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AKT exhibits isoform-specific function in hepatocyte transformation and response to cellular stress
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AKT exhibits isoform-specific function in hepatocyte transformation and response to cellular stress
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Content
AKT exhibits isoform-specific function in hepatocyte transformation
and response to cellular stress
by
Yushan Wang
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCE)
August 2024
Copyright [2024]
Yushan Wang
ii
Acknowledgments
I sincerely extend my appreciation to Dr. Bangyan L Stiles, my advisor during my pursuit
of a master’s degree at USC. Her unwavering support and encouragement in helping me reach my
research goals and develop critical thinking skills have greatly contributed to my education and
future endeavors. The enthusiasm and knowledge I have gained from her have propelled my
studies and will continue to drive my future success. She has always urged me to think critically
like a scientist, fostering my growth and development in profound ways.
Ielyzaveta Slarve has been an invaluable support to me. When I initially joined the lab,
she generously dedicated her time to teaching me lab techniques and providing me with creative
inspiration. I could not have been successful without her help. It is my pleasure to have her as my
mentor. I also want to especially thank Lina He for her patience and abundant advice in training
my animal handling skills. Thanks to Mario Alba, Qi Tang, and Taojian Tu for sharing their
extensive experience and knowledge. Additionally, thanks to Zixin Zong, who became my friend
so that we could enjoy the food and scenery in LA.
I express sincere gratitude to every member of our lab, notably Diala Alhousari, Ebony
Flower, Guo Zhang, and all the lab members, for our perfect teamwork and for creating a joyful
lab environment. Thank you for all your continued support and companionship. I have had the
best two years of my life studying and researching, and I am immensely grateful and proud to be
a part of Dr. Stiles' lab.
iii
Table of Contents
Acknowledgments..........................................................................................................................ii
List of Figures................................................................................................................................v
Abstract..........................................................................................................................................vi
Chapter 1: PI3K/AKT Signaling Pathway and Liver Cancer...........................................................1
1.1 PI3K/AKT Signaling Pathway and its Mechanism and Functions...................1
1.2 PI3K/AKT pathway and Cancer Stem Cells in
Hepatocellular Carcinoma (HCC).............................................................................5
1.2.1 Cancer Stem Cells and HCC.....................................................................5
1.2.2 PI3K/AKT Pathway Regulates CSCs in Liver Cancer..............................6
1.3 AKT Isozymes' Impact on Hepatocellular Carcinoma Progression
via CSC Mediation....................................................................................................8
1.4 Hypothesis and Aims.........................................................................................11
Chapter 2: Akt1 and Akt2 Deletion Regulate the Stem Cell-like Properties in
Immortalized Mouse Hepatocytes...............................................................................12
2.1 Introduction and Rationale................................................................................12
2.2 Results and Discussion......................................................................................13
2.2.1 Akt1 and Akt2 Deficient Cells Exhibit Decreased Proliferation
and Viability in Mouse Hepatocytes................................................................13
2.2.2 Akt1 and Akt2 Deficiency Affects Cell Morphology
and Cell-Cell Communication in Mouse Hepatocytes.....................................16
2.2.3 Akt1 and Akt2 Deficiency Increases HCC Cell Migration
via Epithelial-to-Mesenchymal Transformation (EMT)..................................19
2.2.4 Isoform-Specific Function of Akts Significantly Affect
Tumor Sphere Formation of HCC Cells In Vitro.............................................20
Chapter 3: Effects of Cellular Stresses and Chemotherapy on the Cell Proliferation
of Akt1 and Akt2 Deleted Immortalized Mouse Hepatocytes....................................23
3.1 Introduction and Rationale............................................................................23
3.2 Results and Discussion..................................................................................24
3.2.1 Neither Akt1 nor Akt2 Deficiency Affects Cell Apoptosis
in a Serum-Starved Environment.....................................................................24
3.2.2 Isoform-Specific Akts Function Significantly Affect
Chemotherapy Resistance of HCC Cells In Vitro............................................27
Chapter 4: Discussion...................................................................................................................28
Chapter 5: Methods and Materials ................................................................................................32
5.1 Cell Lines and Cell Culture............................................................................32
5.2 Western Blot..................................................................................................32
5.3 Proliferation Assay........................................................................................33
iv
5.3.1 Proliferation Assay..............................................................................33
5.3.2 MTT Assay..........................................................................................33
5.4 Migration Assay............................................................................................34
5.4.1 Wound Healing Migration Assay........................................................34
5.4.2 Transwell Assay..................................................................................34
5.5 Sphere Formation Assay................................................................................34
5.6 Serum Starvation Assay.................................................................................35
5.7 Confluency Assay..........................................................................................35
5.8 Antibiotic Treatment Assay...........................................................................35
5.9 Chemotherapy Treatment Assays..................................................................36
5.10 Statistics......................................................................................................36
References.....................................................................................................................................37
v
List of Figures
Figure 1. PI3K/AKT signaling pathway..........................................................................................3
Figure 2. Comparison of human AKT isoform domain structures...................................................4
Figure 3. Immunoblotting analysis of AKT proteins in immortalized hepatocytes
Isolated from mice with Akt1 (Akt1-/-
; Akt1-null) or Akt2 (Akt2-/-
; Akt2-null) deletion...................14
Figure 4. The cell growth was evaluated with hemocytometer......................................................15
Figure 5. The cell growth was evaluated with MTT assay.............................................................15
Figure 6. The sizes of WT, Akt-null, and Akt2-null cells in cell culture medium............................16
Figure 7. Effect of serum starvation on cell proliferation at different confluences,
assessed by hemocytometer count.................................................................................................17
Figure 8. Morphological changes of cells under serum-free environment at
70% cell confluency......................................................................................................................18
Figure 9. Morphological changes of cells under serum-free environment at
100% cell confluency....................................................................................................................18
Figure 10. The wound healing assay, migration of WT, Akt1-null and Akt2-null cell....................20
Figure 11. The wound scratch assay is quantified by measuring wound closure...........................20
Figure 12. The Transwell assay, migration of WT, Akt1-null and Akt2-null cells..........................20
Figure 13. Number and size of hepatospheres formed by WT, Akt1-null or Akt2-null cells,
scale bar = 100 μm, original magnification ×100..........................................................................21
Figure 14. Suppressive effect of serum‑starvation on cell proliferation,
assessed by hemocytometer count.................................................................................................25
Figure 15. Antibiotic kill curve for WT, Akt1-null and Akt2-null cell lines...................................26
Figure 16. Growth-inhibitory effects of sorafenib in WT, Akt1-null and Akt2-null cell lines........27
vi
Abstract
Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related
mortalities worldwide. The Phosphoinositide-3-kinase-protein kinase B/Akt (PI3K-PKB/Akt)
pathway plays an important role in regulating stem cells in HCC. The origins of PI3K/Akt
research can be traced back to the discovery in 1977 by Staal and co-workers. Although Akt’s
main functions have been extensively described, particularly its role in promoting metabolism,
proliferation, cell survival, growth, and angiogenesis in response to extracellular signals, how
isoform-specific Akt may regulate the cell’s stem cell-like properties largely remains unknown.
In our in vitro studies, we characterized the role of Akt isoforms in regulating the stem cell-like
phenotype in immortalized mouse hepatocytes lacking either Akt1 or Akt2. Our data
demonstrated that the deletion of Akt1 (Akt1 -/-; Akt1-null) or Akt2 (Akt2-/-
; Akt2-null) significantly
enhanced several stem cell-like properties in mouse hepatocytes. Compared to WT cells, both
Akt1-null and Akt2-null cells demonstrated significantly decreased cell growth. Akt2-null cells
exhibited faster migration in the wound scratch assay, while Akt1-null cells migrated faster
towards a chemoattractant in the transwell assay. Akt1-null cells were significantly more resistant
to sorafenib and formed a significantly higher number of spheres in the sphere formation assay
compared to the WT cells, while Akt2-null cells were highly resistant to antibiotic treatment and
formed far fewer tumorspheres. These results contribute to the understanding of the role of Akt
isoforms in regulating stemness in HCC and may serve as a basis for investigation into isoformspecific inhibitors.
