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Steatosis induces Wnt/β-catenin pathway to stimulate proliferation of hepatic tumor initiating cells and promote liver cancer development
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Steatosis induces Wnt/β-catenin pathway to stimulate proliferation of hepatic tumor initiating cells and promote liver cancer development
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i
Steatosis induces Wnt/β-catenin pathway to stimulate proliferation of
hepatic tumor initiating cells and promote liver cancer development
By
Anketse Debebe
A Dissertation presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY
December 2015
Copyright 2015 Anketse Debebe
ii
Dedication
To my family
iii
Acknowledgements
There is an African proverb that says, “It takes a village to raise a child”.
The same dogma applies in completing PhD. My dissertation would not have
been completed if it weren’t for the contribution of the many people in my life to
whom I owe my utmost gratitude.
I would like to express my sincere gratitude to my advisor Dr. Bangyan
Stiles for her continuous support and guidance throughout the course of my Ph.D
at USC. Her passion for science along with her patience, motivation,
enthusiasm, and immense knowledge helped me to grow scientifically by
allowing me to develop critical thinking abilities, scientific writing and mentoring
skills. I could not have imagined having a better advisor and mentor.
My deepest gratitude goes out to my committee members Dr. Curtis
Okamoto and Dr. Keigo Machida for their encouragement, insightful comments,
and thought provoking questions at different stages of my research. I also would
like to thank Dr. Bogdan Olenyuk and Dr. Kasper Wang for their questions and
discussion during my oral qualifying exam. Their tremendous support helped
propel my research forward.
I want to thank my fellow past and present lab mates Ni Zeng, Richa
Aggarwal, Vivian Galicia, Jennifer Bayan, Lina He, Yang Li, Zhechu Peng, Jing
yu Chen, Joshua Chen, Kelly Yang, Chengyou Jia, Indra Mahajan, Mengchen,
Yating Guo, Fan Fei, Jianyan Huang, and Jing Chen for their helpful ideas,
stimulating discussions and for creating a great collaborative atmosphere in our
lab. They have made my five years at USC so memorable.
iv
I would like to acknowledge Dr. Kahn and his lab with all their help with the ICG-
001 project, Dr. Machida’s lab for providing us with reagents and samples, Dr.
Tsukamoto’s lab for their help with ALT and the USC flow cytometry core for their
technical assistance. I appreciate the financial support from California Institute
for Regenerative Medicine (CIRM) for funding my research for two years.
I also want to thank my friends for their support and for helping me
overcome setbacks and helped me stay focused on my research. My heartfelt
gratitude goes to my family to whom this dissertation is dedicated to, for their
tremendous love, concern, support, and strength. Last but not least, I would like
to thank my husband, Teddy, for being there for me through the good and bad
times. He has been my constant source of motivation and love and I would have
not made it this far without his unwavering love and support. Finally, I want to
thank my son, Lukas, for being my inspiration.
v
Table of Contents
Dedication ………………………………………………………………………... II
Acknowledgements………………………………………………………………. III
Table of Contents………………………………………………………………... V
List of Figures…………………………………………………………………….. VII
List of Tables……………………………………………………………………... VIII
Abstract……………………………………………………………………………. IX
Chapter I: Overview of hepatic steatosis, Wnt/β-catenin and PI3K/AKT
pathway in liver cancer development…………………………………………..
1
I-1 Obesity and Cancer………………………………………………………..
I-2 Role of hepatosteatosis in liver cancer………………………………….. 1
I-3 Liver architecture, statistics and types of liver cancer………………… 2
I-4 Signaling Pathways involved in Liver Cancer………………………….. 3
I-5 PTEN/PI3K/AKT pathways and its role in cancer……………………… 5
I-6 The Wnt/β-catenin signaling pathway…………………………………… 6
Chapter II: Steatosis induces Wnt signal; a niche for liver progenitor cells.. 10
II-1 Introduction and Rationale………………………………………………. 10
II-2 Results…………………………………………………………………….. 11
II-2-1 Steatosis induces Wnt signal…………………………………………. 11
II-2-2 HFD fed mice display fatty liver, hepatic injury, Wnt/β-catenin and
progenitor cell upregulation…………………………
15
II-3 Discussion………………………………………………………………… 20
Chapter III: Inhibition of Wnt/β-Catenin pathway in vitro and in vivo
attenuates proliferation of CD133+ hepatic tumor initiating cells……………
22
III-1 Introduction and Rationale…………………………………………….... 22
III-2 Results…………………………………………………………………….. 23
III-2-1 Tumor initiating cell proliferation is attenuated in β-catenin KD
cells in vitro and in vivo……………………………………………………….
23
III-2-2 β-catenin inhibitor (ICG-001) ameliorates liver TIC proliferation
and reduces expression of progenitor cell
markers………………………….
29
III-2-3 Knocking down β-cateninin liver progenitor cells (β-catenin
lox/lox
;
Sox9-CreERT) results in reduction in progenitor cell and induction in ductal
cell number........................................................................................................
37
III-2-4 Inhibition of Wnt signal blocks human TIC accumulation………….. 43
III-3 Discussion……………………………………………………………… 44
Chapter IV: Calorie Restriction abrogates hepatic steatosis, inhibits TIC
expansion and abolishes tumor development in Pten null livers……………
46
IV-1 Introduction and Rationale……………………………………………… 46
IV-2 Results……………………………………………………………………. 47
IV-2-1 Pten/Akt2 double mutant mice display reduced hepatosteatosis
along with decreased TIC numbers………………………………………….
47
IV-2-2 Calorie Restriction reduces body weight and improves insulin
sensitivity……………………………………………………………………
48
vi
IV-2-3 Calorie Restriction abrogates hepatic steatosis, injury, Wnt/β-
catenin signaling and progenitor cell expansion in Pten null mice…….
53
Chapter V: Overall Discussion…………………………………………………. 59
Chapter VI: Materials and Methods and Protocol Development and
Bibliography……………………………………………………………………….
68
VI-1 Materials and Methods and Protocol Development………………….. 68
VI-2 Bibliography………………………………………………………………. 79
vii
List of Figures
Figure 1. Signaling Pathway and it’s role in survival, proliferation and
metabolism
Figure 2. Liver steatosis induces Wnt/β-catenin signal
Figure 3. Schematic diagram of High Fat Diet (HFD)/Low Fat Diet (LFD)
Feeding
Figure 4. Intraperitoneal glucose tolerance and insulin tolerance test
Figure 5. HFD fed mice have increased body weight (BW) and Liver weight
(LW)
Figure 6. HFD induced steatosis activates Wnt/β-catenin signal
Figure 7. Inhibiting β-catenin downregulates TIC markers and reduce their
growth
Figure 8. Down-regulating β-catenin attenuates TIC proliferation and reduces
hepatic TIC markers in vitro
Figure 9. In vivo downregulation of β-catenin abrogates tumor growth and
expression of hepatic progenitor cell markers
Figure 10. Tumor is undetected when β-catenin downregulated liver progenitor
cells are injected orthotopically in the liver
Figure 11. Inhibiting β-catenin with ICG-001 blocks TIC proliferation and
reduces expression of progenitor cell markers
Figure 12. Illustrated protocol used for ICG-001 treatment
Figure 13. Inhibiting β-catenin with ICG-001 ameliorates TIC proliferation
Figure 14. Inhibiting β-catenin with ICG-001 ameliorates TIC proliferation and
reduces expression of progenitor cell markers in the Pten null liver
Figure 15. DDC induces Wnt and liver Injury
Figure 16. β-catenin loss in Sox9 positive liver progenitor cells leads to reduction
in TIC numbers
Figure 17. Induction of ductal hyperplasia is likely due to differentiation of liver
progenitors and not proliferation of ductal epithelial cells
Figure 18. ICG-001 treatment of human TICs reduces Cyclin D1 and Survivin
expression as well as progenitor cell markers
Figure 19. AKT2 loss abrogates hepatic steatosis, Wnt signaling and TIC
number
Figure 20. Schematic Diagram of Calorie Restriction (CR) and Ad Libitum (AL)
feeding in Pten null and Wild Type mice
Figure 21. CR reduces body weight (BW) and liver weight (LW) in both WT and
Pten null mice.
Figure 22. Intraperitoneal glucose tolerance and insulin tolerance test
Figure 23. Calorie Restriction (CR) blocks lipid accumulation, injury and Wnt/β-
Catenins
Figure 24. CR abrogates reduces liver TICs and blocks tumor formation in Pten
null mice
Figure 25. Schematic representation of the working model that steatosis
promotes tumor progression through Wnt.
viii
List of Tables
Table 1. Tumor incidence in CR and Akt2 deleted Pten null mice vs. controls
Table 2. Diet composition table
Table 3. Primers used for qPCR analysis for mouse genes
ix
Abstract
Obesity is an independent risk for carcinogenesis including liver cancer.
Liver cancer is an aggressive and deadly disease with poor outcome.
Approximately half of hepatocellular carcinoma (HCC) presents progenitor cell
signatures, indicative of the presence of Tumor Initiating Cell (TIC) populations.
Despite many progress in characterizing the physiology of hepatic TICs, a strong
need remains to delineate signaling pathways regulating these cells. Our lab
previously established a mouse model specifically lacking PTEN (phosphatase
and Tensin homologue on Chromosome 10), a critical negative regulator of
PI3K/AKT, in liver hepatocytes. We have shown that loss of PTEN induces fatty
liver mediated hepatic injury followed by TIC expansion and upregulation of
hepatic progenitor cell markers. In addition, we also found factors governing
progenitor niche such as Wnt ligands including Wnt7a and 10a and Wnt receptor
Fzd, as well as β-catenin (a downstream factor of Wnt signaling) are upregulated.
Wnt/β-catenin has been shown to promote self-renewal and proliferation of TICs.
Furthermore, interrupting Wnt/β-catenin pathway using shRNA and small
molecule inhibitor resulted in attenuation of TIC proliferation. This prompted us to
assess what biological mechanism mediates the induction of the Wnt/β-catenin
pathway resulting in TIC proliferation and liver cancer using the Pten null liver
cancer model.
Preliminary data from our lab demonstrates Pten deletion in the liver
results in steatosis and fatty liver disease at 1 month of age and subsequent
development of liver cancer at 9 to 12 months. This observation is in line with the
x
strong link between obesity and liver cancer. Majority of liver cancer patients
experience liver steatosis as a pre-condition before cancer development. The
focus of my doctoral studies is to unveil how fatty liver, a stereotypical feature of
obesity contribute to liver cancer development. The central hypothesis for my
research is hepatic steatosis stimulates TIC activation and liver cancer
development through the Wnt/β-catenin signaling pathway. To test this
hypothesis a non-genetic approach, High Fat Diet (HFD) and Calorie Restriction
(CR) feeding was used to investigate the development and blockage of steatosis
respectively. Indeed, HFD feeding led to an increased lipid accumulation and
liver injury and further induced wnt signals whereas CR not only blocked
steatosis but also led to the reduction of wnt signals and abrogated expression of
hepatic TICs. This data established Wnt/β-catenin as a novel signal induced by
steatosis to promote tumor growth, underlying the increased risk of tumor
development in obese individuals.
In summary, data from these studies established that hepatosteatosis
resulting from Pten deletion or high fat feeding induces activation of Wnt/β-
catenin pathway. Wnt/β-catenin signal induces the expansion of Pten deletion
transformed liver TICs. In Pten null
mice fed a low caloric diet to inhibit
hepatosteatosis, reduced Wnt/β-catenin activation is observed concurrent with
inhibited expansion of TICs and complete blockage of tumorigenesis. Blocking
Wnt/b-catenin signal with pharmacological or genetic approaches inhibits the
proliferation of TICs in vitro, decreases accumulation of TICs and reduces tumor
grafts in vivo. Given that both obesity and liver cancer epidemic is on the rise in
xi
the US, understanding how fatty liver contributes to hepatic tumorigenesis will not
only delineate the interaction between lipid metabolism and cancer development,
but also hold promise for developing effective treatment and eventually
eradicating liver cancer.
1
Chapter I
Overview of hepatic steatosis, Wnt/β-catenin and PI3K/AKT pathway in liver
cancer development
I-1 Obesity and cancer
The prevalence of obesity has increased markedly over the past two
decades with an estimated alarming two-thirds of U. S. adults being overweight
or obese. In recent years, obesity is recognized as a cofactor for cancer
development. One out of three cancer deaths are attributed to individuals who
are overweight or obese. However, how obesity contributes to the increased
cancer risk is unknown. Given that the worldwide obesity epidemic continues to
increase, there is an urgent need to delineate the mechanisms by which obesity
contributes to the development of cancer and its progression. This is essential
for the design of new therapeutic approaches to intervene in this process.
