2011). The phosphorylaton of β-
catenin allows for its recognition by β-TrCP, leading to β-catenin’s ubiquination and subsequent
degradation (Azzolin et al., 2014). During WNT signaling however, extracellular WNT will bind
to the frizzled receptor, leading to its interaction with Dishevelled (DVL) and the subsequent
interaction with AXIN, and its dissociation from the β-catenin destruction complex (Cha et al.,
2004). In the absence of AXIN, there is no longer a scaffold conformation to hold β-catenin next
to GSK3β, allowing β-catenin to remain in its stabilized unphosphorylated form, and allowing it
to accumulate in the cytoplasm and passively translocate into the nucleus (Ding et al., 2005).
13

Once inside the nucleus, β-catenin interacts with various transcription factors due to its lack of a
DNA binding domain. β-catenin can cause an increase in expression of factors such as c-Myc
and Cyclin D1 to promote cellular proliferation (He et al., 1998; Shtutman et al., 1999; Tetsu and
McCormick, 1999). It has also been suggested that β-catenin can interact with YAP1and TBX5
in order to promote the expression of BIRC5, a well known anti-apoptotic factor (Rosenbluh et
al., 2014). Other studies have shown that β-catenin interaction with YAP1 can lead to the
formation of hepatoblastomas in young children (Tao et al., 2014).
The canonical WNT pathway however, has two separate arms which lead to different cell
fates; the CBP pathway that generally maintains proliferation and stemness, and the p300
pathway that leads cells to differentiate (Miyabayashi et al., 2007). Both CBP and p300 are
homologous proteins that share similar zinc finger regions, CREB binding domains,
bromodomains, and glutamine-rich regions (Goodman and Smolik, 2000). While CBP plays a
key role in maintenance of stemness via its KIX domain and HAT activity to increase OCT4,
SOX2, and NANOG expression, p300 plays a role in leading to a more differentiated phenotype
(Feng et al., 2014). p300 uses the KIX domain to competitively inhibit CBP from binding to the
NANOG promoter region, leading to a decreased NANOG expression level and subsequent
decrease in a stemness phenotype (Feng et al., 2014). It has also been shown that p300 can be
phosphorylated at Ser89 to increase its binding affinity to β-catenin, preventing its interaction
with the CBP pathway and allowing for a more differentiated cell phenotype (Miyabayashi et al.,
2007). One proposed mechanism for the more differentiated phenotype is through β-catenin/p300
interaction with the BIRC5 promoter region to inhibit its expression (Ma et al., 2005)
In patients with hepatocellular carcinoma, β-catenin can become dysregulated for a
variety of reasons. One of the main reasons for dysregulation of β-catenin is activation mutations
14

in the phosphorlation regions at Ser/Thr residues (Provost et al., 2003). In HCC, β-catenin is
found to be one of the top mutated genes, with 15.9% of HCC showing β-catenin mutations (Kan
et al., 2013). It is known that patients who are infected with HBV have an increased risk of liver
cancer, and this is thought to be largely due to the effects of HBx (Ng and Lee, 2011). HBx has
been shown to stabilize β-catenin by increasing Src-Kinase activity, leading to the
phosphorylation of GSK3β at Ser9 and its subsequent inactivation (Cha et al., 2004). By this
way, both a significantly increased level of β-catenin can be seen with a coinciding p53
mutation. It has also been hypothesized that the increased Src-Kinase activity caused by HBx can
lead to a more metastatic phenotype, through a Src-Kinase dependent disruption of adheren
junctions (Feng et al., 2009). Hepatocellular adenomas are a rare benign liver mass that has been
linked to increased estrogen levels caused by female contraceptive use (Marquardt and
Thorgeirsson, 2014). Early developing HCAs generally do not contain β-catenin mutations,
however once they acquire an activation mutation in β-catenin (usually in Exon 3) they start to
show more aggressive, malignant tendencies and can become HCC (Pilati et al., 2014). This
gives insight into the potential transformative mechanism by which β-catenin can elicit a
malignant response in normally benign tumor tissue.
1.5 The YAP Pathway and its Interaction with β-catenin
YAP and its transcriptional co-activator TAZ are important components of the Hippo
pathway, which regulates cell proliferation and apoptosis (Moroishi et al., 2015). Two key
regulators of this pathway are LATS1 and LATS2, which phosphorylate both YAP and TAZ,
causing them to become sequestered in the cytoplasm and unable to affect their transcriptional
activity (Moroishi et al., 2015). Most of the regulatory elements that initiate the hippo pathway
and downstream YAP/TAZ come from the ECM (Mo et al., 2014). However, when the Hippo

15

pathway is activated, YAP/TAZ remain hypophosphorylated and move from the cytoplasm to
the nucleus to interact with TEAD transcription factors to induce the expression of both cell
proliferation and anti-apoptosis genes (Mo et al., 2014). During tumor development, niches are
generated that lead to an increase in matrix stiffness and elevated YAP/TAZ activity, increasing
the proliferative and anti-apoptotic ability of tumor cells, as well as affecting cell-cell
interactions (Calvo et al., 2013). This has been shown in vitro by cells grown in high ECM
stiffness showing increased activity of YAP/TAZ, as well as increased nuclear localization, when
compared to cells grown in low ECM stiffness (Aragona et al., 2013). Regulation of YAP/TAZ
can also occur via other pathways such as the WNT pathway. One of the components of the β-
catenin destruction complex, APC, can also lead to TAZ/YAP being sequestered in the
cytoplasm (Moroishi et al., 2015). When the WNT pathway is stimulated, the separation of the
components of the destruction complex allow for β-catenin to become stabilized and translocate
into the nucleus; the loss of APC also prevents YAP/TAZ from being phosphorylated, allowing
Figure 3. Diagram of YAP1 interaction with β-Catenin to increase BIRC5 expression. Adapted from Rosenbluh
et al. (2012).
16

it to also translocate into the nucleus (Azzolin et al., 2014). It has been shown that there may be
some crosstalk between the Hippo pathway and the WNT pathway, as seen by YAP/TAZ
interaction with β-catenin to induce expression of SOX2 and BIRC5 (Figure 3) (Rosenbluh et al.,
2012). However, conflicting evidence has shown that YAP/TAZ can recruit β-TrCP to the
destruction complex, leading to degradation of β-catenin in the absence of WNT signaling, as
well as sequester β-catenin in the cytoplasm to prevent its translocation into the nucleus (Azzolin
et al., 2014; Imajo et al., 2012). While not thoroughly elucidated, it has been suggested that
YAP/TAZ act as negative mediators in the cytoplasm and positive mediators in the nucleus
(Azzolin et al., 2014).
1.6 NANOG Regulation and Role in Stemness
NANOG, along with OCT4 and SOX2, is one of the most transcription factors associated
with the maintenance of cellular stemness (Wang et al., 2013). The NANOG protein is 305
amino acids long, containing an N-terminal, homeobox domain, and C-terminal region (Mitsui et
al., 2003). The N-terminus is the site for transcriptional activity, and as such is regulated via
post-transcriptional modifications like phosphorylation (Oh et al, 2005). The homeobox domain
is the region by which NANOG can bind to DNA (Do et al., 2007). The C-terminus contains 2
transactivation subdomains (Oh et al., 2005). NANOG uses nuclear localization sequences in the
C- and N-terminal regions to move into the nucleus, and uses the nuclear export signal in the
homeobox region to move back into the cytoplasm (Do et al., 2007; Park et al., 2012). There are
12 different NANOG genes, one embryonic gene and 11 psuedogenes (Booth and Holland,
2004). Only the embryonic NANOG gene and the NANOGP8 pseudogene encode for a functional
NANOG protein (Wang et al., 2013). The only difference between the NANOG encoded and
NANOGP8 encoded protein is a switch from Gln253 to His253, respectively (Ibrahim et al.,
17

2012). While it was originally thought that NANOG played a role in embryogenesis and
NANOGP8 played a role in tumorigenesis, recent studies have found that, in colorectal cancer,
the increased levels of NANOG found in tumor cells were derived from both NANOG and
NANOGP8 (Ibrahim et al, 2012; Wang et al., 2013). In various forms of cancer, NANOG has
been shown to be an important factor in cell proliferation by upregulating Cyclin D1 and CDK1,
tumor cell invasion/metastasis by inhibiting E-cadherin and FOXO1 expression, and
chemoresistance by upregulating ABCB1 (Wang et al., 2013).
Two important regulators of NANOG expression are STAT3 and p53 (Wang et al, 2013).
STAT3 has two ways in which it interacts with NANOG to regulate stemness. STAT3 can
directly bind to NANOG and translocate into the nucleus, it can also bind to the enhancer region
of NANOG in order to increase NANOG expression (Bourguignon et al., 2012; Suzuki et al.,
2006). Recent studies have also suggested that STAT3 can modulate NANOG expression via
epigenetic modification (Tang et al., 2012). p53 also plays an important role in NANOG
regulation. Once p53 is phosphorylated at Ser315, it can bind to two consensus motifs in the
NANOG promoter region to prevent the expression of NANOG (Golubovskaya, 2013; Lin et al.,
2005). While the p53-dependent suppression of NANOG has commonly been accepted as a way
to address DNA damage in a cell, it has also been implicated in cancer cells, most notably brain
cancer stem cells (Wang et al., 2013). It has been shown that patients with HCC show elevated
levels of NANOG; in patients infected with HBV, HBx can directly bind to p53 to prevent
nuclear localization, which in turn could potentially prevent the suppression of NANOG and
allow for a more aggressive/invasive form of HBV associated HCC (Golubovskaya, 2013; Lin et
al., 2005; Sun et al., 2013).