1
Chapter 1 PI3K/AKT Signaling Pathway and Liver Cancer
1.1 PI3K/AKT Signaling Pathway and its Mechanism and Functions
The phosphoinositide-3-kinase–protein kinase B/AKT (PI3K-PKB/AKT) pathway was
identified in the early 1980s through extensive efforts to characterize insulin receptor signaling1,2
.
Since then, it has become one of the signaling pathways proven to serve an important role in
regulating cell proliferation, the cell cycle, and apoptosis3
. A number of studies suggest that the
PI3K/AKT signaling pathway is associated with human cancers4,5
, and the rational design of small
molecules targeting the PI3K/AKT signaling pathway is an important option for the treatment of
tumors. In clinical settings, several PI3K inhibitors, such as buparlisib (BKM120)6
, and
copanlisib, are being evaluated for their efficacy in cancer patients, including liver cancer7
.
Similarly, AKT inhibitors like MK-22068
and ipatasertib9
are designed to directly inhibit AKT
kinase activity, preventing it from phosphorylating and activating key substrates involved in cell
survival and growth. Clinical trials have shown that these inhibitors can reduce tumor cell viability
and enhance the effectiveness of other treatments, such as chemotherapy10 and targeted therapies.
For example, combining the PI3K inhibitor copanlisib with sorafenib can synergistically induce
cell death in hepatocellular carcinoma cells, demonstrating the potential for overcoming
chemotherapy resistance and improving patient outcomes.11
PI3K is a member of the lipid kinases family that acts by phosphorylating the 3-hydroxyl
group of the inositol ring of phosphatidylinositol (PtdIns) lipids in the plasma membrane12-13
.
PI3K can be categorized into three classes based on their preference for lipid substrates and
distinct structural features14. Among them, Class I PI3K plays a crucial role in tumors and is the
most widely studied isoform.
Class I PI3K activation begins when cell surface receptors, such as receptor tyrosine
2
kinases (RTKs) or G protein-coupled receptors (GPCRs), bind to their ligands, leading to receptor
autophosphorylation or interaction with G proteins. This creates docking sites for the p85
regulatory subunit of PI3K, which, through its SH2 domains, binds to the phosphorylated
tyrosines on the receptors or adaptor proteins. This binding induces a conformational change that
activates the p110 catalytic subunit of PI3K15. The activated p110 subunit then phosphorylates
PIP2 at the 3-hydroxyl position, converting it into PIP3. PIP3 serves as a secondary messenger,
recruiting and activating proteins with pleckstrin homology (PH) domains, such as AKT and
PDK1, thus initiating signaling pathways involved in cell growth, survival, and metabolism16
(Figure 1).
Over the past decades, studies have shown that PI3K signaling pathway is deregulated in
a wide spectrum of human cancers17
. Hyper-activation of the PI3K/AKT pathway was shown to
contribute to carcinogenesis, proliferation, invasion, metastasis, and drug resistance in tumor
cells18,19
. The PI3K signaling is not only involved in the regulation of the proliferation and
apoptosis of cancer cells but also promotes normal and tumor angiogenesis.
Among the numerous genetic factors involved in the molecular mechanisms of human
cancer, including P53, NFKB, STAT3, and Myc, the PI3K pathway attracts significant attention
due to its central role in multiple cellular processes. Genetic alterations have been identified at
every level of the PI3K signaling cascade. The phosphatidylinositol-4,5-bisphosphate 3-kinase
catalytic subunit alpha (PIK3CA), also known as the p110 alpha protein, is a class I PI3K catalytic
subunit that was initially considered to be highly involved in ovarian cancers20, acting as an
oncogene. In addition, somatic mutations in PIK3CA have also been found in colorectal,
glioblastoma, gastric, breast, lung, and renal cancers21,22
. Mutation of PTEN, which
dephosphorylates PIP3 and down-regulates PI3K activation, has also been reported in primary
3
cancers in the breast, liver23, colon, prostate, uterus, central nervous system and soft tissue. Its
function loss leads to a constitutive increase in PI3K/AKT pathway activity and PTEN alterations,
mutations, and deletions can induce tumorigenesis and other diseases24,25
.
Various stimuli, including growth factors, cytokines, and hormones, can activate
PI3K26,27
. Among the targets that receive signaling from the downstream cascade of PI3K, the
most critical mediator is the serine/threonine kinase AKT15. AKT serves a dominant role in the
signal transduction of the PI3K pathway. First discovered in 198728, AKT has been implicated in
various diseases, including cancer29
. Mammals have three isoforms of AKTs: AKT1, AKT2, and
AKT3
30
. They all share a highly conserved N-terminal pleckstrin homology (PH) domain, a
Figure 1. PI3K/AKT signaling pathway12: Growth factors binding to
receptor tyrosine kinases (RTKs) initiate the pathway by activating
Ras, which subsequently activates PI3K. Activated PI3K converts PIP2
into PIP3, leading to the recruitment of PDK-1 and Akt to the plasma
membrane. PDK-1 phosphorylates Akt, which is further activated by
mTORC2 and DNA-PK. Activated Akt then phosphorylates various
target substrates involved in cellular processes such as growth, survival,
and metabolism. The pathway is negatively regulated by PTEN, PP2A,
and PHLPP, which dephosphorylate PIP3 and Akt, respectively.
4
serine-threonine kinase catalytic domain and a
small carboxy-terminal regulatory domain that
contains a hydrophobic domain (HD) with
homology to other AGC kinases31,32 (Figure 2).
AKT is activated by phosphorylation at two sites
including one in the activation loop (A-loop)
(T308, T309, T305 in AKT1, AKT2, and AKT3,
respectively) in the catalytic domain, and one in
the C-terminal HD (S473, S474, S472 in AKT1, AKT2, and AKT3, respectively)33
. The activation
of the PI3K/AKT pathway is tightly controlled via a highly conserved multistep process.
Activated receptors directly stimulate class 1A PI3Ks bound via their regulatory subunit or
adapter molecules such as the insulin receptor substrate (IRS) proteins. This triggers the activation
of PI3K and conversion by its catalytic domain of PIP2 lipids to PIP3. PKB/AKT binds to PIP3
at the plasma membrane, allowing PDK1 to access and phosphorylate T308 in the “activation
loop,” leading to partial PKB/AKT activation. Concurrent phosphorylate at the c-terminal serine
site leads to full activation of the AKTs.
While AKT1 and AKT2 are abundant in numerous tissues, including the liver, AKT3 is
predominantly expressed in the brain, where it plays a crucial role in maintaining central nervous
system (CNS) cell integrity34,35
. Recent studies have reported the isoform-specific functions of
AKT and their distinct and sometimes opposing roles in metabolism and growth36,37. Mice lacking
Akt1 exhibited abnormal growth with a reduction in body weight38,39
. Conversely, Akt2-deficient
mice exhibited normal growth but a diabetes-like syndrome with an increase in fasting plasma
glucose level, elevated hepatic glucose output and peripheral insulin resistance38,40
. Akt3-deficient
Figure 2. Comparison of human AKT
isoform domain structures.
5
mice showed a reduction in brain weight while maintaining normal glucose homeostasis and body
weight41
. These findings collectively highlight the diverse biological roles played by different
AKT isoforms and their specific contributions to physiological processes.