I-2 Role of hepatosteatosis in liver cancer
Obesity is a major compounding factor for cancer. One out of three
cancer deaths occur in individuals who are overweight or obese [1,2]. This
correlation is particularly strong in liver cancer where male with a body mass
index (BMI) >35 have a 4.52 fold higher incidence of liver cancer vs. those with
BMI between 18.5 and 24.9 [3]. In the clinic, approximately 80% of liver cancer
patients also have underlying liver diseases that display lipid metabolic
dysfunctions [4], a condition that develops in all obese individuals. In this study,
we used liver cancer as a model system to address the contribution of obesity to
cancer.
2
I-3 Liver architecture, statistics and types of liver cancer
The liver is a large organ consisting of four lobes that orchestrate various
metabolic functions. The hepatic lobule is made up of hexagonal arrangement of
hepatocytes radiating away from the central vein. At each vertices of the
hexagon is a portal triad containing the hepatic artery, portal vein and the bile
duct. The liver consists of parenchymal and various non-parenchymal cell all
playing distinct yet significant role. The liver is organized in a zonal fashion
depending on oxygen availability. Hepatocytes in zone 1 region receive the most
oxygenated blood and are responsible for gluconeogenesis, β-oxidation and
cholesterol synthesis while zone 3 hepatocytes get limited oxygen and carry
glycolysis, lipogenesis and drug detoxification functions.
Liver cancer is the third most lethal malignancy worldwide. This year, an
estimated 26,190 new cases of this cancer will arise in the U.S. alone. The
relative 5-year survival rate for liver cancer is less than 17%. The prevalence of
liver cancer continues to rise in western countries and more than 700,000 people
are diagnosed with liver cancer each year throughout the world, highlighting the
importance of liver cancer research[5]. Chronic liver damage caused by viral
infection, alcohol abuse and obesity makes one susceptible to acquiring HCC.
Hepatitis B and C viruses are the common risk factors associated with liver
cancer. Excessive alcohol consumption and viral hepatitis work synergistically in
causing liver damage[6]. Obesity which leads to fatty liver disease and
steatohepatitis increases the risk of getting liver cancer.
3
There are four main types of liver cancer that have been identified.
Heptatocellular carcinoma is the most common kind of liver cancer accounting for
approximately 80% of all liver cancers followed by cholangiocarcinoma also
referred as the bile duct cancer, which affects the ducts that carry bile to the gall
bladder. Angiocarcinoma and hepatoblastoma are the extremely rare types of
liver cancer affecting blood vessels and young children respectively.
Hepatoblastoma originates from immune liver precursor cells and occurs in
infants and children under the age of 3.
I-4 Signaling Pathways involved in Liver Cancer
From a molecular biology perspective, the pathogenesis of liver cancer is
attributed to various cellular signaling pathways. The NF-kB, stat3, MAPK, HGF
receptor MET, PI3K/AKT, and Wnt/β-catenin are some of the signaling pathways
that have been extensively studied to understand liver pathogenesis.
During liver injury, the immune system is activated and it produces
Reactive Oxygen Species (ROS) to induce inflammatory response. One of the
important mechanisms by which ROS mediates inflammation is via NF-kB
activation. NF-κB regulates the transcription of pro-inflammatory cytokines such
as TNFα and IL-1β. TNFα and IL-1β have been shown to stimulate more ROS
production, leading to further NF-κB activation, which perpetuates the
inflammatory cycle increasing the risk of acquiring HCC. HFD induced obesity
increases NF-kB signaling[7] which may further contribute to the amplification of
pro-inflammatory cytokines. Therefore, inactivation of NF-kB in hepatocytes
leads to suppression of HCC[8]. In contrast, ablation of IK-kB an upstream
4
activating signal for NF-kB also led to the progression of HCC upon simultaneous
DEN treatment.
Other signals have also been shown to play a contradictory role in HCC
development. Increased activity of cJun NH2-terminal kinase (JNK) with DEN
injection promotes inflammation followed by tumor development whereas JNK
ablation in hepatocytes also increased tumor burden[9]. The opposing roles in
promoting and suppressing liver cancer development is also evident in stat3 and
HGF receptor, MET. Overexpression of stat3 and MET enhances HCC
development. On the contrary, hepatic specific stat3 and MET knock out mice
that were administered DEN had larger HCC tumors. Wnt/β-Catenin signaling
pathway has various roles in regulating cell growth, development and
differentiation. However, similar paradox has been observed in the role of β-
catenin as either a tumor promoter or tumor suppresser. Hyperactivation of β-
catenin in hepatocytes led to carcinogenesis while hepatic specific deletion of β-
catenin exhibited higher susceptibility to DEN induced hepatocarcinogenesis due
to increased oxidative stress and hepatic inflammation. These seemingly
conflicting data show the complexity of the cell machinery and interdependence
of different signaling molecules. These findings highlight the importance of not
only looking at cell specific gene knock out studies but also looking at the entire
microenvironment that interplays and dictates how this molecules behave as a
result of injury.
5
I-5 PTEN/PI3K/AKT pathways and its role in cancer
Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate a
variety of cellular processes including growth, proliferation, differentiation,
survival, cellular glucose and lipid metabolism [10]. PI3K is a heterodimer that is
composed of a regulatory subunit (p85) and catalytic subunit (p110). The PI3K
pathway gets activated in the presence of growth factors binding to receptors and
phosphorylated at tyrosine residues. This allows PI3K (p85-p110) to be recruited
to the receptor causing activation of the signaling cascade. Activated PI3K
catalyzes the reaction from Ptdlns (4,5) P2 to Ptdlns (3,4,5) P3. It does so by
phosphorylating the hydroxyl group of the 3rd position on the inositol ring of
Phosphatidylinositols (Ptdlns) [11] providing a docking site for AKT. AKT is then
recruited to the membrane and activated to transmit the receptor-binding signal
to its downstream molecules. Activated AKT has a plethora of downstream
functions including the regulation of kinases such as GSK3β,[12] IκB kinases
(IKKα and IKKβ);[13] apoptotic factors such as BAD;[14] MDM2, a ubiquitin
ligase for p53;[15] GTPases such as Rac and Rho;[16] cell cycle inhibitors p21
and p27;[17] and transcription factors such as forkhead transcription family
(FoxO) members[18,19,20] (Figure 1).
The PI3K–AKT axis plays a pivotal role in cell growth, proliferation and
survival and its dysregulation is commonly linked with cancers. Studies over the
past decades have indicated that the constitutive activation of the PI3K–AKT
cascade is associated with a wide spectrum of human cancers, including
glioblastoma, ovarian cancer, breast cancer, hepatocellular carcinoma and lung
6
cancer. PI3K alterations such as p110α amplification and p85α mutation
frequently occurred in ovarian, breast and lymphoid cancers[21,22,23]. Other
PI3K signal activations are due to AKT overexpression and amplification[24]. In
addition, PI3K signal hyperactivation resulting from different types of PTEN
alterations such as mutation, silencing, aberrant transcripts and allelic imbalance
leads to tumor formation[10]. Therefore, both PTEN alterations and PI3K
mutations are associated with tumorigenesis.
PTEN (Phosphatase and Tensin Homologue deleted on chromosome 10)
[25] is a phosphoprotein/phospholipid dual-specificity phosphatase that
antagonizes the activity of PI3K by removing the phosphate from the 3rd position
on (PIP3), leading to reduced PIP3 production and the down-regulation of signals
associated with AKT. PTEN is frequently lost or mutated in various forms of
cancer, and thus identified as a tumor suppressor[26]. Aberrant regulation of
PTEN is found in 30-50% of liver cancer patients. Pten deletion in mice results in
fatty liver and inflammation followed by liver cancer development[25,27]. This
observation is in accordance with the association between obesity and liver
cancer development in humans.
I-6 The Wnt/β-catenin signaling pathway
The Wnt/β-catenin pathway plays a critical role in proliferation,
differentiation, survival and self-renewal. Binding of Wnt to its Frizzled receptor
and co-receptor low density lipoprotein-related protein 5/6 (LRP5/6) activates
disheveled which further inactivates the degradation complex that is comprised of
glycogen synthase kinase 3β (GSK3β), adenomatous polyposis coli (APC) and
7
Axin. β-catenin, thus accumulates and translocates to the nucleus to bind to
lymphoid enhancer-binding factor/T cell factor (LEF/TCF) promoting changes in
transcriptional machinery leading to the activation of several target genes such
as cyclin D1 and survivin. Studies in various organ systems have identified the
Wnt/β-catenin signaling pathway as a crucial regulator of progenitor cell
populations[28,29]. In intestinal crypt, it’s been shown that progenitors
accumulate nuclear β-catenin and activate Wnt target genes that are responsible
for stem cell renewal[30]. Wnt/β-catenin is also implicated to play a role in
hepatocellular carcinoma[31,32]. Disruption of Wnt/β-Catenin pathway is
observed in one third of all hepatocellular carcinomas[33]. Mutations in the β-
catenin gene have been found in 20-40% of HCC patients. In murine liver,
mutation of β-catenin promotes self-renewal and propagation of progenitor
cells[34,35].
Genome-wide analysis of liver cancer samples suggests that the majority
of the human liver cancer specimen display progenitor cell signatures, indicative
of the presence of TIC populations [36,37,38]. Like in other solid tumors, hepatic
tumor initiating cells (TICs) are rare malignant cells that possess tumorigenic
abilities that drive tumor initiation, self-renewal, chemo-resistance, and
metastasis, playing an important role in tumor development and progression
[39]. TICs also known as cancer stem cells (CSCs) possess similar
characteristics to that of normal stem cells such as self-renewal and
differentiation but play an important role in tumor development and progression
and are elevated in chronic liver diseases[39]. Insult to the liver in chronic liver
8
diseases impairs hepatocyte proliferation and regeneration, resulting in TIC
expansion. These cells are believed to reside in the canals of Hering that
connect the bile canaliculus and the biliary tree[40]. Accumulating evidence
supports TICs; hepatic progenitor cells that gain genetic mutation and possess
tumorgenic ability, as key players that drive tumor initiation, self-renewal,
chemoresistance, and metastasis, which explains the reason for failure of
existing therapies[41]. Despite much progress has been made in understanding
and characterizing TICs, the signaling mechanism behind TIC activation and
proliferation remains unknown. The presence of TICs is thought to account for
the high cellular heterogeneity observed with HCC, which leads to the challenge
for therapeutic targeting [42]. While it is unclear whether these TICs originate
from progenitors or dedifferentiated hepatocytes or other sources [43], the
contribution of TICs to generate all tumorigenic and non-tumorigenic cell types
within a tumor is undisputed [44].
In a mouse model where deletion of Pten leads to hepatosteatosis and
later tumor phenotypes [25,27,45,46,47], previous work in our lab demonstrated
that a CD133+/CD45- cell population isolated from Pten null experimental liver
cancer model is capable of grafting multilineage tumors and shows resistance to
chemotherapy which are two hallmarks of progenitor induced tumors[48]. In this
model (Pten
loxP/loxP
; Alb-Cre
+
), we have shown that the presence of the underlying
fatty liver condition is required for the development of liver cancers, which
expands from a population of TICs [45,46,48]. Robust activation of Wnt ligands
and receptors as well as an increase in β-catenin is observed simultaneously.
9
Using this model, my work show here that Wnt/β-catenin activation signal is the
tumor promoting factor induced by lipid accumulation and that this signal is
critical for the expansion of the transformed liver TICs and tumor growth. My
thesis work underlies how steatosis is likely inducing Wnt to signal the
accumulation of TICs, thereby contributing to tumor progression using the Pten
null mouse model.
Figure 1. PI3K/AKT Signaling pathway and its role in survival, proliferation
and Metabolism. Upon ligand mediated receptor tyrosine kinases (RTKs)
activation, PI3K is recruited to the cell membrane where it converts
phosphatidylinositol phosphate 2 (PIP2) to PIP3 further activating AKT.
Activated AKT has a plethora of downstream functions including cell survival,
metabolism, cell growth, and proliferation. PTEN, on the other hand, is a lipid
phosphatase that negatively regulates the PI3K signaling pathway.