18

1.7 Rationale of the Study
By gaining a better insight into the key drivers in HCC development, it may be possible
to design a targeted therapy against HCC. Already drugs are in development, such as ICG-001
which can inhibit the β-catenin/CBP pathway to promote the β-catenin/p300 pathway, as well as
Nabi-HB and Biotest-HCIV to treat circulating tumor stem cells of HBV and HCV origins to
give a better prognosis to patients who have received liver transplants due to HBV or HCV
related HCC (Davis et al., 2005; Dickson et al., 2006; Ma et al., 2005). However, it has yet to be
elucidated as to the crosstalk between HBx, β-catenin, NANOG, and YAP in the development of
HCC. A better understanding of the interactions between these pathways can provide potential
locations for targeted therapy to prevent HBx mediated HCC or increase chemosensitivity of
TICs by targeting the pathways important in the maintenance of their resistance.
1.8 Hypothesis
The purpose of this study was to look at the development of HCC, using HBV as a
model, and keeping β-catenin, NANOG, and YAP1 as the main focus. We hypothesize that β-
catenin and NANOG work together with the YAP1 pathway in order to increase HCC
malignancy, potentially through the regulation of BIRC5. To achieve this goal we want to
determine the β-catenin and NANOG levels in HBx positive and HBx negative mouse livers,
determine any interactions with YAP1 to promote genes crucial to malignancy, and determine in
vivo if what we have seen in vitro and in silico are applicable.

19

2. Materials and Methods
2.1 Western blot and immunohistochemistry
For western blot, mouse liver samples were homogenized by first mincing the tissue,
followed by the addition of RIPA buffer. Protein concentrations of each of the samples were read
on ELISA plates and the concentrations were calculated by comparison to standardized BSA
samples. Once the concentrations were calculated, 30µg was loaded into the wells of a cassette
composed of 5% stacking and 10% separating. The samples were run at 90 volts through the
stacking gel, and then the voltage was adjusted to 130 once it reached the separating gel. After
the samples had run through the gel, they were transferred from the gel to nitrocellulose (GE
Water and Process Technologies) using a semi-dry transfer system at 23 volts for 30min. After
the transfer, the nitrocellulose was stained with Ponceau Red to visualize the bands, followed by
1 hour in blocking solution using a 5% TBST-Milk blocking solution. The primary antibodies
used were mouse anti-active-β-catenin (1:5000, Santa Cruz Biotech), rabbit anti-Nanog (1:1000,
abcam), and mouse anti-β-actin (1:1000). The secondary antibodies used were goat anti-mouse
HRP (1:10000, Santa Cruz Biotech) and goat anti-rabbit HRP (1:10000, Santa Cruz Biotech).
The nitrocellulose was incubated in the primary antibodies overnight at 4°C, followed by
washing in TBST 3 times for 20 min each time. The nitrocellulose was then incubated in the
secondary antibody for 1 hour at room temperature, followed by TBST wash 3 times for 20 min
each time. After the wash, 500µL of Super Signal Stable and 500µL of Super Signal Luminol
was mixed together and the nitrocellulose was allowed to soak in the solution for about 1 min.
The nitrocellulose was then gently dried using kimwipes, being careful not to touch the area
where the bands are located, and visualized on x-ray film using an x-ray developer (Konica
20

Minolta SRX-101A). The band intensity was quantified using ImageJ v.3.91
(http://rsb.info.nih.gov/ij).
For immunohistochemistry, mouse liver samples embedded in paraffin were cut into 5µm
sections and mounted onto slides. One slide from each group was stained with hematoxylin and
eosin. The remaining unstained slides were used for immunohistochemistry staining using
primary antibodies mouse anti-β-catenin (1:250, Santa Cruz Biotech) and rabbit anti-Nanog
(1:250, ab80892, abcam). The slides were rehydrated by soaking 3 times in xylazine for 3 min,
followed by 3 min soaks in 100% EtOH, 90% EtOH, 70% EtOH, 50% EtOH, 3% H
2
O
2
, and
PBS. They were then placed in 1x Citrate Buffer at a temperature of 95°C for 10 mins, and then
taken off the heat and allowed to cool to room temperature. Using a hydrophobic pen, circles
were drawn around each of the samples and blocking buffer was applied to each slide for 1 hour.
The primary antibodies were then added to each slide and allowed to incubate overnight at 4°C.
The following morning, slides were washing 3 times with PBST for 10 min each time. Slides
were then stained with secondary antibodies conjugated to HRP and developed using 3,3’-
diaminobenzidine (DAB) with hematoxylin (invitrogen) and allowed to incubate for 1 hour at
room temperature. After 3 10min washes with PBST, the slides were dehydrated following a
50% EtOH, 70%EtOH, 90%EtOH, 100%EtOH, and 3 times xylazene regimen for 3 mins each
time. The slide coverslips were then mounted by a xylene-based slide mounting media. For
fluorescent images, secondary antibodies goat anti-mouse FITC (1:250, Jackson
ImmunoResearch Lab) and donkey anti-rabbit R-Phycoerythrin (1:250, Jackson
ImmunoResearch Lab) were used and incubated for 1 hour at room temperature in the dark. The
PBST washes were also carried out in the dark. Coverslips were mounted using slide mounting
21

media containing DAPI (Vectashield, Vector) to stain the nuclei of the cells. Slides were left
overnight in 4°C in the dark before visualization using a microscope.
2.2 Quantification of immunohistochemistry staining
To quantify the levels of β-catenin and Nanog staining in the mouse tissue samples, an
immunoreactive score was calculated, as shown by Koomagi et.al. The first of two variables
calculated was the intensity of staining, scored on a scale from 0 to 3. The score of 0 = negative
staining; 1 = weakly positive; 2 = moderately positive; and 3 = strongly positive staining. The
second variable calculated was the extent of distribution of nuclear positive cells, scored on a
scale from 0 to 4. The score of 0 = negative; 1 = positive staining in 0.01 – 1.0% of the cells; 2 =
1.01 – 2.0%; 3 = 2.01 – 3.0%; and 4 = greater than 3.0% positivity. To calculate the
immunoreactive score, the intensity of staining score was multiplied with the extent of
distribution score. Any immunoreactive scores equal to or greater than, 2 were considered to be
positive staining. Images were evaluated by two independent investigators and their two scores
were averaged.
2.3 Mouse strains
Livers from the mouse strains PBS Non-Tg, DEN- Tg05, DEN+ Non-Tg, DEN+ Tg05,
DEN+ mtTg31, DEN+ Tg08, DEN+ mtTg04 were kindly provided by Dr. James Ou (Zheng et
al., 2007). The strains Tg05 and Tg08 harbored wild-type HBV genomes but were two
independent mouse lines. mtTg31 and mtTg04 were also independent mouse lines that had the
HBx negative forms of the HBV genome in Tg05 and Tg08, respectively (Xu et al., 2002).
2.4 Cell lines and passaging
Non-immortalized, non-transformed mouse hepatoblasts were isolated from 12.5 day old
C57Bl/6 mouse embryos. The cells were cultured in DMEM/Ham’s F-12 media (Caisson)
22

containing 50mL FBS, 5mL non-essential amino acids, 5mL nucleosides (Sigma), 5mL
penicillin/streptomycin/glutomycin, 100µL mEGF (Peprotec), and 2.5µL Dexamethasone
(Sigma). The immortalized mouse live progenitor cell like PIL-4 cells were kindly provided by
Dr. Aleksandra Filipovska from the University of Western Australia. These cells were cultured
in Williams E Medium (Gibco) containing 30ng/mL IGF-2, 20ng/mL EGF, 10µg/mL insulin,
and 50mL FBS. Human Embryonic Kidney 293T cells were used in viral vector amplification.
They were grown in DMEM media (Caisson) containing 50mL FBS, 5mL non-essential amino
acids, and 5mL penicillin/streptomycin/glutomycin.
All cell lines were allowed to reach 70 – 80% confluence in 10cm Falcon plates (Fisher
Scientific) before passaging. To passage the cells, the media was removed and the plates were
washed 2 times with 5mL DPBS. 2mL of trypsin was added to each plate and placed into the
37°C 5% CO
2
incubator for 2 minutes to allow the cells to detach (HEK293T cells only required
< 30 seconds). The 2mL trypsin-cell suspension was added to 15mL Falcon centrifuge tubes
(Fisher Scientific), and 6mL of fresh media was added to inactivate the trypsinization process.
The suspensions were then centrifuged at 1200 rpm for 2 minutes. The media was poured out,
being careful not to lose the cell pellet, and about 1mL of fresh media was added to resuspend
the cells. The cell resuspension was then distributed amongst 2 – 5 10cm plates and placed back
into the 37°C 5% CO
2
incubator. Cell cultures were checked within 1 – 2 hours to ensure that
cells were adhering to the plates. Over the course of 24 – 48 hours, cells were checked to ensure
correct morphology, growth rate, and absence of any contamination.
2.5 Plasmids, lentivirus, and retrovirus
Plasmids used were pBABE-YAP1 (#15682, addgene), pMXs-NANOG (#13354,
addgene), and pBABE- β-catenin S33Y HA (A generous gift from Dr. Been Ze'ev at the
23