1.2 PI3K/AKT Pathway and Cancer Stem Cells in Hepatocellular Carcinoma (HCC)
1.2.1 Cancer Stem Cells and HCC
Liver cancer is the sixth most common malignancy and the third major cause of cancer
mortality. In the year 2020 alone, 905,677 new cases of liver cancer were diagnosed, with 830,180
fatalities. Primary liver cancers include hepatocellular carcinoma (HCC), intrahepatic
cholangiocarcinoma (CCA), and combined HCC-CCA tumors (cHCC-CCA)42. HCC accounts for
~90% of primary liver cancer cases, with an overall 5-year relative survival rate of merely
18.4%43
. The treatment of HCC faces multiple barriers. First, HCC tumors usually show strong
resistance to conventional chemotherapy and are usually advanced when detected, making
complete surgical resection difficult. In addition, HCC usually occurs in the livers of patients with
cirrhosis, whose livers have been severely damaged by years of active hepatitis or exposure to
toxins. As a result, patient tolerance to therapy is severely limited.
The existence of CSCs has been proposed to play critical roles in HCC due to their
heightened resistance to chemotherapy. The persistence of CSCs following primary therapy
significantly contributes to tumor recurrence, the emergence of chemoresistance, and poor clinical
outcomes44
. Cancer Stem Cells (CSCs) are a small subpopulation of cells within tumors with
capabilities of self-renewal, differentiation, and tumorigenicity when transplanted into an animal
host, which is also responsible for the maintenance and propagation of the tumor45
. CSCs were
found in several hematopoietic cancer and solid tumors, including HCC. Extensive research has
6
demonstrated that CSCs provide HCC with a proliferative, invasive, and recurrent advantage. The
existence of CSCs in HCC has been demonstrated through the identification of several surface
markers including CD44, CD24, and CD133 46
,
47
. Notably, cells capable of forming spheres via
external culture and fusion cells originating from both cancer cells and stem cells have been
identified as potential CSCs48
. The theories on the origin of CSCs in HCC, however, are
contentious. Investigations into the origin of CSCs in HCC have been employed in vitro culture
and immunodeficient tumor models. These studies propose that CSCs arise from liver progenitor
cells (LPCs), while others suggest they derive from the de-differentiation of mature cells or biliary
cells, triggered by genetic or epigenetic alterations, or induction by pluripotent factors such as
Nanog, Oct4, Yamanaka factor, and Sox2 through reprogramming49
.
1.2.2 PI3K/AKT Pathway Regulates CSCs in Liver Cancer
Accumulating evidence also suggests that CSCs are responsible for chemoresistance and
cancer relapse, as they can self-renew and differentiate into the heterogeneous lineages of cancer
cells in response to chemotherapeutic agents50. CSCs also exhibit the ability to rapidly mediate
toxic efflux and respond to oxidative stress and DNA damage, which further contributes to drug
resistance. For instance, resistance to sorafenib may be linked to Nanog+ CSCs51, while the long
non-coding RNA (lncRNA) THOR inhibits CSCs and enhances HCC sensitivity to sorafenib52
.
The activation of the PI3K/AKT pathway can significantly contribute to drug resistance in liver
cancer stem cells (CSCs) by promoting survival, enhancing efflux transporter activity,
reprogramming metabolism, improving DNA repair, and maintaining stemness53. Activation of
this pathway inhibits apoptotic factors and upregulates cell cycle progression, allowing CSCs to
evade drug-induced cell death. Additionally, the PI3K/AKT pathway enhances DNA repair
7
mechanisms, enabling CSCs to fix drug-induced damage, and promotes stemness-related
transcription factors like NANOG and SOX254, which sustain the CSC phenotype and resistance.
Targeting the PI3K/AKT pathway with specific inhibitors, potentially in combination with other
therapies, offers a promising strategy to overcome CSC-mediated drug resistance and improve
liver cancer treatment outcomes.
The PI3K/AKT pathway also plays a key role in regulating cancer stem cells (CSCs) in
cancer by controlling metabolic pathways. Increased aerobic glycolysis is a crucial hallmark of
cancer metabolism. Many studies have supported the hypothesis that CSCs are more glycolytic
than normal cancer cells55. Similar to normal stem cells, glucose is an essential nutrient for CSCs,
and its presence in the microenvironment significantly increases the number of stem-like cancer
cells in the cancer cell population. Glucose induces the expression of specific genes in CSCs
associated with the glucose metabolism pathway (c-Myc, Glut-1, HK-1, HK-2, and PDK-1),
which contributes to the increase in the CSC population56
.
Liver cancer is often characterized by increased glycolytic activity driven by PI3K/AKT
activation, which is essential for hepatocellular carcinoma (HCC) growth. This pathway controls
multiple aspects of glucose metabolism through both acute post-translational modifications and
prolonged transcriptional effects on glucose transporters and glycolytic enzymes57,58
.
Additionally, PI3K/AKT promotes glycolysis by activating hexokinase (HK) and facilitating the
binding of HK2 to the voltage-dependent anion channel in mitochondria58. The pathway also
indirectly regulates glycolytic enzymes by influencing the expression of AMPK59 and HIF-1
60
.
The activation of the PI3K/AKT signaling pathway is sufficient to promote aerobic glycolysis,
leading to an increase in glucose uptake and utilization, which in turn increases CSC survival and
proliferation, thereby contributing to HCC generation and progression.
8
Additionally, the PI3K/AKT pathway is pivotal in regulating CSCs by modulating lipid
metabolism, which is critical to tumorigenesis by providing energy to cancer cells, sustaining cell
growth, and providing intermediates for biosynthesis61
. Fatty acids and cholesterol, crucial
components of cell membranes, are vital for tumor growth and progression62, while lipid synthesis
and oxidation are essential for CSC maintenance. For example, NANOG, a critical regulator, can
promote mitochondrial fatty acid oxidation (FAO) to meet the energy demands of tumor-initiating
cells (TICs), enhancing their self-renewal, tumor-initiation properties, and hepatocellular
carcinoma (HCC) oncogenesis through metabolic reprogramming from oxidative
phosphorylation (OXPHOS) to FAO. The activation of PI3K/AKT pathway enhances lipid
biosynthesis by activating key enzymes such as ATP citrate lyase, acetyl-CoA carboxylase63, and
fatty acid synthase, and regulates lipid storage and utilization through sterol regulatory elementbinding proteins64,65. This lipid metabolic reprogramming provides CSCs with the necessary
building blocks and energy for rapid proliferation and tumorigenicity, while also contributing to
metabolic flexibility and a supportive tumor microenvironment.
In conclusion, CSCs play a role in HCC metastasis and drug resistance. Therefore,
studying the cellular signaling pathways of CSCs driving HCC will help researchers understand
the mechanisms of HCC generation and increase the efficiency of finding new targets for future
therapeutic development.
1.3 AKT Isozymes' Impact on Hepatocellular Carcinoma Progression via CSC Mediation
The AKT kinase family consists of three highly homologous isozymes: AKT1, AKT2,
and AKT3. AKT1 and AKT2 are widely expressed and involved in a variety of cellular processes,
including the regulation of metabolism and cell growth, whereas AKT3 is expressed
9
predominantly in the brain and testis37. Dysregulation of the PI3K/AKT pathway has been
implicated in tumorigenesis, and each AKT isoform may play a different role in cancer
development and progression. AKT isozymes also play key roles in regulating cancer stem cell
(CSC) properties and epithelial-mesenchymal transition (EMT), which is an important process in
tumor progression and drug resistance66
. For example, it has been found that in breast cancer and
glioma, AKT1 and AKT2 are critical for maintaining CSC-like phenotype and survival, with
AKT1 playing a more prominent role. Conversely, AKT2 deficiency reduces carcinoid stem cell
markers and EMT features, highlighting the specific role of the isoforms in tumorigenesis67. In
addition, the downstream effectors of AKT signaling differ across isoforms, with Foxo/Bim
associated with AKT1-dependent proliferation, while YAP/TAZ signaling is associated with
AKT2-mediated proliferation and stemness maintenance68. In addition, β-catenin is a coparticipant in AKT1 and AKT2 oncogenic signaling pathways, suggesting its involvement in CSC
regulation and EMT transformation.