10
Chapter II
Steatosis induces Wnt signal; a niche for liver progenitor cells
II-1 Introduction and Rationale
Recent studies suggest excessive adiposity due to overconsumption of
energy dense foods increases the risk of developing cancer[49]. According to
various epidemiological studies, endometrial, breast, colon, esophageal, kidney,
pancreatic, gallbladder, and liver cancers have been associated with increased
adiposity[49,50]. Results from various studies indicate that obesity is associated
with a 1.5 to 4 fold increased risk in developing liver cancer in both men and
women[49,51]. Fatty liver is a common pre-condition associated with non-
alcoholic fatty liver disease (NAFLD) induced hepatocellular carcinoma observed
in human patients. These patients experience steatohepatitis, cirrhosis and
inflammation before developing liver cancer. To investigate the role of lipid
accumulation in tumor development, we employed a liver cancer model system
where fatty liver develops prior to tumor formation (Stiles PNAS, Galicia Gastro).
In this model (Pten
loxP/loxP
; Alb-Cre
+
), loss of PTEN, the negative regulator of the
PI3K/AKT signaling pathway, results in liver steatosis starting at 1 months of age
and tumor onset at about 7-8 months of age. Together with loss of PTEN
function, activation of PI3K/AKT signal occurs in more than 80% of liver cancer
patients. PI3K/AKT signaling also mediates insulin regulated lipid metabolism
occurring in diabetes and insulin resistant patients, making this a highly relevant
model for understanding the contribution of lipid accumulation to cancer
development. In this model, we reported previously that fatty liver development
11
is required for tumor progression by crossing the Pten null mice with mice lacking
AKT2, the major isoform of AKT in the liver that regulates lipogenesis (He
AmJPath; Galacia Gastro). A minimum delay of 6 months was observed for
tumor onset when Akt2 was simultaneously deleted with Pten and steatosis was
diminished. Additionally, our lab has discovered that hepatic Pten deletion in
mice not only causes significant fatty liver but also injury and hepatocyte
apoptosis. These conditions provide the driving force for hepatic progenitor cell
proliferation surrounding ductal cells followed by progenitor mediated
carcinogenesis. However, how steatosis and liver injury induces TIC mediated
carcinogenesis is not clear. In this chapter, my goal is to investigate the potential
factors that may be induced by steatosis that contribute to tumorigenesis.
II-2 Results
II-2-1 Steatosis induces Wnt signal
To address how fatty liver development in Pten null mice may promote the
growth of tumors and more importantly how blocking fatty livers (by Akt2 deletion
in this case) is able to inhibit their growth, we performed microarray analysis in 9
month old mice when tumor onset is occurring in the Pten null mice (GSE70501).
In total, 6183 genes are found to be altered more than 2 fold in the Pten null vs.
control livers. Out of this group, the changes of approximately 50% (3002) genes
are due to Akt2 deletion as their expression is changed in the Dm vs. Pm
samples. In order to search for factors secreted due to fatty liver that signals
tumor growth, we screened for growth factors and cytokines that are altered by
Pten deletion and blocked by Akt2 deletion. Out of the 146 growth
12
factor/cytokine genes altered by Pten deletion, 75 are changed back by Akt2
deletion including the top one induced by Pten deletion, Spp1. Spp1 expression
is induced by 14.6 fold with PTEN loss and reduced 7 fold by Akt2 deletion on
top of PTEN loss (Fig 2A). Spp1 encodes a secreted phosphoprotein originally
characterized as a bone morphogenesis protein [52,53]. The major signaling
pathway impacted by Spp1 is the Wnt/b-catenin signal, which both controls Spp1
expression and mediates its downstream effects. We thus analyzed Wnt
signaling gene signatures and found that a number of Wnt signaling genes were
altered with Pten deletion and changed back when Akt2 is deleted (Fig 2A),
including Wnts, the secreted ligands for Wnt signal pathway. Out of the 37 Wnt
signaling genes altered by Pten deletion, 22 are also changed due to AKT2 loss
(59%).
To confirm the array analysis data and verify that Wnt signaling is induced
when steatosis occurs in the Pten null livers, we performed qPCR analysis for the
expression of several Wnt species in the Pten null mice at various ages. We
found that the expression of two of the Wnt species, Wnt 7a (40 and 20 fold) and
Wnt 10a (8 and 12 fold) were robustly induced after chronic steatosis and when
tumors develop at 12 months of age and beyond (Fig 2B). We also observed
increased expression of Fizzled 2 (Fzd 2), a Wnt receptor at 12 months of age (2
folds). Together with these upregulation of Wnt expression in the Pten null livers,
we observed induction of β-catenin, the intracellular effector of Wnt signaling, at
tumor regions and the portal triad area where pre-malignant TIC activation is
observed (Figs 2C-E). Phosphorylation of β-catenin on ser552 is indicative of β-
13
catenin activation [54]. In the Pten null livers, we observed increased β-catenin
ser552 phosphorylation as well as induction of cyclin D1 and survivin,
transcriptional targets of Wnt (Fig 2C). Histologically, β-catenin is expressed
throughout the normal liver (Fig 2D). Its levels are increased in the Pten null
livers (Fig 2C), particularly at the hyperplastic ductal tract where the progenitor
cell niche is located (Fig 2D). When the Pten null mice were crossed with the
BAT-Gal mice carrying a β-catenin transcriptional activity reporter, we found that
transcriptional activity of β-catenin was also induced in a similar region of the
liver structure (Fig 2E). These data confirms that Wnt activity is indeed induced
at the progenitor cell niche following chronic steatosis and when the Pten null
livers start to develop tumors.
14
15
Figure 2. Liver steatosis induces Wnt/β-catenin signal. A. Differentially
expressed genes in Pten null (Pm) livers that are altered by deletion of AKT2
(double mutant, Dm). B. Expression of Wnt ligands and receptors in livers of
Pten null and control mice at different age. n=5. * Indicates significant difference
from controls at P<0.05. C. Immunoblotting analysis of PTEN signal Wnt7a, 10a,
and its downstream targets (Cyclin D1 and Survivin) in TICs. GAPDH and β-actin
are loading controls. D. β-catenin (red) and Dapi stained slides showing β-
catenin staining primarily in hepatocytes in control liver sections and significantly
intense β-catenin staining in ductal structure and portal triad in the Pten null
livers sections. PT, portal triad; HP, hepatocytes. E. β-galactosidase activity was
detected in the periductal area (dark greenish blue staining in left panel) in the
Pten null liver. Right panel, H&E staining in adjacent section.
II-2-2 HFD fed mice display fatty liver, hepatic injury, Wnt/β-catenin and
progenitor cell upregulation
Based on the observation attained so far, our Pten null mice exhibit
chronic hepatic steatosis prior to progenitor cell activation during the
pathogenesis of liver cancer. Therefore, we wanted to see if steatosis is a pre-
condition for progenitor cell proliferation. Thus, we fed wild-type mice with high
fat diet (HFD) for 9 months to induce fatty liver (Fig 3). To verify that the HFD is
working, glucose tolerance test (GTT) and insulin tolerance test (ITT) was
assessed every two months. Starting at 3 months, there was significant
difference in fasting blood glucose level between HFD and LFD mice (Fig 4A).
ITT data also reveals similar trend where HFD mice display insulin resistance
16
compared to LFD fed mice (Fig 4B). Similar to previous findings, our lab
confirms increased fat accumulation upon high fat diet feeding as observed by
H&E, Oil Red O staining, and quantification of hepatic triglyceride levels (Fig 5C).
Our data is also consistent with other observations that show progressive
damage of the liver in response to HFD (Fig 5C). Mice fed with HFD for 1 month
showed increased ALT, AST, and TBil levels[55] and 4 month HFD fed mice also
displayed non-alcoholic steatohepatitis and increased ALT and AST levels[56]
indicating liver injury. Interestingly, our long term HFD feeding (6-9 months) had
a gradual decrease in ALT levels despite it being markedly different than the LFD
fed controls. Some plausible explanations for the reduced ALT levels include
liver regeneration, adaptation of the liver to the chronic high fat diet and/or
damage reduction in response to immune modulation. Furthermore, as early as
one week after the initiation of the diet regimen, HFD fed mice showed significant
increase in body weight compared to the LFD fed mice. Progressive increase in
body weight is observed in the HFD fed mice throughout the course of 9-month
diet treatment (Fig 5A). Moreover, the average body weight and liver weight in
the HFD group is significantly higher than the LFD fed counterparts (Fig 5B).
Following chronic phase lipid accumulation and injury to the liver, we observed
Wnt/β-catenin signaling induction and an increase in pro-oncogenic Wnt/β-
catenin downstream target genes cyclin D1 and survivin alongside an increase in
Wnt 7a, Wnt 10a, Frzd and β-catenin (Fig 6A). This result demonstrates that
steatosis alone is also sufficient to induce these signals without PTEN or AKT2
manipulation.
17
Figure 3. Schematic Diagram of High Fat Diet (HFD) and Low Fat Diet (LFD)
feeding. 3-month old wild type mice were fed HFD or LFD (control) for a
duration of 9 months. IHC, western blot/qPCR and blood analysis was
performed at the end of the diet-feeding regimen.
18
Figure 4. Intraperitoneal glucose tolerance and insulin tolerance test.
Intraperitoneal glucose tolerance (GTT) test and Intraperitoneal insulin tolerance
(ITT) was measured at months 1, 3, 5, 7, and 9 months after the start of HFD
diet. Fasting blood glucose was assessed every month. n=6/group. Data
represented as mean+/- SEM. Significance was indicated by * P<0.05,
**P<0.001
19
Figure 5. HFD fed mice have increased body weight (BW) and liver weight
(LW). A. HFD mice have increased BW compared to LFD mice. B. Final BW
and LW show increased BW and LW respectively in HFD mice compared to LFD
mice. No difference in liver to body weight ratio observe between LFD and HFD
fed mice n=6. C. Steatotic liver is developed in the HFD fed mice. Left, liver TG
levels; Middle, H&E and Oil Red O stained liver sections; Right, Plasma ALT
levels as indication of liver injury. All values are expressed as the mean ± SEM. *
Indicates significant difference from LFD group at P<0.05.
20
Figure 6. HFD induced steatosis activates Wnt/β-catenin Signal. qPCR
analysis of Wnt 7a, Wnt 10a, Fzd, β-catenin, Cyclin D1 and survivin in livers of
HFD and LFD fed mice. n=6. * Indicates significant difference from LFD controls
at P<0.05.
II-3 Discussion
Our finding is corroborated by other studies that show HFD induces wnt
expression. Rats injected azoxymethane, a chemical that induces colon cancer,
and supplemented with 10% corn oil or beef tallow for 44 weeks demonstrated
aberrant crypt foci, increased BrdU incorporation, elevated expression of
21
cytosolic β-catenin and cyclin D1 and increased tumor incidence. In addition,
Wnt2 and Wnt3 expressions as well as Wnt5a expression were increased in rats
fed beef tallow and corn oil respectively with or without azoxymethane treatment.
This study suggests long-term high fat diet enhances cell proliferation through
the Wnt signaling pathway[57]. Another study suggests that Wnt proteins are lipid
modified by palmitoylation, which allows for growth and self-renewal of stem
cells[31]. Together, this data show the molecular mechanism underlying HFD
induced hepatic steatosis and Wnt/β-catenin pathway activation preceding
progenitor cell proliferation. Therefore, hepatic steatosis, independent of its
etiology (Pten deletion or HFD feeding) is required for Wnt mediated progenitor
activation and liver carcinogenesis.
22
Chapter III
Inhibition of Wnt/β-Catenin pathway in vitro and in vivo attenuates
proliferation of CD133+ hepatic tumor initiating cells
III-1 Introduction and Rationale
Wnt/β-catenin plays a crucial role in regulating stem cell/tumor initiating
cell proliferation by establishing niche signals that potentiate the self-renewal of
tissue progenitor cells[58]. In intestinal crypt and hair follicle bulge, the presence of
Wnt and activation of the downstream target β-catenin are necessary for
maintaining the progenitor cell identity[59,60]. PTEN/Akt/β-catenin pathway has
also been identified to regulate self-renewal properties of breast and colon cancer
stem cells [61]. Moreover, Wnt/β-catenin signaling pathway has been crucial in
maintaining proliferation of hepatic progenitor cells under hepatotoxin mediated liver
injury.
Studies including our own demonstrate hepatic Pten loss results in cell
proliferation, survival, and malignancy. Our previous study demonstrates that
the tumors developed in the Pten null mice are dual-lineage expressing both
hepatocyte and cholangiocyte markers (Galacia 2010); and that tumors are most
likely derived from a TIC population (Rountree et al 2009). The CD133 positive
TICs isolated from the 3 month old premalignant Pten null livers gave rise to
tumors and are resistant to chemotherapy (Rountree). These cells also exhibit
progenitor phenotypes in monolayer cultures and readily form spheres when
cultured in suspension (data not shown). Furthermore, our Pten deletion mouse
model displays robust induction of Wnt ligands and receptors as well as β-
23
catenin during proliferation of tumor initiating cells (TICs) and development of
tumors. In this chapter, we determined whether β-Catenin is necessary for
maintaining the proliferation of hepatic tumor initiating cells.