Weizmann Institute, Rehovot, Israel). pCMV-HBx HA was a very generous gift from Dr. Jing-
hsiung Ou (University of Southern California, Department of Molecular Microbiology and
Immunology). A master mix solution of 10µg of YAP1, NANOG, or β-catenin plasmids were
added with 6.5µg of the packaging vector PVpack, 3.5µg of an ectropic envelope vector
pMD2G, 31.5µL of BioT (Bioland Scientific LLC), and 1000µL of serum free DMEM
(Caisson). Each of the master mixes was allowed to sit for 5 min at room temperature, while the
media in each flask of HEK293T cells was replaced with fresh media. After 5 min, 1mL of
complete DMEM was added to the master mixes and 2mL of that was added to one flask of
HEK293T cells. Seventy-two hours later the media was harvested and the virus was concentrated
by ultra-centrifugation, using the SW28 rotor at 20,000rpm for 2 hours at 16°C. The pellet was
then re-suspended in 1mL PBS and stored in -80°C. The shRNA plasmids sh-YAP1 and sh-β-
catenin were mixed with pPAX2, pMD2G, BioT, and serum free DMEM and added into
HEK293T cells using the same procedure that were previously described.
2.6 Luciferase assay
PIL-4 cells were counted using trypan blue and 5 x 10
6
cells were plated in 3mL of media
in a Falcon 6-well plate (Fisher Scientific) and placed into a 37°C 5% CO
2
incubator. Each of the
7 groups was carried out in triplicate: (1) Control, (2) β-catenin, (3) Yap1, (4) Nanog, (5) β-
catenin + Yap1, (6) β-catenin + Nanog, (7) Nanog + Yap1. Twelve hours after plating the cells,
2µg of each of the plasmids, 1.5µL BioT, and 100µL of serum free DMEM were added and
allowed to sit at room temperature for 5 min. A separate mix of 1µg Birc5-luciferase, 100ng
Renilla-luciferase, 1.5µL BioT, and 100µL serum free DMEM was mixed and also allowed to sit
at room temperature for 5 min. After 5 min, 100µL of each of the master mixes was added to
each of the specified wells and the plates were placed back into the incubator. Twenty-four hours
24

post transfection, the media in each of the wells was changed to fresh complete DMEM. After
another 24 hours, the plates were removed from the incubator and washed twice with cold PBS.
After PBS wash, 500µL of 1x passive lysis buffer (Promega) was added to each well and the
plates were placed on a shaker in the 4°C room for 15 min. Each of the wells was transferred to a
labeled eppendorf tube. Into a new test tube, 100µL of luciferase assay buffer compound II
(Promega) compound, followed by 20µL of a sample and read in a Lumat LB 9501 (Berthold)
with a measuring time of 20.0 seconds. After the reading, the test tube was removed from the
machine and 100µL of Stop n’ Glo (Promega) was added and the tube was read again. This
process was repeated or all of the samples, using new test tubes for each sample.
2.7 Colony formation assay
Isolated hepatoblasts from C57Bl/6 mice were seeded into a Falcon 24-well plate (Fisher
Scientific) and infected with the viral vectors 24 hours from initial seeding. Thirteen groups were
created, with each group being carried out in triplicate: (1) Scramble, (2) β-catenin, (3) Nanog,
(4) Yap1, (5) β-catenin + Yap1, (6) β-catenin + Nanog, (7) Nanog + Yap1, (8) HBx +
Scrambled, (9) HBx + sh-Yap1, (10) HBx + sh-β-catenin, (11) HBx + Nanog + Scrambled, (12)
HBx + Nanog + sh-Yap1, (13) HBx + Nanog + sh-β-catenin. HBx was transiently transfected via
BioT transfection (Bioland Scientific LLC) 24 hours from initial seeding. On day 3 post
transfection, 0.5mL of a solution containing 5% agar in PBS with warmed DMEM/Ham’s F-12
media (Caisson; 50mL FBS, 5mL non-essential amino acids, 5mL nucleosides, 5mL
Penicillin/Streptomycin/Glutomycin, 100µL mEGF, and 2.5µL Dexamethasone) in a 1:10 ratio
was added to each of the wells in a new Falcon 24-well plate and allowed to solidify.
Hepatoblasts were then trypsinized and counted using the invitrogen Countess automated cell
counter. 12,500 cells were resuspended in a solution containing 1mL of 3% agarose (Apex
25

BioResearch Products) in PBS with 9mL DMEM/Ham’s F-12 media, and 0.5mL were layered
over the top of the solidified 0.5% agar (Apex BioResearch Products). Each of the thirteen
groups was carried out in triplicate. The plates were placed in a 37ºC humidified incubator for 4
days. Plates were then removed and each well was stained with 0.5mL of 0.005% crystal violet
at room temperature for 2 hours. Each of the wells was then washed in one hour PBS bath cycles
until the colonies could be clearly seen. The wells were photographed and the colonies were
counted via the CellCounter program (Nghia Ho).
2.8 Hepatoblast groups and intrasplenic injection into wild-type mice
Hepatoblasts from C57Bl/6 mice were isolated and played into a Falcon 24-well plate
(Fisher Scientific). To each of the wells, 100µL of each of the created lenti- or retro-viral
particles described in the previous section was added to created 13 groups: (1) Scramble, (2) β-
catenin, (3) Nanog, (4) Yap1, (5) β-catenin + Yap1, (6) β-catenin + Nanog, (7) Nanog + Yap1,
(8) HBx + Scramble, (9) HBx + sh-Yap1, (10) HBx + sh-β-catenin, (11) HBx + Nanog +
Scramble, (12) HBx + Nanog + sh-Yap1, (13) HBx + Nanog + sh-β-catenin. After 24 hours, the
cells from each well were collected via pipetting the media up and down to dislodge the cells
from the plate. Cell number was measured using 10mL of trypan blue and 10mL of the cell
suspension. About 1 x 10
6
cells were resuspended in 0.1mL of media for each group, and this
was used for the intrasplenic injection.
At day -44 and -30, C57Bl/6 mice were injected with retrorsine via intraperitoneal
injection. Stock retrorsine powder was diluted in sterile PBS to a concentration of 7mg/mL.
Using a 27G needle with a 1mL syringe, 100µL of retrorsine (Sigma) solution per 10g mouse
weight was administered (70mg retrorsine/kg mouse weight or about 0.7 – 1.4mg
retrorsine/mouse). Retrorsine is an important pre-treatment as it blocks the native hepatocyte
26

proliferative ability and increases the odds of successful transplantation of cells that are injected.
On the day of the surgery, the mice are anesthetized by intraperitoneal injection of
ketamine/xylazine (100mg/mL concentration that was administered at 0.1mL/20g mouse
weight). Once anesthetized, the mice are shaved on their left side, about 0.5 inches below the rib
case. The shaved area is swabbed with gauze soaked in a Betadine solution (Purdue Products),
followed by gauze soaked in 70% ethanol. This process is thoroughly repeated 2 more times. The
mouse is then placed on a sterile surgical stage with the nose placed into a nose cone that is
administering a constant flow of isoflurane (IsoFlo, Abbot Laboratories). A toe pinch test is
carried out to ensure that the mouse if completely under anesthesia before the surgery
commences. The animal is moved so that the left side of its body is facing upwards, exposing the
shaved and sterilized area. Using forceps, hold the skin away from the body wall and make a 1
centimeter incision (anterior to posterior) using scissors. Once the spleen is exposed, gently
reach under the spleen to gain access to the pancreas. Grasp the pancreas gently and pull in order
to move the spleen out of the incision to make it more accessible for injection. Place cotton
tipped applicator under the spleen, and using a 30G needle with a 1mL syringe, insert the needle
into the tip of the spleen until it is in about half of the spleens length. Slowly inject the 100µL
cell suspension into the spleen and gently remove the needle. Once the splenic injection is done,
grasp the pancreas again and use it to slowly pull the spleen back into the body cavity. Using a 4-
0 synthetic absorbable suture (Ethicon VR494), close the peritoneal wall by using a continuous
running suture. To close the skin, use a 5-0 monofilament non-absorbable suture (Ethicon
8689G) and a simple interrupted suture. Swab the incision area with Betadine and remove the
nose cone from the mouse. Place the mouse on a circulating heated blanket until the mouse
recovers from anesthesia. Place the mice into a clean cage and follow up with intraperitoneal
27

injection of buprenex (0.02 – 0.05mg buprenex/kg mouse weight) both morning and night for
three days. Ketoprofen (5mg Ketoprofen/kg mouse weight) may also be administered to manage
pain in the mice 24 hours and 48 hours post surgery via subcutaneous injection. Sutures should
be removed 14 days post-surgery. To facilitate engraftment and expansion of transplanted cells,
the mice received intraperitoneal injections of CCl
4
(dissolved in mineral oil to a concentration
of 5mL CCl
4
/kg mouse weight) at days 14, 21, and 28 post-surgery. Mice were monitored for 2
months before ultrasound imaging and microCT imaging was carried out at the Animal Imaging
Core of University of Southern California Radiology Department. Studies involving animal use
were conducted in compliance with the Guide for the Care and Use of Laboratory Animals,
published by the National Institutes of Health.
2.9 Statistical analysis
All experiments and data reported, excluding the western blot analysis, were carried out
in triplicate. The data that was presented was the average of the three readings ± the standard
deviation. Statistical significance was a p-value < 0.05, and was carried out using a two-tailed
unpaired student’s t-test.