In vivo, studies analyzing AKT isoform-deficient mouse models demonstrated isoformspecific functions of each AKT. In the germline deletion of AKT isoforms, Akt1 deletion is well
tolerated and can inhibit the development of cancer in various mouse models69,70. By contrast,
Akt2 deletion elicits insulin resistance and does not inhibit cancer development in mouse
models71,72. The major physiological difference between Akt2-/- mice and either Akt1-/-
or WT
mice is the insulin resistance and high blood insulin level in Akt2-/- mice. Up to 10-fold higher
levels of insulin have been found in Akt2-/- mice73. Additionally, two-week-old male Akt1 and
Akt2 knockout mice received a 25 mg/ml DEN intraperitoneal injection to induce
hepatocarcinogenesis, and 12 months later, Akt2 knockout mice with lung metastases exhibited
significantly higher serum insulin levels compared to wild-type mice without lung metastases.
10
The high level of insulin in Akt2-/- mice leads to compensatory activation of Akt1
74, as well as
Erk1/2 activation. Further studies on the effect of AKT isoforms on tumor progression and
emulation of the drug therapy administered after tumor detection show that Tamoxifen-injected
Akt1 or Akt2 whole-body deletion in adult mice induced extremely rapid mortality and a rapid
loss of body weight, demonstrating the critical roles of AKT in maintaining homeostasis. The
study also suggests the role of AKTs in the utilization of fat for fatty acid oxidation and possibly
a block in adipocyte differentiation, as observed in newborn mice carrying a germline deletion of
both Akt1 and Akt275
.
In the liver, the deletion of Akt1 in the livers of Akt2-/- mice induces liver damage followed
by inflammation and HCC development. The serum levels of the liver enzymes alanine
transaminase (ALT) and aspartate transaminase (AST) were markedly elevated in Akt1hep-/-
; Akt2-
/- mice, indicating liver damage75
. Moreover, serum IL-6 levels and liver tumor necrosis factoralpha (TNF-a) mRNA were highly elevated in Akt1hep-/-
; Akt2-/- mice compared with Akt2-/- mice,
suggesting inflammation. In addition, based on gene expression, Akt1hep-/-
; Akt2-/-
liver tumors
appeared to be characterized as undifferentiated and of embryonic origin, resembling a more
primitive stem cell characteristic. The high expression levels of genes, such as IGF2BP1 and,
particularly, IGF2BP3, are proposed to facilitate tumorigenesis. The high expression level of
IGF2BP1 and IGF2BP3 has been observed in many human cancers, including HCC, in which
they have been associated with the induction of cell proliferation, cell survival, and
invasiveness76,77. Both IGF2BP1 and IGF2BP3 are highly and frequently expressed in human
HCC, and their expression is correlated with aggressiveness, invasiveness, and poor
prognosis78,79
. It is unclear however how loss of the AKT2 lead to induced expression of these
genes. Thus, more mechanistic studies to dissect the function of each AKT isozyme in
11
tumorigenesis and stem cell regulation is essential for understanding the potential pathogenic
mechanisms of HCC and improvement of clinical therapeutic treatments.
1.4 Hypothesis and Aims
The PI3K/AKT pathway is one of the major signaling pathways in CSCs involved in the
maintenance of stemness, proliferation, differentiation, EMT, migration, and autophagy. CSCs
play a crucial role in the initiation and metastasis of hepatocellular carcinoma (HCC) due to their
intrinsic abilities for self-renewal and tumor initiation. It is believed that the overexpression of
the PI3K/AKT signaling pathway is involved in the initiation of tumors.
In this project, we aim to investigate the impact of isoform-specific AKT on stem cell-like
properties. We hypothesize that AKT isoforms exhibit distinct regulatory effects on hepatocyte
stemness properties. We applied various in vitro approaches to assess the phenotype of cell lines
with or without isoform-specific AKT. We employed a range of two-dimensional (2D) and threedimensional (3D) assays to investigate the changes in cell stemness properties regulated by AKT1
and AKT2 in immortalized mouse hepatocytes. The results of this study will significantly impact
the understanding of how isoform-specific AKT regulates stem cell-like properties in hepatocytes,
potentially revealing novel mechanisms and therapeutic targets for liver-related diseases.
12
Chapter 2. Akt1 and Akt2 Deletion Regulate the Stem Cell-like
Properties in Immortalized Mouse Hepatocytes
2.1 Introduction and Rationale
Hepatocellular carcinoma (HCC) is a predominant form of liver cancer, posing significant
challenges in clinical management due to its high incidence of recurrence and metastasis. Recent
studies have demonstrated the pivotal role of cancer stem cells (CSCs) in the initiation and
progression of HCC. CSCs are a subpopulation of cancer cells characterized by their ability to
self-renew and drive tumorigenesis. Understanding the molecular mechanisms underlying the
behavior of CSCs is crucial for developing targeted therapies that could effectively impede HCC
progression and improve patient outcomes.
The AKT signaling pathway is a critical regulator of various cellular processes, including
growth, survival, and metabolism. AKT1 and AKT2, two key isoforms of the AKT family, have
been implicated in cancer development and progression. However, their specific roles in liver
cancer, particularly in the context of CSC biology, remain unclear. Deleting Akt1 and Akt2 in
hepatocytes presents a unique opportunity to elucidate their functions and interactions in HCC.
To investigate CSC properties in HCC, we employed migration, invasion, and sphere formation
assays, which reflect CSCs’ enhanced migratory, invasive, and self-renewal capabilities.
Migration assays indicate the potential for metastasis, while invasion assays assess the ability to
penetrate extracellular matrix components, both crucial behaviors of CSCs. Sphere formation
assays measure the ability to grow in non-adherent, three-dimensional environments, a hallmark
of self-renewal. The PTEN/PI3K/AKT pathway, known to regulate these processes, is often
dysregulated in cancers. Loss of PTEN or activation of PI3K/AKT signaling enhances cell
migration, invasion, and sphere formation by promoting motility, extracellular matrix
13
degradation, and stemness80. Thus, these assays combined with insights into the
PTEN/PI3K/AKT pathway provide a comprehensive understanding of CSC characteristics and
their role in HCC progression.
This chapter aims to characterize immortalized mouse hepatocyte cell lines with deletions
in Akt1 and Akt2. By analyzing these modified cell lines, we seek to uncover the contributions of
AKT1 and AKT2 to hepatocyte biology and their potential impact on HCC initiation and
metastasis. This research will provide valuable insights into the differential roles of AKT isoforms
in liver cancer, potentially guiding the development of more precise therapeutic strategies
targeting the AKT pathway in HCC.
2.2 Results and Discussion
2.2.1 Akt1 and Akt2 Deficient Cells Exhibit Decreased Proliferation and Viability in Mouse
Hepatocytes
To characterize the immortalized mouse hepatocyte cell lines with deletions in Akt1 and
Akt2, we first investigated the potential role of isoform-specific AKT in regulating proliferation
and growth rates in mouse hepatocytes. We generated hepatocyte cell lines by immortalizing
primary hepatocytes from mice carrying deletions of Akt1 versus Akt2 using a standard 3T3
protocol81
. Immunoblotting analysis of proteins isolated from the established hepatocytes
validated the deletion of Akt1 and Akt2 in the respective cell lines (Figure 3). We also observed
that the protein level of AKT2 is upregulated in Akt1-null cells compared to wild-type (WT) cells,
suggesting a compensatory mechanism where the cell increases AKT2 expression to
counterbalance the loss of AKT1 and maintain necessary signaling pathways. Conversely, in
Akt2-null cells, the protein level of AKT1 remains similar to that in WT cells, indicating that the
14
loss of AKT2 does not trigger a compensatory increase in AKT1 expression.