III-2 Results
III-2-1 Tumor initiating cell proliferation is attenuated in β-catenin KD cells
in vitro and in vivo
In order to specifically address the effect of β-catenin on the growth and
maintenance of liver TICs, we performed molecular assays to knockdown β-
catenin in cultured TICs. Transient expression of siRNA in cultured TICs reduced
β-catenin protein levels and resulted in the down-regulation of cyclin D1 and
survivin (Fig 7A and 7B). Such downregulation of β-catenin activity inhibited the
expression of EpCAM and K19, both markers of liver TICs, suggesting that the
TIC population in the culture is reduced by introduction of β-catenin siRNA and
suppression of its transcriptional activity (Fig 7C).
Levels of β-catenin start to recover in the siβ-catenin knockdown cells 3-4
days after culturing. In order to achieve long-term knockdown of β-catenin, we
designed two shRNAs to stably knockdown β-catenin so that we can investigate
the effects of inhibiting β-catenin transcriptional activity on TIC identity,
particularly in grafted tumors (Fig 8). Both shRNAs we used were able to inhibit
the mRNA and protein expressions of β-catenin (Fig 8A). We also performed
reporter assays by transfecting the TOP/FLASH reporter construct that contains
the TCF/LEF promoter to these cells. The mutated reporter FOP/FLASH is used
as controls. The TOP/FLASH reporter activity is significantly reduced in both
24
shRNA transfected cells (2.2 and 3 fold respectively) whereas FOP/FLASH
activity did not change (Fig 8A). The expression of survivin and cyclin D1, two
transcriptional targets of β-catenin are also inhibited in shRNA expressing cells,
confirming the specificity of the shRNAs towards β-catenin and its transcriptional
activity. In addition to observing significant inhibition of cell numbers by
introducing the two shRNAs, we performed [
3
H]thymidine incorporation
experiment to confirm that cell proliferation is also inhibited (2-4 folds), likely
contributing to the reduction of cell numbers (Fig 8B). Inhibition of β-catenin
further led to the downregulation of hepatic progenitor cell markers EpCAM, K-
19 and AFP (Fig 8C).
Additionally, we injected these cells subcutaneously to the nude mice.
Expression of shRNA for β-catenin led to significantly attenuated tumor growth
(Fig 9 A&B). Tumor growth started to increase dramatically 4 weeks after
grafting with the scramble RNA transfected cells but remained barely
measurable in the two shRNA transfected samples. When the largest tumor
reached the institutional allowable size, the average tumor weight in the
scrambled shRNA transfected samples are 4 to 5 times the size of that
transfected with β-catenin shRNA (Fig 9B). Morphology of the tumors from the
shβ-catenin transfected samples represents more differentiated tissues with
primarily ductal epithelials and stromas compared to the shScramble transfected
samples with more aggressive TIC phenotypes (Fig 9A). Similar to that of cell
culture, inhibition of β-catenin in xenografted tumors led to downregulation of
progenitor cell markers K-19, AFP, Trop2 and EpCAM (Fig 9C), consistent with
25
its role in maintaining progenitor cell identity. We also performed orthotopic
graftment followed by ultras sound to visualize tumor and found that the shRNA
treated cells exhibit little or no growth (Fig 10).
Figure 7. Inhibiting β-catenin downregulates TIC markers and reduce their
growth. A. Knockdown of β-catenin with si-β-catenin siRNA. B. Expression
levels of Cyclin D1 and Survivin, two target genes of β-catenin are reduced at 72
hours post introduction of si-β-catenin. C. Expression of TIC markers are
reduced when si-β-catenin is introduced. D. Growth curve of liver TICs with or
without si-β-catenin.
26
27
Figure 8. Down-regulating β-catenin attenuates TIC proliferation and
reduces hepatic TIC markers in vitro. A. Confirmation of the effect of β-
catenin targeting shRNAs on the expression of β-catenin, Cyclin D1 and survivin
as well as using TOP-FLASH to indicate inhibition of β-catenin transcriptional
activity. FOP-FLASH, controls. n=3. B. Growth curve and thymidine incorporation
in shRNA expressing cells. n=3. C. qPCR analysis of hepatic progenitor cell
markers in cultured TICs.
28
29
Figure 9. In vivo downregulation of β-catenin abrogates tumor growth and
expression of hepatic progenitor cell markers. A. Representative images of
xenografted tumors from the β-catenin manipulated TICs. H&E images of the
tumors (right). B. Significant reduction in tumor volume and tumor weight is
observed upon β-catenin downregulation. n=6. C. qPCR analysis of xenografted
tumors n=3. * Indicates values that are significantly different from sh-scr group at
P<0.05.
Figure 10. Tumor is undetected when β-catenin downregulated liver
progenitor cells are injected orthotopically in the liver. Ultrasound of the
liver from Pten null mouse shows massive tumor throughout the liver (left panel),
tumor found in the biggest liver lobe where progenitor cells treated with scramble
(control) were initially injected (middle panel), no apparent tumor is detected in
the sh-β-cat 1 treated progenitor cells orthograft.
III-2-2 β-catenin inhibitor (ICG-001) ameliorates liver TIC proliferation and
reduces expression of progenitor cell markers
In order to address whether inhibiting β-catenin indeed attenuates the
expansion of TICs and tumor growth induced by steatosis due to Pten deletion,
30
we used a pharmacological compound ICG-001, a small molecule inhibitor of
Wnt/β-catenin, which specifically blocks the interaction of β-catenin with its
coactivator CBP to inhibit β-catenin/CBP transcriptional activity in vivo. Both the
protein and mRNA expression of cyclin D1 and survivin are significantly inhibited
by increasing dose of ICG-001 treatment (Fig 11A). Inhibiting β-catenin
transcriptional activity using this ICG-001 resulted in cytostasis of the cultured
TIC cells. The cell number in the ICG-001 treated cultures did not change during
the 4 days of culturing whereas vehicle treated cells quadrupled its cell
population during this time frame (Fig 11B). [
3
H] thymidine incorporation
experiment confirms that very little proliferation activity is occurring when ICG-
001 is used to treat the TIC cultures (Fig 11B). ICG-001 has been characterized
for its ability to “differentiate away” TICs, we show here reduced expression of
markers for TIC, i.e. AFP, K19 and EpCAM (Fig 11C), suggesting that inhibiting
β-catenin/CBP activity is likely promoting the differentiation and blocking the
accumulation of cells with progenitor cell characteristics.
To investigate the role of the Wnt/β-catenin pathway in the proliferation of
these TICs during steatosis driven tumor development in the liver, we applied
ICG-001 to the Pten null mice using a continuous delivery osmotic pump system.
Mice were also given low dose DDC to accelerate the expansion of TICs and
shorten the experimental duration (Fig 12). As reported for other tissues,
treatment of ICG-001 inhibited the interaction of β-catenin with its cofactor CBP.
Immunoprecipitation with CBP followed by immunoblotting with β-catenin shows
that ICG-001 treatment results in decreased amount of β-catenin bound to CBP
31
using either total or nuclear liver tissue lysates (Fig 13A). Protein and mRNA
expression of the downstream targets of Wnt/β-catenin, cyclin D1 and survivin
confirmed that ICG-001 treatment reduced the transcriptional activity of β-catenin
in the liver compared to saline treated controls (Figure 13A).
To quantitatively determine the effect of ICG-001 treatment and inhibition
of β-catenin transcriptional activity on liver TICs, we performed FACS analysis to
determine the population change of TICs in ICG-001 vs. vehicle treated livers.
As predicted, DDC treatment increased the population of CD133 and CD49f
double positive cells by approximately 8 folds when compared with the normal
chow (NC) fed animals. Treatment with ICG-001 to inhibit β-catenin activity led
to a 5.6 fold decrease in this cell population (Fig 13B). Histological analysis of
tissue slides confirmed this observation showing reduced cell clusters at the
periductal area where progenitor cell niche is found in the liver (Fig 14A). Ki-67
(Fig 5C) and PCNA staining (Fig 14B) of this periductal regions of the liver exhibit
reduced mitotic activity in the ICG-001 treated groups. To further corroborate
these findings, the expression of markers for liver TICs (AFP, K19, EpCAM and
TROP 2) are all induced by DDC and inhibited when ICG-001 is added to inhibit
β-catenin (Fig 14C). Taken together, our findings indicate that the Wnt/β-catenin
pathway indeed mediate the growth and expansion of liver TICs.
32
Figure 11. Inhibiting β-catenin with ICG-001 blocks TIC proliferation and
reduces expression of progenitor cell markers. A. Both protein and mRNA
levels of Cyclin D1 and Survivin are dose dependently downregulated following
ICG-001 treatment of Pten null TICs. B. Growth curve (left panel) and thymidine
33
incorporation of treated vs. untreated cells (right panel). C. mRNA expression of
TIC markers. * P<0.05, different from vehicle treated controls. n=3.
Figure 12. Illustrated protocol used for ICG-001 treatment. 1 month old mice
were implanted with pump containing ICG-001. The mice were allowed recovery
for 6 days before they were put on two rounds (72 hours each) of DDC
containing diet (0.05% w/w). The mice were euthanized at the end of the
treatment period for TIC analysis.
34
35
Figure 13. Inhibiting β-catenin with ICG-001 ameliorates TIC proliferation.
A. Total liver (Top panel) and nuclear (Second from top panel) lysates from mice
implanted with ICG-001 or saline pumps were immunoprecipitated with CBP and
IgG antibodies and immunoblotted with β-catenin antibody. Bottom two panels,
mRNA and protein expression of β-catenin regulated genes cyclin D1 and
survivin. n=3. B. Quantitative flow cytometry analysis reveals a reduction of
CD133
+
and CD49f
+
progenitor cells upon ICG-001 treatment in Pten null mice.
n=4
36
37
Figure 14. Inhibiting β-catenin with ICG-001 ameliorates TIC proliferation
and reduces expression of progenitor cell markers in the Pten null liver.
A. H&E and Ki-67 staining (brown nuclei) of periductal regions demonstrate that
quiescent TIC niche in ICG-001 treated livers. B. PCNA staining (red) of
periductal regions demonstrates that ICG-001 inhibits TIC proliferation. C. mRNA
expression of hepatic progenitor cell markers is reduced in ICG-001 treated Pten
null mice compared with saline controls. * Indicates values that are significantly
different from Saline group at P<0.05. n=3.
III-2-3 Knocking down β-catenin in liver progenitor cells (β-catenin
lox/lox
; Sox9-
CreERT) results in reduction in progenitor cell and induction in ductal cell
number
To further investigate whether the induction of Wnt/β-catenin by steatosis
may serve as niche signal for liver TICs in vivo, we deleted β-catenin from liver
progenitor cells (β-catenin
lox/lox
; Sox9-CreERT) rendering the progenitor cells
incapable of responding to Wnt as a potential niche factor. To activate the niche,
DDC was given to these mice for 3 months after tamoxifen treatment to induce
deletion of β-catenin. As reported, DDC treatment induces liver injury, increases
Wnt expression (Fig 15), leading to biliary hyperplasia as well as activation of liver
progenitor cell niche highlighted by the appearance of small cells with high nuclear
to cytoplasmic ratio and hyperplasia of the keratin positive ductual cells (Fig 16A
and 15B). In the control livers, β-catenin staining is observed throughout the liver
and more intensive in the activated niche (Fig 16B and Fig 17). When β-catenin is
deleted in the β-catenin
lox/lox
; Sox9-CreERT mice, its staining at the progenitor cell
38
niche disappeared. Hepatocytes retained their β-catenin staining, suggesting the
deletion is specific to SOX9 derived cells and biliary structures at the niche (Fig
16B). We determined the effects of β-catenin deletion on the activation of liver
progenitors by flow cytometric analysis for the CD133 and CD49f positive liver
progenitor populations. More CD133 and CD49f double positive cells were
observed when mice were subjected to DDC. Deletion of β-catenin reduced the
CD133 positive cells by more than 3 fold (Figure 16C). The number of cells positive
for both CD133 and CD49f also decreased moderately, whereas the number of
cells positive for CD49f only did not change consistently. Thus, Wnt is at least part
of the niche signals for maintaining liver progenitors as less progenitors were found
when these cells cannot respond to Wnt signal due to β-catenin loss.