28

3. Results
3.1 HBx plays a role in the regulation of β-catenin and NANOG levels
While β-catenin activation mutations are known to be present in HCCs, previous studies
have shown that having a β-catenin activation mutation alone isn’t enough to cause a
transformation from cirrhotic tissue to TICs (Pilati et al., 2014). In order to evaluate what other
pathways may work together with the WNT/β-catenin pathway to promote tumor occurrence, an
HBV transgenic mouse model was used. HBx ncreases β-catenin levels through the inhibition of
GSK3β via increasing Src-kinase activity, and through an androgen receptor-dependent CCRK
(Cha et al., 2004; Ding et al., 2005). In the DEN+ mtTg31 mouse line that harbors an HBx
negative HBV genome, both NANOG and β-catenin levels are reduced, indicating that HBx is
required for the increased expression of β-catenin in the transgenic mouse liver samples (Figure
4).
3.2 Liver tissue shows more hepatocarcinogenesis in HBx positive group
Histological analysis was carried out on paraffin-embedded mouse liver tissue samples to
evaluate the effects of HBx on tumorigenesis. Consistent with the previous data (Zheng et al.,
2007), the wild-type HBV transgenic mice showed signs of hepatocarcinogenesis with
formations of tumor nodules and signs of fatty liver change (Bharrhan, et al., 2011; Figure 5F),
when compared to the relatively normal morphology of the HBx negative group (Figure 5E). It is
also worth noting that DEN+ Tg05 tissue also showed cellular expansion in the portal triad
region of the liver (Figure 5D), when compared to the PBS Tg05 and DEN+ mtTg31(Figure 5B
and 5C), hinting at a synergistic relationship between HBx and liver damage induced by DEN.

29

3.3 β-catenin and NANOG localize in the nuclei in HBx positive tissue samples
To link the effects of HBx on β-catenin and NANOG levels to the phenotypic changes
seen in the H & E samples, we evaluated if active-β-catenin and NANOG localized in the nuclei
of the tissue samples. Both the wild-type HBV transgenic mice samples showed significantly
higher levels of β-catenin with a large portion localizing in the nucleus (p < 0.05) (Figure 6). The
NANOG levels were also significantly higher in the DEN+ Tg05 and DEN+ Tg08 samples
compared to the DEN+ mtTg31 and DEN+ mtTg04 samples, respectively. For the samples with
nuclear localization (DEN+ Tg05 and DEN+ Tg08), both the β-catenin and NANOG positive
nuclei seemed to overlap. The DEN+ Tg05 sample was positive near the portal triad while the
DEN+ Tg08 sample was positive near fatty liver and dysplastic tissue. To further confirm the
overlap of β-catenin and NANOG in the nuclei we double stained the tissue samples using
fluorescent antibodies and only saw nuclear double positivity in DEN+ Tg05 and DEN+ Tg08
(Figure 7).
3.4 BIRC5 plays an important role in late stage tumorigenesis
Crosstalk between β-catenin and YAP1 promotes the development of the hepatoblastoma
and β-catenin can interact with YAP1 and TBX5 to stimulate BIRC5 expression (Azzolin et al.,
2012; Rossenbluh et al., 2012; Tao et al., 2014). We bioinformatically evaluated to see if
expression levels of BIRC5, β-catenin and NANOG are associated with poor prognosis or
malignant transformation in HCC development. What we saw from the in silico analysis was a
significant relationship between BIRC5 levels and liver cancer precurors (cirrhosis and fibrosis)
compared to HCC (Figure 8). There was also a positive correlation between increasing BIRC5
levels and BCLC stage and tumor grade, as well as higher BIRC5 levels in metastasis sites and
patients who were deceased after 3 years.
30

To understand the mechanism behind the change in BIRC5 levels between LCP and
HCC, we carried out a BIRC5 promoter assay using β-catenin, NANOG, and YAP1 in the mouse
liver progenitor cell line PIL-4. YAP1 and β-catenin + NANOG showed significant increases in
BIRC5-luciferase expression compared to the empty pGL3-Basic control (Figure 9). Potentially,
HCC malignancy could be regulated through YAP1 or β-catenin + NANOG mediated BIRC5
expression.
3.5 β-catenin + Nanog show the highest transformative ability in p53 +/+ hepatoblasts
As β-catenin + NANOG and YAP1 transactivate BIRC5 promoter, we next tested
whether it has malignant transformative abilities in vitro. The data showed that β-catenin +
NANOG had the highest number of colonies, more than triple the amount of YAP1 (Figure 10).
This hinted at the β-catenin + NANOG mediated BIRC5 expression playing a more important
role when compared to the YAP1 mediated BIRC5 expression. As such, the β-catenin +
NANOG group became one of the groups of interest for the following intrasplenic injection
study. The sample 13 groups were intrasplenicly injected into C57Bl/6 mice, and after 2 months
they were scanned using ultrasound and microCT. However, preliminary data shows that the
HBx + Scr and HBx + Nanog + Scr groups had high tumor burden, while the other groups
showed little to no tumor burden (Data not shown). This may be related to HBx’s ability to bind
to the CREB element on the YAP promoter to induce its expression. In this way HBx can carry
out a threefold function of increasing YAP1 expression through its CREB element on the
promoter region, increasing β-catenin stability through its interaction with GSK3β, and
preventing NANOG inhibition by directly binding to p53. However, a larger sample size is
required to validate and results seen in the preliminary ultrasound and microCT.

31

4. Figures

Figure 4. HBx regulation of β -catenin and Nanog levels. Liver samples were kindly provided by Dr. James Ou . Tg05 mouse
sample has a full length 3.2-kb HBV genome and the mtTg31 mouse sample has a 2 point mutations in the HBx gene to
prevent its expression The β -catenin molecular weight was 94kDa, the Nanog molecular weight was 34kDa, and β -actin was
42kDa. Band intensitys in the graph on the left were standardized to the β -actin levels.
32

Figure 5. H&E morphological changes in tissue in the presence or absence of HBx and DEN. (A) Control diet, non-Tg
sample has normal morphology. (B) wt-HBV genome without DEN injection shows normal phenotype. (C) HBx(-)
HBV genome shows normal phenotype. (D) wt-HBV with DEN injection shows expansion of cells near the portal triad.
(E) HBx(-) sample shows normal phenotype. (F) wt-HBV with DEN shows tumor mass as well as fatty liver change and
some slight dysplasia.
33

Figure 6. Comparative analysis between β-catenin and Nanog expression in HBV transgenic mouse models with or
without DEN. DEN+ Tg05 and DEN+ Tg08 both show significantly higher expression of β-catenin and Tg05 has a
significantly higher expression of Nanog. In both samples, both β-catenin and Nanog are localized in the nucleus,
unlike mtTg04 and mtTg31. The * is p-value < 0.05, a two-tailed, unpaired, student’s t-test.
34

Figure 7. Each of the samples was double-stained with β-catenin and Nanog antibodies, followed by secondary antibodies
with FITC, Rho, or DAPI. Images were visualized through a 40x objective and a 10x eye piece, making it 400x
magnification. Removal of HBx significantly reduced Nanog expression levels in both mtTg31 and mtTg04. Only the set
Tg08/mtTg04 showed significantly decreased β-catenin expression. The * is p-value < 0.05, a two-tailed, unpaired,
student’s t-test.
35

Figure 8. Data from in silico analysis using The Oncomine
TM
Platform (Life Technologies, Ann Arbor, MI). BCLC staging
is (1) Stage 0, (2) Stage A, (3) Stage B, (4) Stage C. Tumor grade is listed as (1) well differentiated, (2) intermidately
differentiated, (3) poorly differentiated. HCC vs LCP is hepatocellular carcinoma vs liver cancer precurors (cirrhosis and
fibrosis) BIRC5 levels seen to be more highly expressed in HCC and not the precursors to cancer (Data from Wurmbach
et al., 2007). Similarly we see that BIRC5 expression is more prominent at later stages of cancer shown by BCLC (Data
from Chiang et al., 2008) and tumor grade (Data from Wurmbach et al., 2007). BIRC5 expression levels were higher in
metastasis sites when compared to primary site (Data from Liao et al., 2008), implicating its impact for the establishment
of a new tumor location. Also patients with elevated BIRC5 levels had a lower chance of living past 3 years (Data from
Hoshida et al., 2009). All data presented on BIRC5 has a p-value < 0.05.