Next, we compared the growth potentials of these cells with hepatocytes carrying wildtype Akt. Proliferation assays were performed in
wild-type (WT), Akt1-null, and Akt2-null cell
lines, with cell counts conducted using a
hemocytometer. As shown in Figure 4, the
deletion of either AKT significantly decreased
the proliferation of hepatocytes compared to the
wild-type (WT) control. The cell count in the
Akt1-null and Akt2-null cell lines is
approximately 40% to 70% of that observed in
WT cells, respectively. This data indicates a notable reduction in cell proliferation in the absence
of AKT, suggesting both AKTs contribute to the growth of hepatocytes. To improve the rigor of
this experiment, we further confirmed whether Akt deletion results in impaired cell growth. We
conducted MTT assays 24 hours after seeding to assess cell proliferation/survival (Figure 5). The
results also revealed a significant reduction in MTT absorbance in Akt1/2 knockout cells
compared to control cells. Notably, the Akt1-null cells also had significantly reduced MTT
absorbance readings compared with the Akt2-null cells. Together, these proliferation assays
suggest that both AKT1 and AKT2 are essential for efficient cell growth in mouse hepatocytes,
while Akt1 may play a more significant role in this function.
Figure 3. Immunoblotting analysis of AKT
proteins in immortalized hepatocytes
isolated from mice with Akt1 (Akt1-/-
; Akt1-
null) or Akt2 (Akt2-/-
; Akt2-null) deletion.
Glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) is a loading control.
15
Figure 4. The cell growth was evaluated with
hemocytometer count. **P≤0.01,
***P≤0.001, ****P≤0.0001.
Figure 5. The cell growth
was evaluated with MTT
assay. **P≤0.01,
***P≤0.001, ****P≤0.0001.
16
2.2.2 Akt1 and Akt2 Deficiency Affects Cell Morphology and Cell-Cell Communication in
Mouse Hepatocytes
During the characterization of the cell lines, we observed that Akt deficiency not only
impacted cell growth but also altered their morphology and cell-to-cell communication regarding
growth interpretation. Notably, both Akt1-null and Akt2-null cells display elongation compared to
the WT cells (Figure 6). Specifically, the average length of WT cells measures approximately 110
micrometers, while Akt1-null cells exhibit a length of around 280 micrometers, and Akt2-null cells
have an average length of about 220 micrometers. This suggests a substantial increase in cell
elongation in the absence of either AKT1 or AKT2, indicating an alteration in cellular
morphology and potentially an earlier onset of contact inhibition compared to WT cells, with
Akt1-null cells showing a roughly 2.5-fold increase in length and Akt2-null cells showing a
roughly 2-fold increase.
Concurrent with the morphology change, we also observed isoform-specific effects of cell
confluency on serum starvation-induced cell death (Figure 7). Specifically, Akt1-null cells showed
significantly reduced viability when maintained at lower cell confluency levels compared to
higher cell confluency levels. In both WT and Akt2-null cell lines, cell viability was significantly
decreased at both 70% and 100% confluence in response to serum starvation. On the other hand,
while the Akt1-null cells are more sensitive to serum starvation at 70% confluence, they did not
Figure 6. The sizes of WT, Akt-null, and Akt2-null cells in cell culture medium.
17
display a significant reduction of cell viability at 100% confluence. Closer analysis of the cells
suggests that cell contact may contribute to this sensitivity of Akt1-null cells at 70% confluency
and lack thereof at 100% confluency. At 70% confluency, Akt1-null cells exhibited a dispersed
growth pattern with noticeable gaps between cells, contrasting with the clustered growth observed
at 100% confluency (Figure 8, 9). The lack of direct cell-to-cell contact at lower densities may
have resulted in reduced intercellular communication. Thus, the survival of Akt1-null cells in
stressful environments may be more dependent on cell-to-cell communication, potentially
explaining why these cells are more severely impacted at lower densities compared to higher
densities.
Figure 7. Effect of serum starvation on cell proliferation at different confluences,
assessed by hemocytometer count. **P≤0.01, ***P≤0.001, ****P≤0.0001.
18
2.2.3 Akt1 and Akt2 Deficiency Increases HCC Cell Migration via Epithelial-toFigure 8. Morphological changes of cells under serum-free
environment at 70% cell confluency.
Figure 9. Morphological changes of cells under serum-free
environment at 100% cell confluency.
19
Mesenchymal Transformation (EMT)
To investigate the impact of isoform-specific AKT function on cell migration, we applied
the 2D-cultured wound healing assay to assess the migratory ability of Akt knockout cells. Equal
numbers of cells were seeded, and the monolayer was then mechanically scratched to create a
wound, which was monitored for closure over 2, 4, and 6 hours. While both WT and Akt1-null
cells exhibited migratory behavior through moving into the wound area at 4 and 6 hours post
scratching, Akt2 knockout cells exhibited significantly faster migration as indicated by the
significantly less time needed to close the wound. By 6 hours, 71% of the scratch gaps in the Akt2
knockout cells were closed when only 24% and 27% were closed in the WT and Akt1-null cells,
respectively (Figure 10, 11). These findings suggest that the deficiency in Akt2 could enhance cell
migration in mouse hepatocytes.
Furthermore, we examined the migration activity of Akt knockout cells in a 3D cultured
trans-well migration assay. Here, cells were placed in the upper chamber with the serum-free
DMEM, while 10% FBS was added as a chemoattractant to the lower chamber to stimulate the
migration. After 24 hours of incubation, we observed active cell migration in WT control cells,
while Akt1 knockout cells displayed notably higher migration activity (Figure 12). In a contrast,
the Akt2 knockout cells revealed lower migration activity compared to WT cells. These findings
collectively suggest that both AKT1 and AKT2 are essential for regulating cell migration.
20
2.2.4 Isoform-Specific Function of AKTs Significantly Affect Tumor Sphere Formation of
HCC Cells In Vitro
High migration and clonality are distinctive features of cancer stem cells (CSCs) and are
particularly evident in circulating liver CSCs, which actively promote the formation of new
colonization sites82,83. Our data here show that AKT1 and AKT2 have the isoform-specific
capability to mediate cells migration and invasion. Thus, we decided to further explore the impact
Figure 10. The wound healing assay,
Migration of WT, Akt1-null and Akt2-null
cells, magnification x100.
Figure 11. The wound scratch assay
is quantified by measuring wound
closure. **P≤0.01, ***P≤0.001,
****P≤0.0001.
Figure 12. The Transwell assay, Migration of WT, Akt1-null and Akt2-
null cells, magnification x200.
21
of AKT1 and AKT2 on the clonogenic and stemness properties of HCC cells by doing a sphere
formation assay.
Using a criterion of sphere size >100μm, we observed a significant increase in the number
of spheres formed by Akt1-null cells compared to the control wild-type cells (Figure 13). 48hrs
after starting the sphere formation, Akt1-null cells formed approximately 25 ±2.28 ((mean ± SE)
spheres, which is a 2-fold increase relative to the 13.67 ± 1.28 (mean ± SE) spheres formed by
WT cells, a more than 2-fold increase. This data indicates an upregulation of the clonogenic and
stemness phenotypes in these cells, suggesting that the loss of AKT1 led to an upregulated selfrenewal capability in the mouse hepatocytes. Conversely, the Akt2-null cells were not able to form
any spheres that fit the sphere criteria. Thus, the presence of AKT2 may be needed to sustain
clonogenic and stemness properties in these cells.