As we analyzed the histological phenotype of the liver, we were surprised to
find that the β-catenin
lox/lox
; Sox9-CreERT livers displayed a greater biliary
hyperplasia phenotype than the controls (Fig 16A). We thus tested the posibility
that β-catenin may independently control ductal cell growth and loss of β-catenin
actually provided growth advantages for the ductal cells, allowing them to
proliferate, leading to hyperplasia (Fig 17A). Our data showed, however, that very
few of the CK positive ductual cells are positive for ki67 staining (Fig 17C),
suggesting that they have not progressed through the cell cycle in the last few days
of the mouse’s life. The hyperplastic ductal cells are also not a result of “recovery”
of β-catenin deletion as was observed with β-catenin
lox/lox
; Alb-Cre
+
mice [62] as
they are still negative for β-catenin (Fig 16A). Thus, it is likely that loss of β-catenin
and loss of Wnt signal input permitted differentiation of the progenitor cells,
39
producing more differentiated progenies, the ductal cells (Fig 17A, possibility 2).
This observation, while needs to be confirmed by lineage tracing analysis, is
consistent with observations that activation of progenitor cells leads to their
differentiation and depletion, consistent with the role of Wnt/β-catenin as niche
signals in the liver to maintain progenitor cell identity.
Figure 15. DDC induces Wnt and liver Injury. A. Expression of Wnt 7a and 10a.
n=3, * P<0.05 compared with controls. Data expressed as the mean ± SEM. B.
High magnification for morphology of control and β-catenin deletion mice treated
with DDC.
40
41
Figure 16. β-catenin loss in Sox9 positive liver progenitor cells leads to
reduction in TIC numbers. A. Representative H&E (left panel) and Keratin
(green) +β-catenin (red) stained (right panel) images of Control and β-
catenin
loxP/loxP
; Sox9-CreERT+Tam mice (β-catenin null) treated with DDC.
Control (n=3) and β-catenin null (n=4) B. β-catenin staining demonstrates
deletion of β-catenin is restricted to the Sox9+ cell progenitor cell niche area but
not in hepatocytes in β-catenin null mice. Red: β-catenin; Blue: DAPI. n=3 C.
Top, representative flow cytometric plot of CD133
+
and Cd49f
+
cells in control
and β-catenin null mice fed with DDC. Bottom panel, quantification of the flow
cytometric analysis. a, significantly different from control at P<0.05.
42
Figure 17. Induction of ductal hyperplasia is likely due to differentiation of
liver progenitors and not proliferation of ductal epithelial cells. A. Two
possibilities for how β-catenin deletion may induce ductal hyperplasia. Possibility
1 is that β-catenin is necessary to inhibit ductal cell replication and loss of β-
43
catenin allowed this replication. Possibility 2 is that loss of β-catenin rendered
self-renewal of liver progenitor cells inefficient and at the same time, permitted
their differentiation into ductal cells. B. β-catenin levels in total liver lysates. C.
Representative images of staining of liver tissues sections from mice lacking β-
catenin in the sox9 positive cells (β-catenin null). Green, CK for ductal cells; Red,
Ki67 for proliferating cells; Blue, Dapi for nuclei. Right panel, quantification of the
staining counted from 2 sections each sample, 5 randomly selected field per
section. * P<0.05 compared with controls.
III-2-4 Inhibition of Wnt signal blocks human TIC accumulation
To address whether blocking Wnt signal will also inhibit activation of
human TICs (hTICs), we obtained hTICs from primary tumors surgically removed
from HCC patients. Human TICs were isolated using CD133 as a surrogate
marker. In cultured hTICs, treatment with ICG-001 to block β-catenin/CBP
transcriptional activity led to complete inhibition of Cyclin D1 and survivin
expression (Fig 18A). qPCR analysis on TIC marker AFP and Keratin-19
showed that their expressions are significantly inhibited by ICG-001 treatment
(Fig 18B). Thus, blocking the β-catenin/CBP interaction with ICG-001 in vivo
may serve as a viable approach to block the accumulation of liver TICs and
attenuate tumor growth.
44
Figure 18. ICG-001 treatment of human TICs reduces Cyclin D1 and
Survivin expression as well as progenitor cell markers. A. Cyclin D1 and
Survivin expression B. AFP and Keratin-19 (K-19) mRNA levels n=3. *
Significantly different from vehicle controls (Con) at P<0.05.
III-3 Discussion
To specifically address the role of β-catenin signaling in progenitor cell
proliferation, we designed β-Catenin targeted shRNA to block the activity of
Wnt/β-catenin signaling. We have demonstrated the effect of downregulating β-
catenin on the proliferation potential of the CD133+ liver progenitor cells in tissue
culture using growth curves and thymidine incorporation analysis and in vivo
using xenograft and orthograft studies. We have also shown that blocking the
transcriptional activity of β-catenin using a small molecular inhibitor, ICG-001,
effectively attenuates the growth of TICs in vitro and in vivo. ICG-001 binds
specifically to transcriptional co-activator CREB-binding protein (CBP), a cofactor
needed for the transcriptional action of β-catenin and the induction of Wnt
targets. Binding of ICG-001 to CBP prevents it from binding to β-catenin and
thus disrupts the transcriptional complex and inhibits the activation of Wnt
45
targets. Because we observed robust activation of β-catenin in our Pten null
mice leading to expansion of liver TICs, we hypothesized that deletion of β-
catenin in liver progenitor cells will result in attenuation of TIC numbers.
Therefore, we selectively deleted β-catenin in Sox9 progenitor cells (β-
catenin
lox/lox
; Sox9-CreERT). β-catenin deletion not only caused reduction in
progenitor cells but also promoted expansion of ductal cells most likely due to
progenitor cell differentiation. Taken together, this data suggest that the Wnt/β-
catenin pathway mediates proliferation of hepatic TICs.
46
Chapter IV
Calorie Restriction abrogates hepatic steatosis, inhibits TIC expansion and
abolishes tumor development in Pten null livers
IV-1 Introduction and Rationale
Calorie Restriction (CR) also referred, as ‘undernutrition without
malnutrition’ is a potent widely used dietary intervention for inhibiting cancer. CR
is a dietary regimen by which there is moderate restriction of food intake while
providing essential nutrients and vitamins while limiting total energy intake by 20-
40% relative to ad libitum (AL) controls. Major outcomes of CR without
malnutrition are delayed aging and extended lifespan in a variety of species
including yeast, fish, mice, rats, and monkeys[63,64]. There is also promising
evidence that humans benefit from CR such as improved insulin sensitivity
leading to reduced diabetes, improved memory, reduced cardiovascular risk as
well as increased longevity. In addition to increasing life span, the hormetic effect
of CR is protection against cancer in mammals[65]. Moreschi (1909) and Rous
(1914) were the first to demonstrate that CR prevented the growth of
transplanted tumor compared to AL fed mice[66]. Later, Tannenbaum and
colleagues confirmed the incidence of tumors in mice was reduced when mice
received CR diet[67]. CR without malnutrition has been found to reduce the
levels of various anabolic hormones, growth factors and inflammatory cytokines.
Furthermore, reduction in oxidative stress and cell proliferation as well as
increase DNA repair process has been observed[65].
Previously our study showed that Pten deletion in liver resulted in fatty
47
liver phenotype due to excess accumulation of lipid in the liver. CR has been
found to suppress fatty acid synthesis and increases fatty acid oxidation[68].
Preliminary data from our lab also indicates CR reduces liver steatosis by
activating β-oxidation pathway in Pten null mice model (Ajeetha Rajan). In this
chapter, we hypothesize that calorie restriction reduces liver steatosis, liver injury
and reduced hepatic progenitor cell proliferation ultimately resulting in reduced
tumor burden.
IV-2 Results
IV-2-1 Pten/Akt2 double mutant mice display reduced hepatic steatosis
along with decreased TIC numbers
To explore whether this Wnt/β-catenin niche signal for TIC indeed
mediates the effect of steatosis, we first evaluated the TIC populations in the
Pten/Akt2 double mutant livers. We have reported [46] that AKT2 loss blocked
the steatosis development in the liver (Fig 19A). In these Pten/Akt2 double
mutant livers, Wnt signals are attenuated compared to the Pten null livers (Fig
19B). While Pten deletion led to increases in TICs, Akt2 deletion attenuated this
increase (Fig 19C). The number of CD133
+
CD49f
+
TICs observed in the Dm
liver is more than 3 fold less than that observed in the Pten null livers (Fig 19C).
This decrease in TIC population also occurs concurrently with significant
inhibition of tumor burden in the Pten/Akt2 double mutant mice (Table 1). In
addition, expression for markers of TICs are also significantly attenuated in the
Pten/Akt2 double mutant mice [45].
48
Figure 19. AKT2 loss abrogates hepatic steatosis, Wnt signaling and TIC
number. A. Oil Red O staining displays reduced fatty liver phenotype in
Pten/Akt2 double mutant (Dm) mice compared to Pten null (Pm) mice. B. AKT2
loss also results in reduction of Wnt 7a, Wnt 10a, n=5. C. Representative flow
cytometric plot of TICs in Pten null vs. Pten/Akt2 double mutant mice. a,
significantly different from control. b, significantly different from Pm. P<0.05.
IV-2-2 Calorie Restriction reduces body weight and improves insulin
sensitivity
Previously our study showed that Pten deletion in liver resulted in fatty
liver phenotype due to excess accumulation of lipid in the liver. On the other
hand, CR has been found to suppress fatty acid synthesis and increases fatty
acid oxidation[68]. To further substantiate the role of fatty liver and induction of
49
the Wnt/β-catenin pathway to increase the mitotic activity of progenitor cells, we
put Pten null mice under CR. Body weights of the four groups of mice Pten
loxp/loxp, Alb-Cre- (WT), Pten loxp/loxp, Alb-Cre + (Mutant) under calorie
restriction (CR) or ad libitum (AL) diets were compared. Body weight and food
intake was measured weekly for a period of 10 months (Fig 20). Before the start
of the diet regimen, there was no difference in body weight between all four
groups. However, two weeks after the start of the calorie restriction diet,
significant reduction in body weight is observed in CR groups compared to AL
groups despite their genotype differences (Fig 21A). Our data supports previous
data that show CR is associated with reduced body weight[69]. Final body weight
also reveals Mut/AL mice weigh less than WT/AL mice whereas WT/CR and
Mut/CR mice display similar body weights (Fig 21A). Liver weight of the mice
corresponds to their body weight in terms of significant reduction in the calorie
restricted groups compared to the ad libitum mice in the WT and mutant mice.
However, a 6-fold increase of liver weight was observed in the Pten null mice
compared to the control mice in the AL group (Fig 21B). This result is in
accordance with previous data that shows Pten loss in the liver increases its
weight[27,70].
Its also been consistently shown that CR improves insulin sensitivity in
both rodents and humans[71,72]. The effect of CR on blood glucose was studied
using glucose and insulin tolerance tests at regular intervals. In both, GTT and
ITT data, CR mice display reduction in blood glucose in either control or mutants
compared to WT/AL mice. In fact, for the ITT experiment, insulin levels had to be
50
reduced to 0.4 units and 0.3 units for the 8
th
and 10
th
month readings respectively
because CR mice became too insulin sensitive during the course of the study.
Injection of 0.5 units insulin caused the CR mice to be hypoglycemic. Thus, we
reduced the amount of insulin that was injected, which explains the increase in
overall glucose levels at months 8 and 10 (Fig 22).
Figure 20. Schematic Diagram of Calorie Restriction (CR) and Ad Libitum
(AL) feeding in Pten null and Wild Type mice
51
Figure 21. CR reduces body weight (BW) and liver weight (LW) in both WT
and Pten null mice. A. Average BW starting at 2 weeks after start of diet
regimen till final BW (13months) resulted in significant reduction in BW in CR
groups compared to AL controls. Open bar, (WT/AL); closed bar, (WT/CR);
striped bar, (Mut/AL); Checkered bar, (Mut/CR). B. LW was significant in both
calorie restricted groups compared to AL groups in their respective genotypes. C.
CR reduced the percentage of liver weight to body weight ratio in the mutant
group compared to the AL mutant. Both diet groups in the wild type mice exhibit
lover liver to body weight. Data presented as mean +/- SEM. * indicates values
that are significantly different at P≤0.05. n= WT/AL=11, WT/CR=10, Mut/AL=6,
Mut/CR=7.
52
53
Figure 22. Intraperitoneal glucose tolerance and insulin tolerance test.