36

Figure 9. BIRC5-Luciferase assay carried out in mouse liver progenitor cells, PIL-4. Expression vectors were transiently
transfected into the PIL-4 cells and the luciferase measurements were taken 48 hours after initial transfection. The
BIRC5-luciferase readings were standardized to the SV40-Renilla-Luciferase. YAP1 and β-catenin + NANOG were the
only groups that saw significant increase in BIRC5 promoter activity when compared to the empty control vector. The *
is p-value < 0.05, a two-tailed, unpaired, student’s t-test.

37

Figure 30. Colony formation assay was carried out using C57Bl/6 hepatoblasts and lenti- or retro-viral constructs were
used to stably transfect each of the groups. β-catenin + Nanog showed the highest amount of colonies and had a significant
amount more than Nanog on its own. This could potentially be due to Nanog being able to recruit CBP/p300 to β-catenin.
The * denotes that β-catenin + Nanog had a significantly higher amount of average colonies than any other group (p-value
< 0.05, two-tailed, unpaired, student’s t-test).
38

Supplementary Figure 1. β -catenin and Nanog stained control DEN- Non--Tg and DEN- Tg05 tissue samples. In both
immunohistochemistry staining (A) and immunofluorescent staning (B) samples show little to no expression of β-catenin
and Nanog. Quantification for both panels (A) and (B) are seen in Figures 7 and Figures 8, respectively.

39

5. Discussion
Here we have shown that HBx is crucial for regulating the β-catenin and NANOG levels
in HBV transgenic mice. The mice that were HBx negative showed little β-catenin and NANOG
expression, but conversely those mice that had a full length HBV genome had a much higher
expression levels. Mice also showed significantly increased levels of nuclear localization of β-
catenin and NANOG with the presence of HBx. The areas of localization generally centered
around fatty liver tissue that had dysplastic characteristics, as well as areas where the cell
population by the portal triad seemed to have expanded. The genotoxic carcinogen DEN was
required by and required HBx intervention in order to facilitate its effects on the liver. This was
seen in the decreased levels of β-catenin and NANOG that had localized in the nucleus in the
absence of either HBx or DEN injection. Despite HBx’s ability to regulate both β-catenin and
NANOG, it seems that an initiation event is required before both components move into the
nucleus and regulate their downstream targets. We are currently looking into the possibility as to
whether or not a p53 mutation can be a substitute for the effects of DEN injection. By this
method of using p53 -/- with HBx-mediated β-catenin stabilization, the results are still applicable
to a clinical setting, as having both p53 -/- and β-catenin -/- together are extremely rare in most
HCC (Tornesello et al., 2013).
Hepatocellular carcinoma can be a complex issue to solve with many interconnected
pathways. In the case of a simultaneous HBV infection, the HBx protein can add another layer of
complexity. It has been shown that HBx can not only stabilize the β-catenin levels by modulating
GSK3β activity through Src-kinase, but it can also regulate p53 activity by directly binding to,
and inhibiting its function (Cha et al., 2004; Ding et al., 2005; Elmore et al., 1997).
40

With the recent study that had found a correlation between the WNT/ β-catenin pathway
and the YAP1 pathway interacting to lead to hepatoblastoma development, it lead us to evaluate
the probability of similar pathways working for HCC (Tao et al., 2014). In silico analysis of
clinical samples showed that BIRC5 was statistically relevant (p < 0.05) to higher BCLC staging
and tumor grade, as well as metastasis and death at 3-years. A previous study by Rosenbluh et al.
showed that β-catenin, YAP1, YES, and TBX5 interact with the BIRC5 promoter region to
increase BIRC5 expression. Our BIRC5 promoter assay hinted at YAP1 and β-catenin +
NANOG being the best groups for increased BIRC5 expression. Colony formation assay
confirmed that β-catenin and NANOG highly likely work together in order to give hepatoblasts
TIC-like characteristics, as β-catenin or NANOG together with YAP1 or alone did not produce
the same amount of colonies. One potential explanation for the increase in β-catenin’s ability to
promote stemness with the NANOG group only is that NANOG has been found to recruit both
CBP and p300 to facilitate stemness pathways (Fang et al., 2014). By this way NANOG could be
promoting β-catenin to follow the CBP pathway and promote downstream cell stemness factors.
Preliminary ultrasound and microCT imaging in intrahepatically injected mice hinted at HBx and
HBx + NANOG to be the most malignant groups in vivo. It can be speculated that due to HBx’s
effects on a broad range of cellular functions, that it is by another pathway that tumors formed.
However it is likely due to HBx’s effects on either the β-catenin, YAP1, or both pathways, as the
groups that had sh-YAP1 and sh-β-catenin showed no tumors and relatively small amounts of
colonies. However more mice per group are required to increase the power of the statistics.
To summarize the information that has been shown, HBx can regulate β-catenin and
NANOG (likely through its regulation of GSK3β for β-catenin and p53 for NANOG), which
work together with YAP1 in order to lead to the development of HCC through, potentially, the
41

activation of BIRC5. HBx itself can induce YAP1 expression through the CREB element on the
YAP1 promoter region. However, based on preliminary data HBx interaction with YAP1 is not
likely a major pathway; however, YAP1 interaction with β-catenin and NANOG seems to play
an important role, as removing β-catenin from the HBx and HBx + NANOG models decreased
colony formation and tumor incidence. More data is required to provide statistically significant
proof, with the projected future experiments being repeating the in vivo studies using the same
groups but with the addition of sh-BIRC5, expanding the study to include p53 -/- groups to
determine if p53 -/- can substitute for the genotoxic damage done by DEN, and addition of
spheroid formation assay to observe whether or not anchorage independent growth occurs in the
groups of interest. Further studies will be required to correctly pinpoint the connections between
these pathways, as well as using dual-lineage tracing to determine exactly what cells HCC is
derived from and at what time points we see the transformation occur. With more time and data,
we may be able to elucidate the major pathways required for HCC development, and develop
drugs to target those pathways to provide better treatment and hope for late stage HCC patients.

42

6. References

Ahn, SM , Jang, SJ, Shim, JH, Kim, D, Hong, SM, Sung, CO, Baek, D, Haq, F, Ansari, AA, Lee,
SY, Chun, SM, Choi, S, Choi, HJ, Kim, J, Kim, S, Hwang, S, Lee, YJ, Lee, JE, Jung, WR, Jang,
HY, Yang, E, Sung, WK, Lee, NP, Mao, M, Lee, C, Zucman-Rossi, J, Yu, E, Lee, HC, Kong, G.
(2014). Genomic portrait of resectable hepatocellular carcinomas: Implications of RB1 and
FGF19 aberrations for patient stratification. Hepatology. 60, 1972–1982.

AJCC Cancer Staging Handbook. 7 ed. Chicago, IL: American Joint Committee on Cancer,
2010.

American Cancer Society. Cancer Facts & Figures 2015. Atlanta: American Cancer Society;
2015.

Aragona, M, Panciera, T, Manfrin, A, Giulitti, S, Michielin, F, Elvassore, N, Dupont, S, Piccolo,
S. (2013). A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation
by actin-processing factors. Cell 154, 1047–1059.

Azzolin, L, Panciera, T, Soligo, S, Enzo, E, Bicciato, S, Dupont, S, Bresolin, S, Frasson, C,
Basso, G, Guzzardo, V, Fassina, A, Cordenonsi, M, Piccolo, S. (2014). YAP/TAZ incorporation
in the β-Catenin destruction complex orchestrates the Wnt response. Cell. 158, 157 – 170.

Beasley, RP, Chien, CS, Chen, CJ, Hwang, LY. (1982). Geographic distribution of HBsAg
carriers in China. Hepatology. 2, 553 – 556.

Beck, J, Nassal, M. (2007). Hepatitis B virus replication. World J Gastroenterol. 13, 48 – 64.

Bharrhan, S, Koul, A, Chopra, K, Rishi, P. (2011). Catechin suppresses an array of signaling
molecules and modulates alcohol-induced endotoxin mediated liver injury in a rat model. PLoS
ONE. 6, 1 – 9.

Booth, HA, Holland, PW. (2004). Eleven daughters of NANOG. Genomics. 84, 229 – 238.

Bourguignon, LY, Earle, C, Wong, G, Spevak, CC, Krueger, K. (2012). Stem cell marker
(Nanog) and Stat-3 signaling promote MicroRNA-21 expression and chemoresistance in
hyaluronan/CD44-activated head and neck squamous cell carcinoma cells. Oncogene. 31, 149 –
160.

Bruix, J, Sherman, M, American Association for the Study of Liver Diseases. (2011).
Management of hepatocellular carcinoma: an update. Hepatology. 53, 1020 - 1022.

Buddeberg, G, Schimmer, BB, Spahn, DR. (2008). Transfusion-transmissible infections and
transfusion-related immunomodulation. Best Pract Res Clin Anaesthesiol. 22, 503 – 517.