Previous studies have shown that the deletion of both hepatic Akt1 and Akt2 could lead to
liver injury, inflammation, and unexpected spontaneous HCC development, which may have
significant implications for cancer therapy. Our data support these findings, as Akt1-null cells
exhibit higher stemness properties compared to Akt2-null and wild-type (WT) cells, while Akt2-
Figure 13. Number and size of hepatospheres formed by WT, Akt1-null, or Akt2-null cells,
scale bar = 100 μm, original magnification ×100. **P≤0.01, ***P≤0.001, ****P≤0.0001.
22
null cells demonstrate an increased level of migration only in a 2D cultured environment. This
suggests that hepatic Akt1 inhibition induces liver injury that could potentially promote HCC.
23
Chapter 3. Effects of Cellular Stresses and Chemotherapy on the
Cell Proliferation of Akt1 and Akt2 Deleted Immortalized Mouse
Hepatocytes
3.1 Introduction and Rationale
Cellular stress refers to any disruption in cellular process that leads to an imbalance in the
cellular homeostasis. Cells can respond to stress through various mechanisms, from activating
survival pathways to initiating programmed cell death to eliminate damaged cells, which depends
on their abilities to appropriately respond to environmental or intracellular stress stimuli84
. These
environmental stimuli include exposure to toxins, radiation, extreme temperature, or physical
trauma, while intracellular stress stimuli may involve conditions like oxidative stress, nutrient
deprivation, DNA damage from radiation or chemicals, or protein misfolding in the endoplasmic
reticulum85
. For example, hypoxia, respiratory poisons, and xenobiotics can lead to mitochondrial
stress86, while nutrient deprivation can trigger autophagy, a cellular process where cells degrade
and recycle their own components to sustain survival87
. Chemotherapeutic reagents are designed
to target rapidly dividing cells, such as cancer cells, by interfering with processes like DNA
replication or cell division. However, these agents can also cause damage to healthy cells, leading
to various forms of cellular stress88. This stress can trigger DNA damage responses, activate
apoptotic pathways, or induce cellular senescence89
.
While this cellular adaptation mechanism allows the cells to cope with stress and hostile
environments, studies have also demonstrated their role in promoting cancer cell survival,
therapeutic resistance, and malignant progression, as well as the paralysis of antitumor immune
responses. Given the isoform-specific roles of AKT in the transformation of hepatocytes (Chapter
24
2), we aim to investigate the isoform-specific roles of AKT in regulating the response of
hepatocytes to cellular stress in this chapter.
Sorafenib is a multi-kinase inhibitor that has been widely used as a first-line therapy for
hepatocellular carcinoma (HCC) since its approval in 2007
90
. Despite its effectiveness, resistance
to sorafenib in HCC is a significant challenge, leading to disease progression and reduced patient
survival. To overcome this, combination therapies involving sorafenib and other agents have been
explored to enhance therapeutic efficacy, especially where monotherapy falls short due to drug
resistance or tumor heterogeneity91. Notable strategies include pairing sorafenib with immune
checkpoint inhibitors like nivolumab or atezolizumab to enhance anti-tumor immune responses92
,
and with other targeted therapies such as regorafenib and lenvatinib to target complementary
pathways93. Additionally, combining sorafenib with chemotherapeutic agents like doxorubicin
and cisplatin, anti-angiogenic agents like bevacizumab94, and epigenetic modulators like HDAC
inhibitors are under investigation95. Integrating sorafenib with radiotherapy and natural
compounds like curcumin also shows promise in improving outcomes96
. Given the roles of AKT
isoforms in regulating CSCs' response, we aim to investigate the isoform-specific roles of AKT
in regulating sorafenib sensitivity and explore the potential of combining AKT inhibitors with
sorafenib for enhanced therapeutic effects.
3.2 Results and Discussion
3.2.1 Neither Akt1 nor Akt2 Deficiency Affects Cell Apoptosis in a Serum-Starved
Environment
To address the effects of losing one or the other AKT on cell death, we assessed the impact
of serum starvation on cell viability. Wild-type (WT), Akt1-null, and Akt2-null cell lines were
25
cultured in serum-free DMEM for varying durations of 24, 30, and 36 hours. Cell viability was
evaluated using a hemocytometer under a microscope. As a control representing optimal growth
conditions, WT, Akt1-null, and Akt2-null cells were also cultured in DMEM media supplemented
with 10% FBS. In the WT cell lines, there is a notable decrease in cell viability across all time
points, consistently showing an extremely high level of statistical significance (p < 0.0001).
Surprisingly, neither Akt1-null nor Akt2-null
cells also exhibit a significant decrease in cell
viability at any time point. This suggests that
the deficiency of neither AKT1 nor AKT2
impacts cell apoptosis in a serum-free
environment.
Serum starvation has been known to
induce apoptosis-mediated cell death in various
human and mouse cell lines97,98
. Previous
studies have identified AKTs as critical
survival kinases that play key roles in
preventing cell apoptosis. Both AKT1 and
AKT2 activation have been shown to inhibit
apoptosis. For example, it has been speculated
that AKT can inhibit apoptosis by maintaining
Bcl-x function and preventing cytochrome c
release from mitochondria99. Therefore, it
would be expected that Akt-deficient cell lines would be more sensitive to apoptosis under serum
Figure 14. Suppressive effect of
serum‑starvation on cell proliferation, assessed
by hemocytometer count. **P≤0.01,
***P≤0.001, ****P≤0.0001.
26
starvation conditions. However, our results indicate no significant difference in apoptosis
sensitivity between WT, Akt1-null, and Akt2-null cells under serum starvation (Figure 14). This
lack of difference might be due to the extended duration of starvation that exceeds the effective
protective range of AKT-mediated apoptosis. To address this issue, future experiments will use
shorter serum starvation periods to better assess the isoform-specific role of AKT in regulating
apoptosis.
We subsequently employed G418 to investigate whether the knockdown of isoformspecific Akts could affect the sensitivity of these cells to antibiotic resistance. WT, Akt1-null, and
Akt2-null cells were exposed to increasing doses of G418 for 6 days. On Day 6, the number of
live cells was counted using a hemocytometer under a microscope (Figure 15). Surprisingly, our
results revealed a significant increase in resistance to G418 antibiotic treatment in Akt2-null cells.
While both WT and Akt1-null cells died to the antibiotic at a concentration of 1 mg/ml, Akt2-null
cells demonstrated remarkable resilience, requiring a concentration of 2 mg/ml to kill all cells.
Thus, the metabolic function of AKT2 in hepatocytes may have affected the ability of these Akt2-
null cells to metabolize antibiotics.
Figure 15. Antibiotic kill curve for WT, Akt1-null, and Akt2-null cell
lines, assessed by hemocytometer count.
27
3.2.2 Isoform-Specific AKTs Function Significantly Affect Chemotherapy Resistance of
HCC Cells In Vitro
To understand the impact of isoform-specific Akt knockout on chemotherapy drug
resistance, WT, Akt1-null, and Akt2-null cells were treated with increasing doses of sorafenib for
48 hours. The cell viability was measured using crystal violet staining and was measured with a
plate reader at 570 nm absorbance (Figure 16). The results show that Akt1-null cells displayed
more resistance to sorafenib treatment whereas Akt2-null cells are more sensitive. At 10 µM
concentrations up to, sorafenib treatment significantly reduced cell viability in WT and Akt2-null
cells. The Akt1-null cells however did not respond to sorafenib until 20 µM doses. This resistance
is observed in all sorafenib concentrations at and above 10uM concentration. In contrast, Akt2-
null cells exhibited increased sensitivity to sorafenib treatment. A notable decrease in cell
viability was observed even at a concentration of 5 µM when neither the WT nor Akt1-null cells
were responding. This result suggests that AKTs may be the potential targets for modulating
sorafenib sensitivity, and AKT2-specific inhibitors could be an important addition to sorafenib
combination therapies, potentially extending their efficacy.