Intraperitoneal glucose tolerance (GTT) test and Intraperitoneal insulin tolerance
(ITT) was measured at months 1, 3, 6, 8, and 10 months after the start of CR
diet. n= WT/AL=11, WT/CR=10, Mut/AL=6, Mut/CR=7. Data represented as
mean+/- SEM. Significance was indicated by *# P<0.05. # WT/AL vs WT/CR;
*Mut/AL vs Mut/CR.
IV-2-3 Calorie Restriction abrogates hepatic steatosis, injury, Wnt/β-catenin
signaling and progenitor cell expansion in Pten null mice
Using a non-genetic approach, caloric restriction (CR, 40% reduction in
calories), to reduce steatosis in the Pten deletion induced liver steatosis (Fig 23),
we observed more than 50% reduction in the number of cells positive for both
CD133 and CD49f (Fig 24). Like Akt2 deletion, CR diet completely blocks the
development of steatosis in the Pten null mice. In the Pten null livers, triglyceride
(TG) content is more than 10 fold higher than that of the controls (862 vs.
76.5mg/mg DNA) (Fig 23A). The lipid content in the CR fed animals (control and
Pten null) are 57.1 and 55.6mg/mg DNA respectively, similar to the ad libitum fed
control mice. CR also significantly reduced the body and liver weight of both
control and Pten null mice (Fig 23A and Fig 24). Together with this inhibition of
liver steatosis, plasma ALT, the indicator of liver injury is also attenuated in the
CR vs. ad lib. fed Pten null mice (Fig 23B). Consistent with the role of Wnt/β-
catenin as niche factor induced by steatosis, CR treatment reduced the
expression of Wnt 7a, 10a, Fzd2 in the Pten null liver (Fig 23C). The inhibition of
54
Wnt10a is the most pronounced (both mRNA and protein expression) when CR
is fed to the Pten null mice. The two transcriptional targets of β-catenin, survivin
and cyclin D1 are also both coordinately inhibited by CR treatment (Fig 23D).
Protein levels of survivin are only detectable in the Pten null liver lysate but not in
either of the control groups or the CR fed Pten null group. These data suggest
that Wnt signal, which is induced upon deletion of Pten in the liver, is inhibited by
feeding the mice CR diet.
When compared to the ad lib fed Pten null mice, the cell population
positive for both CD133 and CD49f is reduced by 3 fold when the mice were
subjected to CR diet (Fig 24A). Though this number failed to reach statistical
significance due to high variations observed in the ad lib fed Pten null mice,
consistent with the concept of tumor heterogeneity, the percentage of CD133
single positive TICs are significantly lowered (approximately 50%) by CR
treatment (Fig 24A). Concurrent with the reduction of TIC population,
expressions of EpCam, K19, AFP and Trop2, markers for liver TICs are also
reduced (3-5 folds) dramatically by CR treatment in Pten null mice (Fig 24B).
Collectively, these data suggests that blocking fatty liver formation with CR or
Akt2 deletion led to reduction of hepatic TICs.
Since TIC populations were significantly reduced by CR, we evaluated the
CR and AL mice for tumor incidence. At 12 months and beyond, all Pten null
mice develops mixed lineage tumors in the liver (Table 1, 7 out of 7 in this cohort
of study). Multiple tumor nodules are visible on the livers of these mice (Fig
24C). Histological analysis of liver sections confirms that these tumors are
55
composed of both hepatocyte and cholangiocytes (Fig 24C), as we reported
before [45]. At this age, none of the Pten null mice fed on CR diet (0 out of 7)
developed tumors (Table 1). These data are consistent with our earlier
observation that deleting the metabolic regulator, Akt2, which diminishes
steatosis attenuates tumor growth and confirms that steatosis is necessary for
tumor progression [45]. It has been reported that established tumors are not
responsive to CR treatment if their PI3K signaling is activated, likely because CR
signals primarily by reducing circulating insulin/IGF-1 levels [73]. Our
observation suggests that inhibition of Wnt by reducing steatosis may have
overridden the PTEN loss activation signal at least at initiation of tumor growth to
establish tumors. Wnt, thus, may serve as an early competency factor to
promote the activation of TICs and establishment of tumors.
Consistent with induction of AKT phosphorylation by HFD, CR inhibited
phosphorylation of AKT (Ser 473) in the control mice (Fig 24D). In the Pten null
mice, however, AKT phosphorylation is significantly higher due to loss of PTEN.
CR is unable to affect p-AKT levels in these mice, consistent with the published
literature showing that CR is unable to suppress PI3K signal in tumors when
PTEN is lost [73]. Levels of Erk1/2 however are attenuated by CR in both
control and Pten null livers. Whether this downregulation of Erk may contribute
to the inhibition of Wnt is not known. AMPK activity measured by its
phosphorylation did not change.
56
57
Figure 23. Calorie Restriction (CR) blocks lipid accumulation, injury and
Wnt/β-Catenin signaling. A. Liver tissue sections were collected from 13 month
old control (Con) and Pten null (Pm) mice fed with ad libitum (AL) or Calorie
restriction (CR) diet. Left panel, H&E staining (top) and Oil Red O staining
(bottom). Right panel, average body weight. n=6-11. B. Liver triglyceride (TG)
and serum ALT levels. C. qPCR (left panel) and western blot (right panel)
analysis of Wnt signaling pathway. D. mRNA and protein expression levels of
Cyclin D1 and survivin. a, difference from Con/AL; b, difference from Con/CR; c,
different from Pm/AL. P<0.05.
58
Figure 24. CR abrogates reduces liver TICs and blocks tumor formation in
Pten null mice. A. Analysis of TIC populations in AL (n=6) and CR (n=7) fed
Pten null mice. * Significantly different from AL at P<0.05. B. Hepatic TIC
markers are induced in Pm/AL mice and blocked with CR. n=6-11. C.
Representative images of tumors and H&E stained liver sections. D. Western
blot analysis of AKT, ERK and AMPK pathways.
59
Chapter V: Overall Discussion
Recent advances in tumor metabolism have significantly improved our
understanding on how glucose metabolisms may contribute to tumor growth.
Lipid metabolic dysfunction, on the other hand, has received far less attention
even though tumor mortality is higher in obese individuals. Obesity increases the
risk of a number of cancers including colon, mammary, pancreas, and liver
[3,74], etc. Statins, drugs used to lower cholesterol levels, have been shown to
reduce the risk of colon cancer by 47% [75,76]. Our recent work established that
the tumor suppressor PTEN regulated PI3K/AKT pathway regulates both the
positive and negative transcriptional regulators of de novo lipogenesis [46,77].
Using animal model lacking PTEN and the metabolic kinase AKT2 [45], we
established that this pro-lipogenic effect is necessary for tumorigenesis occurring
in these mice. In the current study, we showed that accumulation of lipids in
hepatocytes establishes tumor-promoting signals by inducing Wnt/β-catenin
signal, a niche factor for TICs in a number of tissues (Fig 8G). Through induction
of Wnt, accumulation of lipids signals expansion of TICs and promotes tumor
progression. When we inhibited Wnt signals in cultured TICs using shRNA
against β-catenin or in vivo using a pharmacologic reagent ICG-001 or by
deleting β-catenin, TIC accumulation and tumor growth were both blocked.
Finally, inhibiting steatosis with genetic or dietary approaches reduces Wnt,
inhibits the accumulation of TIC and blocks tumorigenesis. Taken together, our
data show that lipogenesis, or at least accumulation of lipids in hepatocytes, is
needed to establish the niche factors, Wnt that promote tumors. This study, thus,
60
provides mechanistic link for whether and how obesity may contribute to
tumorigenesis.
Like all progenitor cells, intrinsic properties give TICs the capability of
being the progenitors for tumors. Extrinsic factors, however, are needed to
determine cell fate decisions for self-renewal vs. differentiation at any given time.
The concept of “niche factors” was first established based on C. Elegans and
Drosophila genetic studies together with observations in hematopoiesis and
other systems where the presence of putative stem cells were identified [78].
Since then, multiple signaling molecules/pathways have been identified to play
roles in maintaining the “niche” [79]. Decline in “niche factor” signals leads to
reduction in progenitors while permitting their differentiation. Increases in “niche
factor” signals allow accumulation of progenitors while preventing their
differentiation. A number of factors have been identified to be niche factors for
progenitor cells in the liver including Notch, Sonic Hedgehog and transforming
growth factor b as well as Wnt/β-catenin signaling. Interestingly, these factors
are also highly correlated with tumor development in the liver.
To delineate if de novo lipogenesis by Pten deletion or HFD feeding
contributes to steatosis mediated wnt induction in activating our progenitor cell
pool wild-type mice were given high fat diet (HFD). Our analyses demonstrate
HFD enhances steatosis, lipotoxic injury and activates wnt signaling and its
downstream genes cyclin D1 and survivin as well as hepatic progenitor cell
markers. This data substantiates the role of wnt activation and progenitor cell
proliferation in response to hepatic steatosis independent of fatty liver etiology.
61
Our findings are relevant because obesity induced fatty liver disease is a major
factor for acquiring liver cancer and understanding the molecular alteration
responsible for initiation and exacerbation of this disease is critical.
Our data show that both high fat diet feeding and Pten loss induced steatosis
both stimulate the expression of Wnt, one of the putative niche factors. The Wnt
family of soluble factors is characterized as niche signals that potentiate the self-
renewal of tissue progenitor cells [58]. Work on embryonic stem cells showed that
Wnt binding to coactivators such as CBP is associated with undifferentiated
phenotypes of stem cells [80]. Wnt signals through both canonical signaling
pathway that involves β-catenin and a non-canonical pathway that goes through
several kinase cascades. In intestinal crypt and hair follicle bulge, the presence of
Wnt and activation of the downstream target β-catenin are necessary for
maintaining progenitor cell identify [59,60]. β-catenin is one of the most highly
correlated genes with human liver cancer [81]. Wnt binding to its receptor stablizes
the b-catenin protein, allowing it to move to the nucleus where it associates with
Lef/Tcf transcriptional factors and participates in gene transcription. In Pten null
livers as well as HFD fed mice, the increases in Wnt expression resulted in
induction of downstream genes of β-catenin transcriptional activity as well as
increases in markers of progenitor cells, suggesting that this signal is sustaining the
identity of the TICs. When Wnt signal is blocked by β-catenin knockdown,
pharmacological inhibition of β-catenin, or β-catenin deletion in SOX9 positive cells,
the TICs are unable to accumulate in vitro or in vivo. Thus, Wnt is likely one of the
major niche factors regulating the identity of these TICs during steatosis promoted
62
tumor development in the Pten null liver. Supporting this observation, blocking
steatosis in the Pten null liver inhibits Wnt expression and TIC accumulation.
By definition, TICs are cells capable of contributing to all cell types in the
tumor while maintaining their own identity through self-renewal. The portal triad
area of the liver has been identified as the progenitor cell niche based on models
of hepatic injury. The appearance of small oval-shaped cells with high nuclear to
cytoplasmic ratio was identified in the portal triad area following chronic injury to
the liver [82]. The appearance of these facultative progenitor cells correlates with
increases in a cell population expressing stem cell markers. Isolated progenitors
using these cell surface markers such as CD133, CD49f, CD44, or CD24 have
been demonstrated to display progenitor cell properties including clonogenic
growth and cellular transplantation capacity [83]. In the Pten null liver cancer
model, the appearance of small progenitor-like cells was observed following
chronic steatosis and injury [45,48]. CD133 is a pentaspan transmembrane
glycoprotein that has been shown to be transiently upregulated during liver
regeneration. In addition, CD133+ Hepatocellular carcinoma (HCC) cells
exhibited tumorigenic properties including self-renewal, unlimited proliferative
capacity and pluripotency[84].
Moreover, the CD133+ cells isolated from the Pten null model were shown
to be the tumor forming cells or TICs though CD49f single positive cells appears
to correlate with ductal hyperplasia phenotype when b-catenin is deleted. Akt2
deletion and CR both inhibited the appearance of these cells and the
accumulation of CD133 single and CD133+/CD49f double positive TICs. A
63
common effect of Akt2 deletion and CR is that both approaches blocked the
steatosis and injury observed, and both inhibited Wnt signaling in the Pten null
liver. Thus, steatosis is likely inducing Wnt to signal the accumulation of these
TIC cells, thereby contributing to tumor progression.
How steatosis induces Wnt signaling is not understood. In adipose tissue,
Wnt produced by preadipocytes or infiltrating macrophages due to swelling
adipocytes in obese individuals blocks the differentiation of preadipocytes [85].