Caldwell S, Park SH. (2009). The epidemiology of hepatocellular cancer: from the perspectives
of public health problem to tumor biology. J Gastroenterol. 44, 96 – 101.
43

Calvo, F, Ege, N, Grande-Carcia, A< Hooper, S, Jenkins, RP, Chaudhry, SI, Harrington, K,
Williamson, P, Moeendarbary, E, Charras G, Sahai, E. (2013). Mechanotransduction and YAP-
dependent matrix remodelling is required for the generation and maintenance of cancer-
associated fibroblasts. Nat Cell Biol. 15, 637–646.

Cha, M, Kim, C, Park, Y, Ryu, W. (2004). Hepatitis B virus x protein is essential for activation
of Wnt/β-Catenin signaling in hepatoma cells. Hepatology. 39, 1683 – 1693.

Chen, DS. (1993). Hepatitis to hepatoma: Lessons from type B viral hepatitis. Science. 262, 369
– 370.

Chiang, DY, Villanueva, A, Hoshida, Y, Peix, J, Newell, P, Minguez, B, LeBlanc, AC,
Donovan, DJ, Thung, SN, Solé, M, Tovar, V, Alsinet, C, Ramos, AH, Barretina, J, Roayaie, S,
Schwartz, M, Waxman, S, Bruix, J, Mazzaferro, V, Ligon, AH, Najfeld, V, Friedman, SL,
Sellers, WR, Meyerson, M, Llovet, JM. (2008). Focal gains of VEGFA and molecular
classification of hepatocellular carcinoma. 68, 6779 – 6788.

Colagrande, S, Regini, F, Taliani, GG, Nardi, C, Inghilesi, AL. (2015). Advanced hepatocellular
carcinoma and sorafenib: Diagnosis, indications, clinical and radiological follow-up. World J
Hepatol. 7, 1041-1053.

Davis, GL, Nelson, DR, Terrault, N, Pruett, TL, Schiano, TD, Fletcher, CV, Sapan, CV, Riser,
LN, Li, Y, Whitley, RJ, Gnann, JW Jr; Collaborative Antiviral Study Group. (2005). A
randomized, open-label study to evaluate the safety and pharmacokinetics of human hepatitis C
immune globulin (Civacir) in liver transplant recipients. Liver Transpl. 11, 941 – 949.

Di Costanzo, GG, Tortora, R. (2015). Intermediate hepatocellular carcinoma: Howto choose the
best treatment modality. World J Hepatol. 7, 1184 – 1191.

Dickson, RC, Terrault, NA, Ishitani, M, Reddy, KR, Sheiner, P, Luketic, V, Soldevila-Pico, C,
Fried, M, Jensen, D, Brown, RS Jr, Horwith, G, Brundage, R, Lok, A. (2006). Protective
antibody levels and dose requirements for IV 5% Nabi Hepatitis B immune globulin combined
with lamivudine in liver transplantation for hepatitis B-induced end stage liver disease. Liver
Transpl. 12, 124 – 133.

Ding, Q, Xia, W, Liu, J, Yang, J, Lee, D, Xia, J, Bartholomeusz, G, Li, Y, Pan, Y, Li, Z, Bargou,
RC, Qin, J, Lai, C, Tsai, F, Tsai, C, Hung, M. (2005). Erk associates with and primes GSK-3β
for its inactivation resulting in upregulation of β-Catenin. Mol Cell. 19, 159 – 170.

Do, HJ, Lim, HY, Kim, JH, Song, H, Chung, HM. (2007). An intact homeobox domain is
required for complete nuclear localization of human Nanog. Biochem Biophys Res Commun.
353, 770 – 775.

44

Elmore, LW, Hancock, AR, Chang, SF, Wang, XW, Chang, S, Callahan, CP, Geller, DA, Will,
H, Harris, CC. (1997). Hepatitis B virus X protein and p53 tumor suppressor interactions in the
modulation of apoptosis. Proc Natl Acad Sci USA. 94, 14707– 14712.

European Association for the Study of the Liver & European Organisation for Research and
Treatment of Cancer. (2012). EASL-EORTC clinical practice guidelines: management of
hepatocellular carcinoma. J. Hepatol. 56, 908 – 943.

Fairley, CK, Read, TR. (2012). Vaccination against sexually transmitted infections. Curr Opin
Infect Dis. 25, 66 – 72.

Fang, F, Xu, Y, Chew, KK, Chen, X, Ng, HH, Matsudaira, P. (2014). Coactivators p300 and
CBP Maintain the Identity of Mouse Embryonic Stem Cells by Mediating Long-Range
Chromatin Structure. Stem Cells. 32, 1805 – 1816.

Farazi, PA, DePinho RA. (2006). Hepatocellular carcinoma pathogenesis: from genes to
environment. Nat Rev Cancer. 6, 674 – 687.

Feng, H, Li, X, Niu, D, Chen, WN. (2009). Protein profile in HBx transfected cells: a
comparative iTRAQ-coupled 2D LC-MS/MS analysis. J Proteomics. 73, 1421 – 1432.

Golubovskaya, VM. (2013). FAK and Nanog cross talk with p53 in cancer stem cells. Anticancer
Agents Med Chem. 13, 576 – 580.

Goodman, RH, Smolik, S. (2000). CBP/p300 in cell growth, transformation, and development.
Genes Dev. 14, 1553 – 1577.

Guichard, C, Amaddeo, G, Imbeaud, S, Ladeiro, Y, Pelletier, L, Maad, IB, Calderaro, J, Bioulac-
Sage, P, Letexier, M, Degos, F, Clément, B, Balabaud, C, Chevet, E, Laurent, A, Couchy, G,
Letouzé, E, Calvo, F, Zucman-Rossi, J. (2012). Integrated analysis of somatic mutations and
focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma. Nat
Genet. 44, 694–698.

He, TC, Sparks, AB, Rago, C, Hermeking, H, Zawel, L, da Costa, LT, Morin, PJ, Vogelstein, B,
Kinzler, KW. (1998). Identification of c-MYC as a target of the APC pathway. Science. 281,
1509 – 1512.

Hemming, AW, Cattral, MS, Reed, AI, Van der Werf, WJ, Greig, PD, Howard, RJ. (2001). Liver
transplantation for hepatocellular carcinoma. Annals of Surgery. 233, 652 - 659.
Hoshida, Y, Nijman, SM, Kobayashi, M, Chan, JA, Brunet, JP, Chiang, DY, Villanueva, A,
Newell, P, Ikeda, K, Hashimoto, M, Watanabe, G, Gabriel, S, Friedman, SL, Kumada, H, Llovet,
JM, Golub, TR. (2009). Integrative transcriptome analysis reveals common molecular subclasses
of human hepatocellular carcinoma. Cancer Res. 69, 7385 – 7392.
45

Hoshida, Y, Toffanin, S, Lachenmayer, A, Villanueva, A, Minguez, B, Llovet, JM. (2010).
Molecular classification and novel targets in hepatocellular carcinoma: recent advancements.
Semin Liver Dis. 30, 35 - 51.

Hughes, RA. (2000). Drug injectors and the cleaning of needles and syringes. Eur Addict Res. 6,
20 – 30.

Ibrahim, EE, Babaei-Jadidi, R, Saadeddin, A, Spencer-Dene, B, Hossaini, S, Abuzinadah, M, Li,
N, Fadhil, W, Ilyas, M, Bonnet, D, Nateri, AS. (2012). Embryonic NANOG activity defines
colorectal cancer stem cells and modulates through AP1- and TCF-dependent mechanisms. Stem
Cells. 30, 2076 – 2087.

Imajo, M, Miyatake, K, Iimura, A, Miyamoto, A, Nishida, E. (2012). A molecular mechanism
that links Hippo signaling to the inhibition of WNT/β-catenin signaling. EMBO J. 31, 1109–
1122.

Innacone, M, Sitia, G, Ruggeri, ZM, Guidotti, LG. (2007). HBV pathogenesis in animal models:
recent advances on the role of platelets. J Hepatol. 46, 719 – 726.

Kan, Z, Zheng, H, Liu, X, Li, S, Barber, TD, Gong, Z, Gao, H, Hao, K, Willard, MD, Xu, J,
Hauptschein, R, Rejto, PA, Fernandez, J, Wang, G, Zhang, Q, Wang, B, Chen, R, Wang, J, Lee,
NP, Zhou, W, Lin, Z, Peng, Z, Yi, K, Chen, S, Li, L, Fan, X, Yang, J, Ye, R, Ju, J, Wang, K,
Estrella, H, Deng, S, Wei, P, Qiu, M, Wulur, IH, Liu, J, Ehsani, ME, Zhang, C, Loboda, A,
Sung, WK, Aggarwal, A, Poon, RT, Fan, ST, Wang, J, Hardwick, J, Reinhard, C, Dai, H, Li, Y,
Luk, JM, Mao, M. (2013). Whole-genome sequencing identifies recurrent mutations in
hepatocellular carcinoma. Genome Res. 23, 1422–1433.