Chapter 4: Discussion
Figure 16. Growth-inhibitory effects of sorafenib in WT, Akt1-null and
Akt2-null cell lines. Cell viability is measured by crystal violet
staining. **P≤0.01, ***P≤0.001, ****P≤0.0001.
28
Liver cancer ranks as the sixth most prevalent cancer globally. It is the 5th most common
cancer in men and the 9th most common cancer in women. In 2020, an estimated 830,180 people
around the world died from the disease100
. The development of synchronous metastasis by patients
with HCC even post-curative surgical resection is a medical challenge in liver oncology
clinics101,102 While the majority of these cases are intrahepatic metastatic, it is estimated that about
13.5–42% are extrahepatic metastasis for patients with HCC103,104
. The relatively limited
therapeutic options for managing metastatic and late-stage HCC necessitate the discovery of
reliable targetable or druggable disease-specific biomarkers, and give clinical and/or translational
relevance to the findings of this present study. Here, we used 2D and 3D cell culture assays to
investigate the effects of AKT1 and AKT2 on regulated cell stemness properties during liver
cancer progression. Our objective was to discover unique isoform-specific alterations in stemness
properties upon Akt1 vs. Akt2 deletion.
Based on the results of in vitro data, we discovered that isoform-specific Akt knockout can
regulate cell proliferation, resistance, and sensitivity to stresses environment and drug resistance
to different extends. This study demonstrates that (i) Both AKT1 or AKT2 regulate the growth of
hepatocytes; (ii) AKT1 and AKT2 differentially regulates cellular response to stressed
environment conditions; (iii) Both AKT1 and AKT2 loss affect the tumor transformation
phenotypes of the hepatocytes. While the loss of AKT1 function significantly increases HCC cell
migration and sphere formation. AKT2 appears to be important for the invasion properties of the
cells. Finally, we demonstrated that (iv) AKT1 loss results in resistance to Sorafenib
chemotherapy, while deletion of AKT2 leads to more sensitivity to Sorafenib. Additionally,
hepatocytes lacking AKT2 may hinder their ability to metabolize antibiotics, leading to resistance
to G418 treatment.
29
The AKTs have been considered to be a contributor to HCC progression and tumor
development. They do this by controlling the growth, proliferation, and survival in tumor cells,
on the one hand, and by modulating the tumor microenvironment. A study showed that the AKT1-
mediated phosphorylation of mTORC2 is crucial for triggering hepatocarcinogenesis in humans
and mice, as it contributes to cellular growth through c-Myc activation105
. On the other hand,
elevated expression of AKT2 correlated with histopathological differentiation, portal invasion,
and the number of tumor nodules in HCC patients, while AKT1 did not correlate with any of these
clinicopathological features106
. Thus, it is important to clarify the roles each AKT plays in the
specific tumor types. This study contributes to the understanding of the role of AKT isoforms in
regulating tumor cell transformation in HCC and may serve as a basis for investigation into
isoform-specific inhibitors.
We demonstrated that AKT1 knockout cells’ survival largely relies on the confluency
level, which implies that the loss of the AKT1 impairs the cell's ability to effectively communicate
with a neighboring cell, which is essential for tissue function and ultimately for the organism to
which it belongs107
. This observation is consistent with the roles of AKTs as a survival kinase
mediating growth factor signal of cellular survival. However, it also suggests that the AKT1-
regulated apoptosis may be context-dependent on the cellular environment. This finding is
consistent with recent a study showing that Akt1 deletion resulted in accelerated apoptosis at low
concentrations of IL-3, while the expression of constitutively active Akt1 was sufficient to delay
apoptosis in response to IL-3 withdrawal108
. Through the IL-3 signal pathway’s role in promoting
cell proliferation and survival, AKT1 might be the factor that contributes to the maintenance and
expansion of CSC populations within tumors.
Although AKT1 is more widely considered to play a more important role in cell survival
30
and proliferation, a recent study conducted in breast cancer cells also highlighted the role of AKT1
as a negative regulator of EMT and metastasis109
. Previous studies by Arboleda and colleagues110
have indicated the importance of AKT2 in metastasis in breast and ovarian cancer cells. Here, our
results show that AKT2 is necessary for sustaining cell migration in a 3D cultured transwell assay
and a wound healing assay. Since EMT has been proposed to be the basis for tumor invasion and
metastasis, this data suggests that AKT2 could play a crucial role in sustaining EMT and
promoting tumor cell migration and stemness. By inhibiting AKT2 specifically, we may
potentially disrupt the mesenchymal phenotype essential for tumor invasion and metastasis. Cells
lacking AKT2 also displayed a reduced ability to form spheres, further supporting the role of
AKT2 in sustaining cancer cell stemness properties.
Finally, AKT1 and AKT2 also appear to affect the ability of the hepatocytes to respond to
drug treatment. While Akt2-null cells are significantly more resistant to G418 antibiotic
treatment, this may be an effect of its metabolic regulatory activity. Similar to the need for AKT2
to sustain cancer cell stemness properties, AKT2 also appears to be required for more resistance
to Sorafenib, the multi-kinase inhibitor used as first-line therapy in HCC. CSCs have been
indicated to display many characteristics of embryonic or tissue stem cells and developmental
signaling pathways such as Wnt, HH, and Notch that are highly conserved embryonically and
control the self-renewal of stem cells111,112
. Therefore, activation of these pathways may play an
important role in the expansion of CSCs and hence the resistance to therapy113
. Future studies will
focus on dissecting the interactions between AKT2-regulated signals and these stemness signals
to determine how AKT2 controls cancer cell transformation.
In conclusion, our study demonstrates that the AKT isoforms exhibit distinct regulatory
effects on the stem cell-like properties of immortalized mouse hepatocytes. Akt1-null cells show
31
heightened stemness properties compared to Akt2-null and WT control cells, suggesting that
AKT2 plays a critical role in regulating cell transformation and the initiation and development of
HCC through the regulation of CSCs in the liver. Similar to Akt1, Akt2 deletion also
downregulates the growth rate of cells, shows a higher migration level in wound healing assays,
and exhibits greater resistance to antibiotic treatment compared to WT cells, indicating that Akt1
could also play a role in regulating metastasis in HCC. Since the mechanisms by which AKT1 or
AKT2 lead to increased stemness properties in HCC remain unclear, investigating the cellular
signaling pathways involved in this process is essential for elucidating the role of isoform-specific
AKTs in regulating cancer stem cells (CSCs) and understanding their contribution to the initiation,
maintenance, and progression of HCC.
32
Chapter 5: Methods and Materials
5.1 Cell Lines and Cell Culture
Immortalized wild-type control (Control), Akt1-null (Akt1–/–
) and Akt2-null (Akt2
–/–
)
hepatocytes were established from 1-month-old Akt1
wt/wt Akt2
wt/wt; Alb-Cre-
, AKT1loxP/loxP; AlbCre+
and Akt2
loxP/loxP; Alb-Cre+ mouse liver, respectively. The cell lines were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 1% penicillin/streptomycin, 5 mg/ml insulin, 10 ng/ml EGF in a humidified 5% CO2
atmosphere at 37°C.