This leads to accumulation of lipids in mature adipocytes and underlies the
adipocyte centric insulin resistance mechanism and leads to obesity related
inflammation. Non Alcoholic Fatty Liver Disease (NAFLD), which is commonly
associated with obesity, encompasses histological features including hepatic
steatosis, steatohepatitis, fibrosis and cirrhosis and is often accompanied by
infiltrations of inflammatory cells including macrophages [86]. In the Pten null
liver, we observed induction of Wnt accompanying lipid accumulation. Severe
inflammatory cell infiltration is also observed in these livers (data not shown). CR
and Akt2 deletion, which blocked tumor growth, inhibits fatty liver and hepatic
injury as well as the accompanying inflammatory cell infiltrations. Thus, Wnt
may be produced by infiltrating macrophages as a response to swelling of lipid-
laden hepatocytes, similar to what was observed for adipocytes. In MDA-MB-
231 breast cancer cells, adiponectin, an adipokine produced by adipocyte has
been shown to regulate Wnt-β-catenin signal and through which affects the
growth, invasion and metastasis of these cells [87]. Our CR study failed to
observe any changes in p-AMPK with Pten deletion or CR treatment. Thus, the
64
involvement of adiponectin, which induces AMPK phosphorylation, is unlikely.
Other signals, such as leptin may also crosstalk with Wnt/β-catenin. Specifically
what kind of adipokines is produced by lipid-laden hepatocytes and whether they
play a role in regulating the Wnt signal remains to be determined.
Additional studies are required to determine if other signaling molecules
that interact with PI3K pathway such as mTOR and SIRT-1 play roles in the
steatosis induced Wnt signal. Although CR is able to inhibit AKT phosphorylation
in the control mice, it was not able to overcome the AKT phosphorylation induced
by PTEN loss. Thus, CR likely abolished tumorigenesis independent of IGF-
1/PI3K signaling. Recently, it was demonstrated that short term CR cannot cause
tumor remission in established tumors if PI3K signals are induced [73]. Our
experiment using a long term CR protocol starting before the onset of tumors
showed that CR is able to block tumor development independent of PI3K status.
Our data favor the role of Wnt in the process of tumorigenesis as Wnt RNA and
protein (particularly Wnt 10a) are both robustly induced by Pten deletion and
completely inhibited with CR. In a different model where tumorigenesis is
inhibited by attenuating AKT2 signaling, downregulation of Wnt also occurs with
inhibition of tumor development, supporting a role of Wnt in the steatosis induced
tumorigenesis. In this scenario, Wnt likely serves as an early competency factor
that allows the initial establishment of tumors, i.e. activation of the TIC niche.
Once tumors reach a certain size and “niche” is no longer confined to Wnt
gradient, CR may not be able to have an effect and tumors with PI3K activation
are allowed to propagate. Our preliminary analysis suggests a potential role of
65
Erk in this Wnt mediate tumor regulatory effect. Erk is minimally induce by Pten
loss in the liver but significantly attenuated by CR which blocked activation of b-
catenin and Wnt. Wnt3a has been shown to induce Erk [54]. Through
phosphorylating GSK3b, Erk may potentially regulate the stability of b-catenin.
How this induction of Erk by Wnt may influence the transcriptional activity of β-
catenin in TICs is not clear.
In the pathogenesis of many cancers including HCC, it has traditionally
been believed that long-term, persistent injury of damaged mature cells induce
an accumulation of multiple genetic or epigenetic alterations, which ultimately
lead to cancer[88]. However, the recent “cancer stem cell/TIC hypothesis”
proposes the involvement of a minor population of cells with self-renewal
capability and ability to repopulate a tumor mass after traditional cancer
treatments[89]. In some cases of HCC, the development of tumors has been
attributed to the propagation of these stem/progenitor cells[88]. Whether the
TICs are derived from liver progenitors or dedifferentiated mature liver cells is up
to debate [43]. In homeostatic and injured livers, lineage tracing studies using
Cre recombinase based expression of reporter markers have led to inconsistent
conclusions for the contribution of the facultative progenitors to liver
regeneration. Studies using Sox9-CreERT to label the putative progenitors have
found that 80-90% of hepatocytes become labeled with reporter in non-injured
livers following prolonged periods of chase [90]. Using other promoters to drive
the Cre recombinase, other studies have found only 1-2% or no contribution of
progenitors to hepatocyte regeneration in different models of liver injury [91,92].
66
Hepatocytes, however, have the ability to transdifferentiate to other lineages
upon stimulation or injury. In the Pten null livers, whether TICs are derived from
the facultative progenitors or reprogrammed from hepatocytes due to the primary
mutation occurring in these cells is unknown. Tumor cells, though, are positive
for Sox9 (data not shown).
In summary, our findings show that Wnt/β-catenin signaling is induced by
steatosis to promote tumorigenesis in the liver. This finding is highly relevant to
the understanding of the strong correlations between obesity and cancer.
Obesity, which occurs in approximately half of the US population, is now
recognized as a confounding factor for cancer-related death [1,2]. It’s estimated
approximately 44% of men and 37% of women will develop cancer during their
lifetime[93]. This number continues to increase because of diet and lifestyle
changes. Liver cancer is also on the rise due to rising obesity epidemic.
Therefore, this study provides a molecular mechanism for how obesity may
cause increased cancer risk and further establishes a feasible target for
therapeutic interventions in select populations.
67
Figure 25. Schematic representation of the working model that steatosis
promotes tumor progression through Wnt.
68
Chapter VI: Materials and Methods and Protocol Development
Materials and Methods
Animals
Targeted deletion of Pten in the liver was achieved by crossing Pten
loxP/loxP
mice
with Alb-Cre
+
mice
as reported previously[25]. Pten and Akt2 double mutants
(Dm) were generated by crossing the Pten
loxP/loxP
;Alb-Cre
+
(Pten null, Pm) mice
with the Akt2
-/-
mice. Control animals are Pten
loxP/loxP
; Alb-Cre
-
. For reporting β-
catenin transcriptional activity, the Pten
loxP/loxP
;Alb-Cre
+
mice were crossed with
BAT-Gal mice carrying 7 repeats of the b-gal promoter luciferase reporter
construct. Targeted deletion of β-catenin in the progenitor was achieved by
crossing Ctnnb
loxP/loxP
mice (Jackson) with Sox9-CreER
+
mice[94]. Deletion of β-
catenin is induced by subcutaneous injection of Tamoxifen (5mg/40g BW, Sigma
Aldrich) for 3 doses every other day in 5 days at 1 month of age (b-cat null). Cre-
mice injected with tamoxifen were used as controls. Nude mice (Stain NU/J)
used for xenograft were purchased from the Jackson laboratory. All animals
were housed in a temperature, humidity, light-controlled room (12-h light/dark
cycle), allowing free access to food and water. All experimental procedures were
conducted according to the Institutional Animal Care and Use Committee
(IACUC) guidelines at the University of Southern California.
Diet Feeding
For the high fat diet (HFD) experiment, mice were given 60 kcal% fat diet (06414-
Harlan laboratories) whereas low fat diet 10.5 kcal% of fat diet as control (98247
Harlan laboratories) for 9 months (Table 2). For Calorie Restriction (CR)
69
experiment, control and Pten null mice were allocated to receive either free
access to food (Ad libitum, AL) or Calorie restriction (CR) diet. Calorie restriction
was defined as reducing daily caloric intake (calories/day) by 40% than average
AL intake with a diet that includes similar relative proportions of micronutrients for
CR and AL groups (provided by TestDiet). These groups were further divided into
wild type Pten (Pten loxp/loxp; Alb-Cre-) or Pten null (Pten loxp/loxp; Alb-Cre+).
To determine food consumption, mice were fed normal chow for one week and
weight was ranked lowest to highest and alternatively assigned to AL control
group or CR group. The CR mice were restricted to 60% of AL intake gradually
over 3-week period (80%, 70%, and 60%).
Diet was started at 3 months of age and continued for 10 month duration.
All CR mice were fed between 1500 and 1600 h each day and food intake of both
groups was measured daily and body weight was measured weekly.
Additionally, Intraperitoneal glucose (GTT) and Insulin (ITT) tolerance tests were
performed every alternate month. GTT (2g/kg glucose) was performed after 16
hours fasting while ITT (0.5U/kg; Novolin-R), unless otherwise stated, was done
after 4-6 hours of fasting. Blood glucose was measured at frequent time
intervals. All experiments were conducted according to the Institutional Animal
Care and Use Committee of the University of Southern California research
guidelines.
70
Cell Culture
Mouse hepatic TIC cell line was established by isolating the CD133
+
non-
parenchymal cell population [48]. Human TICs were obtained from surgically
removed HCC tumors by isolating the CD133
+
non-parenchymal cell population
and maintained as described [95]. For sphere formation, mouse TICs were
seeded in polyHEMA-coated 6-well plate at a density of 5×10
4
cells/ml using a
culture media (DMEM/F12, Insulin 5ng/mL, Nicotinamide 5 mL, EGF 20 ng/mL,
phenobarbitol 2 mM). Medium was changed every 3 days and spheres were
passaged every seven days.
Reagents
Primer sequences, siRNA and shRNA sequences against b-catenin are listed on
supplemental Table 3. Housekeeping genes included glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA. For Western blot,
membranes were probed with antibodies for PTEN, pAKT (Thr 308 and Ser 473),
AKT, ERK1/2, pAMPK, pGSK3β, cyclin D1 and survivin (Cell Signaling Beverly,
MA), keratin (Milipore, Billerica, MA), AFP (Epitomic, Burlingame, CA), Wnt 7a
(Novus Biologicals, Littleton, Co), Wnt 10a (Abcam, Cambridge, MA), and β-
catenin (Sigma-Alderich, St Louis, Mo). Antibody for pSer552-b-catenin is a gift
from Dr. Linheng Li. The membranes were also probed with β-actin (sigma) and
GAPDH (Cell Signaling) for loading control.
For immunofluorescence staining b-catenin was purchased from BD
Biosciences, Ki67, and PCNA are from Thermo fisher scientific. Ki67 (Vector
71
laboratories), cytokeratin (Dako) and FITC-conjugated secondary antibody Alexa
Fluor 594 (Invitrogen) were used for frozen section staining. Lipofectamine 2000
Transfection Reagent is from Invitrogen. ICG-001 is synthesized as previously
described[96]. Diet containing 3,5-dietoxycarbonyl-1,4 dihydrocollidine (DDC) is
purchased from Newco Distributors Inc.
Microarray analysis
mRNAs were extracted from liver tissues from 9 months old control, Pten null, or
Dm mice (n=3-5). mRNA samples were submitted to Penn State Hershey
University Microarray Functional Genomics Core facility for mouse mRNA
Microarray analysis for gene expression using Illumina single color array
platform. Unpaired Student’s t test was used to analyze differentially expressed
genes between Pten null vs. control, and Pten null vs. DM samples.
Biochemical Assays
For determining β-catenin transactivation activity, TICs were co-transfected with
shRNA β-catenin constructs and with either TOPFLASH or FOPFLASH
luciferase. Cells were harvested 48 hours after transfection. Transfection
efficiencies were normalized using Renilla luciferase activity. Luciferase assays
were performed using the Dual-Luciferase Reporter Assay System (Promega).
Folch method was used to extract hepatic lipid by adding chloroform/methanol
(2/1). The supernatant was used for TG assay and the pellets were used for
DNA extraction as reported. Final TG content was determined using Triglyceride
72
(GPO) Reagent Set (Thermo). Plasma ALT was determined using ALT Reagent
(Raichem, San Diego, CA) as previously described.
Quantitative polymerase chain reaction and Protein Electrophoresis and
Co-Immunoprecipitation
Total RNA (2ug) extracted from liver tissues or cells using Trizol reagent
(Invitrogen) was used for quantitative PCR analysis using ΔΔCt method. Protein
lysates (50 µg) prepared with RIPA buffer were loaded for each sample for
electrophoresis using polyacrylamide gels. For co-immunoprecipitation, 1 mg
protein total liver or liver nuclear lysate was diluted in CoIP buffer (150mM NaCl,
1.5mM MgCl2, 10% glycerol, 0.5%NP-40, 20mM Tris-HCl, PH 8.0, 1mM EDTA,
5mM DTT, protease inhibitor cocktail) mixed, and incubated with 2ug of anti-CBP
(A-22: SC-369) Santa Cruz or normal rabbit IgG (Santa Cruz) at 4°C overnight.