Kekule, AS, Lauer, U, Weiss, L, Luber, B, Hofschneider, PH. (1993). Hepatitis B virus
transactivator HBx uses a tumour promoter signalling pathway. Nature. 361, 742 – 745.

Kim, SY, Kim, JC, Kim, JK, Kim, HJ, Lee, HM, Choi, MS, Maeng, PJ, Ahn, JK. (2008).
Hepatitis B virus X protein enhances NFκB activity through cooperating with VBP1. BMB Rep.
41, 158 – 163.

Klein, NP, Schneider, RJ. (1997). Activation of Src family kinases by hepatitis B virus HBx
protein and coupled signaling to Ras. Mol Cell Biol. 17, 6427 – 6436.

Lee, MH, Yang, HI, Liu, J, Batrla-Utermann, R, Jen, CJ, Iloeje, UH, Lu, SN, You, SL, Wang,
LY, Chen, CJ, R.E.V.E.A.L.-HBV Study Group. (2013). Prediction models of long-term
cirrhosis and hepatocellular carcinoma risk in chronic hepatitis B patients: Risk scores
integrating host and virus profiles. Hepatology. 58, 546 – 554.

Li, S, Mao, M. (2013). Next generation sequencing reveals genetic landscape of hepatocellular
carcinomas. Cancer Lett. 340, 247 - 253.

46

Liao, J, Wu, Z, Wang, Y, Cheng, L, Cui, C, Gao, Y, Chen, T, Rao, L, Chen, S, Jia, N, Dai, H,
Xin, S, Kang, J, Pei, G, Xiao, L. (2008). Enhanced efficiency of generating induced pluripotent
stem (iPS) cells from human somatic cells by a combination of six transcription factors. Cell
Res. 18, 600 – 603.

Lin, CL, Kao, JH. (2012). Risk stratification for hepatitis B virus related hepatocellular
carcinoma. J Gastroenterl Hepatol. 28, 10 – 17.

Lin, T, Chao, C, Saito, S, Mazur, SJ, Murphy, ME, Appella, E, Xu, Y. (2005). p53 induces
differentiation of mouse embryonic stem cells by suppressing Nanog expression. Nat Cell Biol.
7, 165 – 171.

Llovet, JM, Brú, C, Bruix, J. (1999). Prognosis of hepatocellular carcinoma: the BCLC staging
classification. Semin Liver Dis. 19, 329 - 338.

Llovet, JM, Ricci, S, Mazzaferro, V, Hilgard, P, Gane, E, Blanc, JF, de Oliveira, AC, Santoro, A,
Raoul, JL, Forner, A, Schwartz, M, Porta, C, Zeuzem, S, Bolondi, L, Greten, TF, Galle, PR,
Seitz, JF, Borbath, I, Häussinger, D, Giannaris, T, Shan, M, Moscovici, M, Voliotis, D, Bruix, J.
(2008). Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 359, 378 - 90.

Llovet, JM, Villanueva, A, Lachenmayer, A, Finn, RS. (2015). Advances in targeted therapies
for hepatocellular carcinoma in the genomic era. Nat Rev Clin Oncol. Advanced Online
Publication, 1 – 17.

Lok, ASF, McMahon, BJ. (2007). Chronic hepatitis B. Hepatology. 45, 507 – 539.

Ma, H, Nguyen, C, Lee, KS, Kahn, M. (2005). Differential roles for the coactivators CBP and
p300 on TCF/β-catenin-mediated surviving gene expression. Oncogene. 24, 3619 – 3631.

Marquardt, JU, Thorgeirsson, SS. (2014). Next-generaton genomic profiling of hepatocellular
adenomas: A new era of individualized patient care. Cancer Cell. 25, 409 – 411.

Metcalfe, C, Bienz, M. (2011). Inhibition of GSK3 by Wnt signaling – two contrasting models. J
Cell Sci. 124, 3537 – 3544.

Mitsui, K, Tokuzawa, Y, Itoh, H, Segawa, K, Murakami, M, Takahashi, K, Maruyama, M,
Maeda, M, Yamanaka, S. (2003). The homeoprotein Nanog is required for maintenance of
pluripotency in mouse epiblast and ES cells. Cell. 113, 631 – 642.

Miyabayashi, T, Teo, JL, Yamamoto, M, McMillan, M, Nguyen, C, Kahn, M. (2007). Wnt/β-
catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency. Proc Natl
Acad Sci. 104, 5668 – 5673.

Mo, JS, Park, HW, Guan, KL. (2014). The Hippo signaling pathway in stem cell biology and
cancer. EMBO Rep. 15, 642–656.

47

Moroishi, T, Hansen, CG, Guan, KL. (2015). The emerging roles of YAP and TAZ in cancer.
Nat Rev Cancer. 15, 73 – 79.

Murakami, Y, Saigo, K, Takashima, H, Minami, M, Okanoue, T, Brechot, C, Paterlini-Brechot,
P. (2005). Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related
hepatocellular carcinomas. Gut. 54, 1162 – 1168.

Nault, JC, De Reyniès, A, Villanueva, A, Calderaro, J, Rebouissou, S, Couchy, G, Decaens, T,
Franco, D, Imbeaud, S, Rousseau, F, Azoulay, D, Saric, J, Blanc, JF, Balabaud, C, Bioulac-Sage,
P, Laurent, A, Laurent-Puig, P, Llovet, JM, Zucman-Rossi, J. (2013). A hepatocellular
carcinoma 5-gene score associated with survival of patients after liver resection.
Gastroenterology. 145, 176 - 187.

Ng, SA, Lee, C. (2011). Hepatitis B virus x gene and hepatocarcinogenesis. J Gastroenterol. 46,
974 – 990.

Oh, JH, Do, HJ, Yang, HM, Moon, SY, Cha, KY, Chung, HM, Kim, JH. (2005). Identification of
a putative transactivation domain in human Nanog. Exp Mol Med. 37, 250 – 254.

Park, SW, Do, HJ, Huh, SH, Sung, B, Uhm, SJ, Song, H, Kim, NH, Kim, JH. (2012).
Identification of a putative nuclear export signal motif in human NANOG homeobox domain.
Biochem Biophys Res Commun. 421, 484 – 489.

Park, Y.H., Shin, H.J., Kin, S.U., Kim, J.M., Kim, J.H., Bang, D.H., Chang, K.T., Kim, B.Y.,
Yu, D.Y. (2013). iNOS promotes HBx-induced hepatocellular carcinoma via upregulation of
JNK activation. Biochem. Biophys. Res. Commun. 435, 244 – 249.

Paterlini, P, Poussin, K, Kew, M, Franco, D, Brechot, C. (1995). Selective accumulation of the X
transcript of hepatitis B virus in patients negative for hepatitis B surface antigen with
hepatocellular carcinoma. Hepatology. 21, 313 – 321.

Pilati, C, Letouzé, E, Nault, JC, Imbeaud, S, Boulai, A, Calderaro, J, Poussin, K, Franconi, A,
Couchy, G, Morcrette, G, Mallet, M, Taouji, S, Balabaud, C, Terris, B, Canal, F, Paradis, V,
Scoazec, JY, de Muret, A, Guettier, C, Bioulac-Sage, P, Chevet, E, Calvo, F, Zucman-Rossi, J.
(2014). Genomic profiling of hepatocellular adenomas reveals recurrent FRK-activating
mutations and the mechanisms of malignant transformation. Cancer Cell. 25, 428 – 441.

Poon, RT, Fan, ST. (2003). Evaluation of the new AJCC/UICC staging system for hepatocellular
carcinoma after hepatic resection in Chinese patients. Surg Oncol Clin N Am. 12, 35 - 50.

Provost, E, Yamamoto, Y, Lizardi, I, Stern, J, D’Aquila, TG, Gaynor, RB, Rimm, DL. (2003).
Functional correlates of mutations in beta-catenin exon 3 phosphorylation sites. J Biol Chem.
278, 31781- 31789.

Rosenbluh, J., Wang, X., Hahn, W.C. (2014). Genomic insight into WNT/β-catenin signaling.
Trends Pharmacol. Sci. 25, 103 – 109.
48

Scholer-Dahirel, A, Schlabach, MR. (2011). Maintenance of adenomatous polyposis coli (APC)-
mutant colorectal cancer is dependent on Wnt/β-catenin signaling. Proc Natl Acad Sci. 108,
17135–17140.

Seeger, C, Mason, WS. (2000). Hepatitis B virus biology. Microbiol Mol Biol Rev. 64, 51 – 68.

Shibata, T, Aburatani, H. (2014). Exploration of liver cancer genome. Nat Rev Gastroenterol
Hepatol. 11, 340 – 349.

Shtutman, M, Zhurinsky, J, Simcha, I, Albanese, C, D’Amico, M, Pestell, R, Ben-Ze’ev, A.
(1999). The cycling D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci.
96, 5522 – 5527.

Subramaniam, S, Kelley, RK, Venook, AP. (2013). A review of hepatocellular carcinoma (HCC)
staging systems. Chin Clin Oncol. 2, 1 – 12.