5.2 Western Blot
Cellular protein lysates were isolated using RIPA lysis buffer (25 mM Tris-HCl [pH 7.6],
150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with Halt™
Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Protein concentration
was measured using BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA). Protein
electrophoresis and Western blotting were performed using a Mini Trans-Bolt Cell system (BioRad). Equal amounts of protein from tissue lysates were added to a 5X protein loading dye
solution before being put through a 10% Tris-glycine SDS-PAGE. The proteins were then
transferred from the gel to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories,
Hercules, CA, USA) for immunoblotting. PVDF membranes were blocked using 0.1% Tween-20
and 5% nonfat milk added in phosphate buffer saline (PBST). Blots were probed with anti-AKT1
(2938s, Cell signaling), anti-AKT2 (3063s, Cell signaling) and anti-GAPDH (G9545, Sigma)
antibodies. The membrane was then treated with HRP (horseradish peroxidase)-linked secondary
antibodies (GE Healthcare Chicago IL) following PBST washing. Solutions with HRP substrate-
33
enhanced chemiluminescence solutions (Thermo Fisher Waltham, MA) were used to detect
signals. The protein blot membranes were visualized with ECL Western Blotting Substrate
(Thermo Fisher Scientific) and images were taken with X-ray film or ChemiDoc Imaging System
(Bio-Rad). Sample, tryptic peptides with amino acid sequences specific to individual proteins
were employed.
Table 1. Antibodies used in Western Blot
Antibodies Source Manufacturer Catalog
numbers
Primary AKT1 Rabbit Cell signaling
Technology
2938s
AKT2 Rabbit Cell signaling
Technology
3063s
GAPDH Rabbit Sigma
Technology
G9545
Secondary Anti-rabbit Donkey Ge Healthcare NA9340-1ML
5.3 Proliferation Assay
5.3.1 Proliferation Assay
Cells (2.5 x 104
per well) were plated in 12-well plates (Corning) with Dulbecco’s
Modified Eagle’s Medium (Mediatech) medium containing 10% FBS. After incubation at 37 °C
for 24h, 48 h, and 72h, cell numbers were counted by using a hemocytometer.
5.3.2 MTT Assay
The cell proliferation was determined by the MTT (Vybrant® MTT Cell Proliferation
Assay Kit (V-13154)) uptake method. Approximately 2 x 104
cells were seeded in each well of a
96-well plate and incubated for 16 h. On the next day, the cells were exposed to the following
treatments: Various concentrations of wh-4 only, 10 µM sorafenib only, or a combination of both
34
drugs. The treatment was carried out at 37˚C for 24h, 48 h and 72h. Finally, MTT (5 mg/ml; cat.
no. M6496; Vybrant® MTT Cell Proliferation Assay Kit) was added to each well and incubated
at 37˚C for 4 h. The absorbance was measured using a plate reader at 570 nm.
5.4 Migration Assay
5.4.1 Wound Healing Migration Assay
Cells were seeded in 60mm plates (Corning) with Dulbecco’s Modified Eagle’s Medium
(Mediatech) medium containing 10% FBS and cultured to 95–100% confluence. A scratch along
the median axis of the confluent adherent cells layer was then made with a sterile transparent
pipette tip114. Cell migration, viz-a-viz scratch wound healing images were captured at 0, 3h, and
6h after the scratch was made, under a microscope115
.
5.4.2 Transwell Assay
Cells (4 × 104
) were seeded in the 24-transwell plate (Corning) with an 8 μm pore
membrane in the upper chamber of the transwell system containing serum-free DMEM medium.
The lower chamber of the transwell chamber contained medium with 10% FBS. After incubation
at 37 °C for 24 h, the migrated cells were stained with crystal violet dye, air-dried and
photographed under a microscope. Images were analyzed with NIH ImageJ software116
.
5.5 Sphere Formation Assay
Cells (2.5 x 104
per well) were plated in ultra-low-attachment 24-well plates (Corning)
containing DMEM-F-12. After incubation at 37 °C for 48 h, formed spheres (>100 μm) were
counted and photographs were taken114
.
35
5.6 Serum Starvation Assay
For each cell line, cells were seeded in 24-well plates (Corning, Corning, NY, USA) with
Dulbecco’s Modified Eagle’s Medium (Mediatech) medium containing 10% FBS and cultured to
95–100% confluence. Cells were then starved by serum-free DMEM medium. Cell numbers were
counted by using a hemocytometer at 24h, 30h, and 36h after the starvation was started.
5.7 Confluency Assay
For each cell line, 50000 cells were seeded in 24-well plates (Corning, Corning, NY, USA)
with Dulbecco’s Modified Eagle’s Medium (Mediatech) medium containing 10% FBS and
cultured to 70% and 100% confluence. Cells were then starved by serum-free DMEM medium
for 16 hours. After the starvation, photos were taken and cells were harvested for cell number
counting by using a hemocytometer.
5.8 Antibiotic Treatment Assay
For each cell line, 5 x 104
cells were seeded in 24-well plates (Corning, Corning, NY,
USA) with Dulbecco’s Modified Eagle’s Medium (Mediatech) medium containing 10% FBS and
cultured to 25–50% confluence. Replace the FBS-contained DMEM with serum-free DMEM with
a range of antibiotic concentrations. Include untreated control cells. Monitor the cells daily using
a microscope and observe the percentage of surviving cells. Every 2 days replace medium with
freshly prepared. The minimum antibiotic concentration to use is the lowest concentration that
kills 100% of untreated control cells in 2–15 days from the start of antibiotic selection.
36
5.9 Chemotherapy Treatment Assay
For each cell line, 5 x 104
cells were seeded in 24-well plates (Corning, Corning, NY,
USA) with Dulbecco’s Modified Eagle’s Medium (Mediatech) medium containing 10% FBS and
cultured to 25–50% confluence. Replace the FBS-contained DMEM with a new FBS-contained
DMEM with a range of Sorafenib concentrations and incubate for 48 hours. The sensitivity of the
cell lines to chemotherapeutic drugs was measured by crystal violet staining. After the staining,
1ml of Methanol was added to dissolve the cells, and the optical density of each well’s absorbance
was measured at 590 nm.
5.10 Statistics
All data are statistically analyzed by using Excel (Microsoft) and Prism. All assays were
performed at least thrice in triplicate. Values are expressed as the mean ± standard deviation (SD).
Comparisons between groups were estimated using Student’s t-test for cell line experiments.
Differences between individual groups were analyzed by Student’s t-test when two-tailed p values
less than 0.05 were considered statistically significant differences.
37
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Abstract (if available)
Abstract
Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortalities worldwide. The Phosphoinositide-3-kinase-protein kinase B/Akt (PI3K-PKB/Akt) pathway plays an important role in regulating stem cells in HCC. The origins of PI3K/Akt research can be traced back to the discovery in 1977 by Staal and co-workers. Although Akt’s main functions have been extensively described, particularly its role in promoting metabolism, proliferation, cell survival, growth, and angiogenesis in response to extracellular signals, how isoform-specific Akt may regulate the cell’s stem cell-like properties largely remains unknown. In our in vitro studies, we characterized the role of Akt isoforms in regulating the stem cell-like phenotype in immortalized mouse hepatocytes lacking either Akt1 or Akt2. Our data demonstrated that the deletion of Akt1 (Akt1 -/-; Akt1-null) or Akt2 (Akt2-/-; Akt2-null) significantly enhanced several stem cell-like properties in mouse hepatocytes. Compared to WT cells, both Akt1-null and Akt2-null cells demonstrated significantly decreased cell growth. Akt2-null cells exhibited faster migration in the wound scratch assay, while Akt1-null cells migrated faster towards a chemoattractant in the transwell assay. Akt1-null cells were significantly more resistant to sorafenib and formed a significantly higher number of spheres in the sphere formation assay compared to the WT cells, while Akt2-null cells were highly resistant to antibiotic treatment and formed far fewer tumorspheres. These results contribute to the understanding of the role of Akt isoforms in regulating stemness in HCC and may serve as a basis for investigation into isoform-specific inhibitors.
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AKT exhibits isoform-specific function in hepatocyte transformation and response to cellular stress
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