Immunohistology
Liver tissues were preserved in 10%Zn-formain or OTC. Hemotoxylin and Eosin
(H&E) staining was performed on all liver samples for morphology analysis. Liver
sections were also stained for various antibodies using standard protocol as we
previously reported. All immunochemically stained slides were counterstained
with hemotoxylin and immunoflurescent stained slides also co-stained with DAPI.
To visualize β-galactosidase and lipids, chemical reactive labeling and Oil Red O
stains were performed as described.
73
Cell Growth and Thymidine incorporation Analysis
Two shβ-Catenin sequences were used (Table 3). 1.0 x10
4
TIC cells were
seeded in 6 well plates and allow overnight. Cells were transfected with
shScramble (control) and shβ-Catenin preceding puromycin selection. Medium
was refreshed every two days until the conclusion of the experiment. Cells from
three wells each were collected and counted at 24 hr intervals for five days for
growth curve. For thymidine incorporation, TICs were incubated with 0.5 µCi/well
[3H]thymidine for 48hr post drug selection. 10% trichloroacetic acid was used to
precipitate the DNA and radioactivity was determined using Perkin Elmer
Luminescence Counter after washing.
Tumor Formation Assay
For xenograft model, one million TIC cells stably transfected with shscramble
(control), shβ-catenin 1 and shβ-catenin 2 were injected subcutaneously onto the
back of nude mice. Tumor growth is monitored daily.
For ICG-001 treatment, Mini osmotic pumps (Alzet model # 1004) containing
ICG-001 or saline were implanted sub-cutaneously at the back of the Pten null
C57BL/6 at 1 month of age. After 6 days recovery diet containing 0.05% DDC
was administered for 72 hours followed by normal chow for 72 hours twice
(supplemental Fig 5). Blood samples were collected through cardiac puncture
and perfused liver was collected for histo-chemical and biochemical analysis.
74
Statistical Analysis
Statistical differences between control and Pten mutant groups are determined
using Student’s t tests. Multigroup comparisons were performed using ANOVA
analysis followed by Newman Keuls pairwise comparison test. A p value of less
than 0.05 was considered significant. Data are presented as mean ± standard
error of the mean.
Protocol Development
Orthografting and Ultrasound
Prior to orthografting, mice were injected ip with 70 mg/kg retrorsine
(sigma Aldrich), an alkaloid compound that exerts a strong and persisitent
blockage of native hepatocyte proliferation and augments competitive advantage
of transplanted cells.
To prepare retrosine of final concentration of 5mg/ml, first dissolve the
powder (initial concentration 10mg/m/) in 1M HCL. Add a few drops at a time
and vortex vigorously to thoroughly dissolve. To neutralize the PH, we then add
NaOH till PH=7. Final injection 70mg/kg of reterosine and a final concentration of
5mg/ml calculate to become 14ul/g of mouse. Retrosine was injected twice (30
day and 15 day) prior to liver transplantation of cells.
One day prior to cell injection, shave the stomach area (doing this helps
prevent hair contamination on the day of surgery). On day of surgery, 500,000
TICs were dissolved in 50ul PBS and transplanted into the liver through a mini-
laparatomy incision after mice were properly anesthetized. The biggest liver lobe
was retracted and clamped during TIC injection and held for 1-3 minutes till the
75
blood clots. The abdomen was then sutured using 4-0 vicryl. Iodine was
administered at the suture site to prevent infection. Mice were monitored there
after for proper healing.
Four and eight months after injection of TICs mice were monitored for the
growth of liver tumors using ultrasound (the facility is available at USC). After
mice were shaved, we were able to visualize a small tumor in the scramble
(control) mouse at 8 months after TIC injection but not visible tumor was detected
when cells were treated with shβ-catenin 1. A 12 month old tumor bearing
mouse was used as control for ultrasound.
Liver progenitor cell isolation and flow cytometry protocol
To perform liver progenitor cell isolation, the first step is cell dissociation. Under
sterile conditions, the liver is dissected out and rinsed in PBS. We then cut 1
small piece for paraffin embedding, 1 small piece for OTC and 1 small piece will
be kept for protein and RNA isolation. Equal amount of liver from each
respective mouse will then be minced with razor blades for cell isolation. The
minced liver tissue will then be collected in 10 ml digestive solution (Dispase
1mg/ml and DNase 50ug/ml) and incubated in 37c water bath for a total of 50
minutes. During incubation, the tube containing the tissue is shaken to mix the
sendemented tissue with the digestive solution. Every 10 minutes, 5ml solution
is collected and put in a different tube that contains 25ml serum containing
culture medium and 5 more ml digestive solution is added to the tissue. Once all
cells are digested, they are passed through a 70uM sterile filter. To separate
large parenchymal cells from the small cells that contain progenitor cells,
76
differential centrifugation is performed at 100xg for 3 minutes. 2 ml ammonium
chloride is then added to the cells in suspension and put on ice, away from light
for 10 minutes to lyse red blood cells. The cells are then washed 2x in
DMEM/F12 medium. To further enrich the progenitor cell population, MACS
apparatus from Miltenyi Biotec is used. 1x 107 washed and centrifuged cells are
resuspended in bead binding solution that contains 80ul DMEM/F12 and 20ul
anti CD 45 bead and incubated at 4C for 15 minutes. The cells are then washed
with DMEM/F12 and centrifuged at 400g for 10 minutes and applied to prepped
miltenyi biotec column and magnetic separator. The column is then washed to
serum free medium 3x, 3ml each. The flow through is collected with contain the
CD45- (non-blood cell) population. Cells are spun at 1500rpm for 5 mins and
are now enriched for liver progenitors without blood cell contamination. The cells
are now ready for flow cytometric analysis.
Flow Cytometry Analysis
After blocking for 30 minutes, one million cells were incubated for 30 minutes
with primary antibodies: CD133 (Miltenyi-Biotec), Streptavidin eluor 450 (e-
Bioscience), Anti-Human/mouse CD49f-APC (e-bioscience), Anti-mouse CD133-
FITC (e-Bioscience). Cells were then washed in PBS and analyzed using Cyan
(Bechman Coulter) and Calibur. Further analysis was conducted using Flow-Jo
program.
77
Table 1. Tumor incidence in CR and Akt2 deleted Pten null mice vs. controls
Table 2. Diet composition table
Kcal % LFD HFD
CHO 67 21
FAT 10.5 60
Protein 23 80
Control Pten null
Ad lib (13 months) 0/10 7/7
CR (3months+10months
CR)
0/10 0/7
Akt2 wild type (>12months) 0/10 26/26
Akt2 deleted (>12 months) 0/10 6/48
78
Table 3. Primers Used for Real-Time PCR and sequence for shβ-catenin
Gene Forward Primer (5’ to 3’) Reverse Primer (5’ to 3’)
Wnt 7a CGACTGTGGCTGCGACAAG CTTCATGTTCTCCTCCAGGATCTTC
Wnt
10a
GACTCCACAACAACCGTGTG CCTACTGTGCGGAACTCAGG
Fzd2 CTCAAGGTGCCGTCCTATCTC
AG
GCAGCACAACACCGACCATG
Cyclin
D1
TCCGCAAGCATGCACAGA GGTGGGTTGGAAATGAACTTCA
Survivi
n
ACTACCGCATCGCCACCT GACGGTTAGTTCTTCCATCT
EpCA
M
AGGGGCGATCCAGAACAACG ATGGTCGTAGGGGCTTTCTC
AFP ATCGACCTCACCGGGAAGAT GAGTTCACAGGGCTTGCTTCA
K-19 CCGGACCCTCCCGAGATTA CTCCACGCTCAGACGCAAG
Trop2 CTGACCTAGACTCCGAGCTG CCAACCCATCTGGTCTGAGG
GAPD
H
GTCGGTGTGAACGGATTTGG GACTCCACGACATACTCAGC
18S AAACGGCTACCACATCCAAG CAATTACAGGGCCTCGAAAG
shb-
catenin
1
GATCCGATGTTGACACCTCCC
AAG
TTCAAGAGACTTGGGAGGTGT
CAA
CATCTTTTTTGGAAA
AGCTTTTCCAAAAAAGATGTTGACA
CC
TCCCAAGTCTCTTGAACTTGGGAG
GTG
TCAACATCG
shb-
catenin
2
GATCCGGCTTTCCCAGTCCTTC
AT
TCAAGAGATGAAGGACTGGGA
AA
AGCCTTTTTTGGAAA
AGCTTTTCCAAAAAAGGCTTTTCCC
AG
TCCTTCATCTCTTGAATGAAGGACT
GG
GAAAAGCCG
79
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Abstract (if available)
Abstract
Obesity is an independent risk for carcinogenesis including liver cancer. Liver cancer is an aggressive and deadly disease with poor outcome. Approximately half of hepatocellular carcinoma (HCC) presents progenitor cell signatures, indicative of the presence of Tumor Initiating Cell (TIC) populations. Despite many progress in characterizing the physiology of hepatic TICs, a strong need remains to delineate signaling pathways regulating these cells. Our lab previously established a mouse model specifically lacking PTEN (phosphatase and Tensin homologue on Chromosome 10), a critical negative regulator of PI3K/AKT, in liver hepatocytes. We have shown that loss of PTEN induces fatty liver mediated hepatic injury followed by TIC expansion and upregulation of hepatic progenitor cell markers. In addition, we also found factors governing progenitor niche such as Wnt ligands including Wnt7a and 10a and Wnt receptor Fzd, as well as β-catenin (a downstream factor of Wnt signaling) are upregulated. Wnt/β-catenin has been shown to promote self-renewal and proliferation of TICs. Furthermore, interrupting Wnt/β-catenin pathway using shRNA and small molecule inhibitor resulted in attenuation of TIC proliferation. This prompted us to assess what biological mechanism mediates the induction of the Wnt/β-catenin pathway resulting in TIC proliferation and liver cancer using the Pten null liver cancer model. ❧ Preliminary data from our lab demonstrates Pten deletion in the liver results in steatosis and fatty liver disease at 1 month of age and subsequent development of liver cancer at 9 to 12 months. This observation is in line with the strong link between obesity and liver cancer. Majority of liver cancer patients experience liver steatosis as a pre-condition before cancer development. The focus of my doctoral studies is to unveil how fatty liver, a stereotypical feature of obesity contribute to liver cancer development. The central hypothesis for my research is hepatic steatosis stimulates TIC activation and liver cancer development through the Wnt/β-catenin signaling pathway. To test this hypothesis a non-genetic approach, High Fat Diet (HFD) and Calorie Restriction (CR) feeding was used to investigate the development and blockage of steatosis respectively. Indeed, HFD feeding led to an increased lipid accumulation and liver injury and further induced wnt signals whereas CR not only blocked steatosis but also led to the reduction of wnt signals and abrogated expression of hepatic TICs. This data established Wnt/β-catenin as a novel signal induced by steatosis to promote tumor growth, underlying the increased risk of tumor development in obese individuals. ❧ In summary, data from these studies established that hepatosteatosis resulting from Pten deletion or high fat feeding induces activation of Wnt/β-catenin pathway. Wnt/β-catenin signal induces the expansion of Pten deletion transformed liver TICs. In Pten null mice fed a low caloric diet to inhibit hepatosteatosis, reduced Wnt/β-catenin activation is observed concurrent with inhibited expansion of TICs and complete blockage of tumorigenesis. Blocking Wnt/-catenin signal with pharmacological or genetic approaches inhibits the proliferation of TICs in vitro, decreases accumulation of TICs and reduces tumor grafts in vivo. Given that both obesity and liver cancer epidemic is on the rise in the US, understanding how fatty liver contributes to hepatic tumorigenesis will not only delineate the interaction between lipid metabolism and cancer development, but also hold promise for developing effective treatment and eventually eradicating liver cancer.
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Creator
Debebe, Anketse
(author)
Core Title
Steatosis induces Wnt/β-catenin pathway to stimulate proliferation of hepatic tumor initiating cells and promote liver cancer development
School
School of Pharmacy
Degree
Doctor of Pharmacy / Doctor of Philosophy
Degree Program
Molecular Pharmacology and Toxicology
Publication Date
11/26/2015
Defense Date
10/14/2015
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
fatty liver,liver cancer,OAI-PMH Harvest,obesity,tumor initiating cells,Wnt/β-catenin
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application/pdf
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Language
English
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Electronically uploaded by the author
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Stiles, Bangyan (
committee chair
), Machida, Keigo (
committee member
), Okamoto, Curtis (
committee member
)
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anketsed@gmail.com,kassa@usc.edu
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https://doi.org/10.25549/usctheses-c40-201414
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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...
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Tags
fatty liver
liver cancer
obesity
tumor initiating cells
Wnt/β-catenin