Sun, C, Sun, L, Jiang, K, Gao, DM, Kang, XN, Wang, C, Zhang, S, Huang, S, Qin, X, Li, Y, Liu,
YK. (2013). NANOG promotes liver cancer cell invasion by inducing epithelial-mesenchymal
transition through NODAL/SMAD3 signaling pathway. Int J Biochem Cell Biol. 45, 1099 –
1108.

Suzuki, A, Raya, A, Kawakami, Y, Morita, M, Matsui, T, Nakashima, K, Gage, FH, Rodriguez-
Esteban, C, Izpisúa Belmonte, JC. (2006). Nanog binds to Smad1 and blocks bone
morphogenetic protein-induced differentiation of embryonic stem cells. Proc Natl Acad Sci. 103,
10294 – 10299.

Sze, KMF, Chu, GKY, Lee, JMF, Ng, IOL. (2013). C-Terminal Truncated Hepatitis B virus X
protein is associated with metastasis and enhances invasiveness by c-Jun/Matrix
Metalloproteinase Protein 10 activation in hepatocellular carcinoma. Hepatology. 57, 131 – 139.

Tang, Y, Luo, Y, Jiang, Z, Ma, Y, Lin, CJ, Kim, C, Carter, MG, Amano, T, Park, J, Kish, S,
Tian, XC. (2012). Jak/Stat3 signaling promotes somatic cell reprogramming by epigenetic
regulation. Stem Cells. 30, 2645 – 2656.

Tao, J, Calvisi, DF, Ranganathan, S, Cigliano, A, Zhou, L, Singh, S, Jiang, L, Fan, B,
Terracciano, L, Armeanu-Ebinger, S, Ribback, S, Dombrowski, F, Evert, M, Chen, X, Monga,
SP. (2014). Activation of β-catenin and Yap1 in human hepatoblastoma and induction of
hepatocarcinogenesis in mice. Gastroenterology. 147, 690 – 701.

Tarafdar, A, Dobbin, E, Corrigan, P, Freeburn, R, Wheadon, H. (2013). Canonical Wnt signaling
promotes early hematopoietic progenitor formation and erythroid specification during embryonic
stem cell differentiation. PLoS ONE. 8, 1 – 15.

Tetsu, O, McCormick, F. (1999). Beta-catenin regulates expression of cycling D1 in colon
carcinoma cells. Nature. 398, 422 – 426.
49

Tian, Y, Yang, W, Song, J, Wu, Y, Ni, B. (2013). Hepatitis B virus x protein-induced aberrant
epigenetic modifications contributing to human hepatocellular carcinoma pathogenesis. Mol Cell
Biol. 33, 2810 – 2816

Tornesello, ML, Buonaguro, L, Tatangelo, F, Botti, G, Izzo, F, Buonaguro, FM. (2013).
Mutations in TP53, CTNNB1 and PIK3CA genes in hepatocellular carcinoma associated with
hepatitis B and hepatitis C virus infection. Genomics. 102, 74 – 83.

Vauthey, JN, Lauwers, GY, Esnaola, NF, Do, KA, Belghiti, J, Mirza, N, Curley, SA, Ellis, LM,
Regimbeau, JM, Rashid, A, Cleary, KR, Nagorney, DM. (2002). Simplified staging for
hepatocellular carcinoma. J Clin Oncol. 20, 1527 - 1536.

Villanueva, A, Llovet, JM. (2014). Mutational landscape of HCC—the end of the beginning. Nat
Rev Clin Oncol. 11, 73–74.

Villanueva, A, Llovet, JM. (2011). Targeted therapies for hepatocellular carcinoma.
Gastroenterology. 140, 1410–1426.

Wang, ML, Chiou, SH, Wu, CW. (2013). Targeting cancer stem cells: Emerging role of Nanog
transcription factor. OncoTargets Ther. 6, 1207 – 1220.

World Health Organization. Hepatitis B fact sheet N°204. Geneva: World Health Organization;
2015.

Wurmbach, E, Chen, YB, Khitrov, G, Zhang, W, Roayaie, S, Schwartz, M, Fiel, I, Thung, S,
Mazzaferro, V, Bruix, J, Bottinger, E, Friedman, S, Waxman, S, Llovet, JM. (2007). Genome-
wide molecular profiles of HCV-induced dysplasia and hepatocelular carcinoma. Hepatology.
45, 938 – 947.

Xu, Z, Yen, B, Lanying, W, Madden, CR, Tan, W, Slagle, BL, Ou, JJ. (2002). Enhancement of
hepatitis B virus replication by its X protein in transgenic mice. J Virol. 76, 2579 – 2584.

Yan, H, Zhong, G, Xu, G, He, W, Jing, Z, Gao, Z, Huang, Y, Qi, Y, Peng, B, Wang, H, Fu, L,
Song, M, Chen, P, Gao, W, Ren, B, Sun, Y, Cai, T, Feng, X, Sui, J, Li, W. (2012). Sodium
taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D
virus. eLife. 1, 1 – 28.

Ye, SL, Takayama, T, Geschwind, J, Marrero, JA, Bronowicki, JP. (2010). Current approaches
to the treatment of early hepatocellular carcinoma. The Oncologist. 15, 34 – 41.

Zhang, T, Zhang, J, You, X, Liu, Q, DU, Y, Gao, Y, Shan, C, Kong, G, Wang, Y, Yang, X, Le,
L, Zhang, X. (2012). Hepatitis B virus x protein modulates oncogenes yes-associated protein by
CREB to promote growth of hepatoma cells. Hepatology 56, 2051 – 2059.

50

Zheng, Y, Chen, WL, Louie, SG, Yen, B, Ou, JJ. (2007). Hepatitis B virus promotes
hepatocarcinogenesis in transgenic mice. Hepatology. 45, 16 – 21.

Abstract (if available)

Abstract Hepatocellular carcinoma (HCC) is the fifth most common type of cancer. The mortality rate continues to rise every year. Hepatitis B virus (HBV) is a major risk factor associated with the development of HCC, due to its ability to regulate cell stemness and proliferation factors, and cause liver cirrhosis and constant inflammation. As sorafenib treatment is not curative, tumor-initiating stem-like cells (TICs) contributes to the low survival rates of HCC patients. Gaining a better understanding of the mechanisms that are involved in the activation of TIC regulators will provide rationale for targeted treatments for those HCC patients. Here we show that HBV X Protein (HBx) can upregulate both β-catenin and NANOG pluripotency transcription factor expression, where they then translocate into the nucleus upon activation. Coexpression of constitutively-active β-catenin or YAP1 with NANOG transactivate Survivin (Birc5) promoter in a liver progenitor cell line. Coexpression of constitutively active β-catenin with NANOG promotes self-renewal ability in hepatoblasts. Expression of HBx in the presence or absence of NANOG expression in hepatoblasts promotes tumor development in immune-competent mice transplanted with hepatoblasts. Non-invasive live animal imaging using ultrasound and microCT method confirmed tumor development. Taken together, coexpression of constitutively active β-catenin with NANOG promotes self-renewal ability and HBx protein plays a key role for oncogenic transformation of hepatic progenitor cells.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Roles of epithelial-mesenchymal transition and niche in tumorigenesis of tumor-initiating cells

PDF

Polycomb repressive complex 2 subunit stabilizes NANOG to maintain self-renewal in hepatocellular carcinoma tumor-initiating stem-like cells

PDF

Steatosis induces Wnt/β-catenin pathway to stimulate proliferation of hepatic tumor initiating cells and promote liver cancer development

PDF

Identification of molecular mechanism for cell-fate decision in liver; &, SARS-CoV replicon inhibitor high throughput drug screening

PDF

Tri-specific T cell engager immunotherapy targeting tumor initiating cells

PDF

Mechanisms of nuclear translocation of pyruvate dehydrogenase and its effects on tumorigenesis of hepatocellular carcinoma…

PDF

Targeting mitochondrion-nucleus PDH1 transfer to suppress self-renewal and epigenetic NANOG reprogramming of tumor-initiating cells

PDF

Role of TLR4 and AID in lymphomagenesis induced by obesity and hepatitis C virus

PDF

Musashi-2 promotes c-MYC expression through IRES-dependent translation and self-renewal ability in hepatocellular carcinoma

Asset Metadata

Creator Nakagawa, Chad Isamu (author)

Core Title Hepatitis B virus X protein regulation of β-catenin and NANOG and co-regulatory role with YAP1 in HCC malignancy

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 07/30/2015

Defense Date 06/18/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag HBx,HCC,hepatitis B virus X protein,hepatocellular carcinoma,NANOG,OAI-PMH Harvest,TICs,tumor initiating cells,YAP1,β-catenin

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Machida, Keigo (committee chair), Ou, J.-H. James (committee member), Tsukamoto, Hidekazu (committee member)

Creator Email cnakagaw@alumni.uci.edu,cnakagaw@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-615884

Unique identifier UC11303307

Identifier etd-NakagawaCh-3759.pdf (filename),usctheses-c3-615884 (legacy record id)

Legacy Identifier etd-NakagawaCh-3759.pdf

Dmrecord 615884

Document Type Thesis

Format application/pdf (imt)

Rights Nakagawa, Chad Isamu

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA