Download LKB1 Deficient Non-small Cell Lung Cancer Cells are Vulnerable to

Texas Medical Center Library
DigitalCommons@TMC
UT GSBS Dissertations and Theses (Open Access)
Graduate School of Biomedical Sciences
12-2014
LKB1 Deficient Non-small Cell Lung Cancer Cells
are Vulnerable to Energy Stress Induced by ATP
Depletion
Chao Yang
Follow this and additional works at: http://digitalcommons.library.tmc.edu/utgsbs_dissertations
Part of the Cancer Biology Commons, Cell Biology Commons, and the Medicine and Health
Sciences Commons
Recommended Citation
Yang, Chao, "LKB1 Deficient Non-small Cell Lung Cancer Cells are Vulnerable to Energy Stress Induced by ATP Depletion" (2014).
UT GSBS Dissertations and Theses (Open Access). 516.
http://digitalcommons.library.tmc.edu/utgsbs_dissertations/516
This Thesis (MS) is brought to you for free and open access by the
Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has
been accepted for inclusion in UT GSBS Dissertations and Theses (Open
Access) by an authorized administrator of DigitalCommons@TMC. For
more information, please contact [email protected].
LKB1 Deficient Non-small Cell Lung Cancer Cells are Vulnerable to
Energy Stress Induced by ATP Depletion
by
Chao Yang, B.S
APPROVED:
Approval Sheet
_____________________
John V. Heymach, M.D., Ph.D.
Supervisory Professor
________________________
Gary E. Gallick, Ph.D.
_____________________
Varsha V. Gandhi, Ph.D.
_____________________
Faye M. Johnson, M.D., Ph.D.
_____________________
Dennis Hughes, M.D., Ph.D.
APPROVED:
_____________________
Dean, The University of Texas Graduate School of Biomedical Sciences at
Houston
i
LKB1 Deficient Non-small Cell Lung Cancer Cells are Vulnerable to
Energy Stress Induced by ATP Depletion
A
THESIS
Presented to the Faculty of
The University of Texas
Health Science Center at Houston
and
The University of Texas
MD Anderson Cancer Center
Graduate School of Biomedical Sciences
In Partial Fulfillment
of the Requirements
for the Degree of
MASTER OF SCIENCE
by
Chao Yang, B.S
Houston, Texas
December 2014
ii
Abstract
LKB1 Deficient Non-small Cell Lung Cancer Cells are Vulnerable to Energy
Stress Induced by ATP Depletion
Chao Yang
Advisory Professor: John V. Heymach, M.D., Ph.D.
Lung cancer is the second most frequent cancer in United States, which
represents about 13.5% of new cancer cases every year. It accounts for about
27.2% of all cancer related deaths, which is more than the sum of deaths
caused by prancretic, breast and colorectal. On average, only about 16% of
lung cancer patients survive beyond 5 years. LKB1 is the third most mutated
gene in lung cancer. It has been shown that LKB1 is mutated in at least 15% to
30% of NSCLC. Tumor with LKB1 mutation is associated with poor
differentiation, high metastasis and worse response to chemotherapy. The
development of targeted therapies has greatly improved the prognosis of a
small subtype of lung cancer. However, there is no targeted therapy for this
subtype and less effective chemotherapy is still being used as the first line
treatment.
In normal cells, LKB1 regulates cellular energy by balancing energy production
and consumption. Because tumor cells need vast amounts of energy for their
growth, they must adapt alternative strategies to amplify their energy production.
During low energy condition in normal cells, LKB1 signals cellular proteins to
decrease energy consumption, often resulting in decreased cell growth.
However, in LKB1 deficient tumor cells, there is no signal to slow down cell
growth. As such, these tumor cells have a disrupted balance between energy
iii
consumption and production, and LKB1 deficient tumor cells are able to escape
the normal growth inhibitory signals, resulting in unrestricted cell growth. I
hypothesize that targeting cellular pathways regulating energy production in
LKB1 deficient NSCLC will inhibit the growth of these tumor cells. 8chloroadenosine (8-Cl-Ado) is a compound which depletes cellular ATP. In this
study, we have identified that 8-Cl-Ado is a potential therapeutic agent for
targeting tumors with LKB1 deficiency. To determine the role of LKB1 and 8-ClAdo in cellular metabolism, we generated isogenic cell lines with either LKB1
stable re-constitution or knockdown. Cells with LKB1 re-constitution showed
decreased sensitivity to 8-Cl-Ado. Similarly, cells with LKB1 stable knockdown
displayed increased sensitivity to 8-Cl-Ado. Additionally, analysis on cellular
ATP and reactive oxygen species (ROS) levels showed that 8-Cl-Ado caused a
greater reduction in ATP and increase in ROS in LKB1 deficient cells compared
to cells with wild-type LKB1. Furthermore, a metabolic study demonstrated that
LKB1 deficiency is associated with reduction in mitochondrial membrane
potential. It also results in a phenotype with increased anaerobic respiration but
decreased oxidative phosphorylation. These findings indicate that LKB1 can
regulate the balance between anaerobic and aerobic respiration. This shifts
cells toward a less efficient energy production mechanism, rendering a potential
vulnerability of LKB1 deficient NSCLC to metabolic disruption.
iv
Table of Contents:
Approval Sheet .............................................................................................................. i
Abstract ........................................................................................................................ iii
Table of Contents: ........................................................................................................ v
List of Figures ............................................................................................................. vii
CHAPTER 1: INTRODUCTION ..................................................................................... 1
1.1 Overview ............................................................................................................... 2
1.2 Non-small cell lung cancer .................................................................................... 2
1.3 LKB1 in lung cancer .............................................................................................. 3
1.4 LKB1 signaling ...................................................................................................... 4
1.5 Metabolism and cancer ......................................................................................... 6
1.6 LKB1-AMPK in cell metabolism .......................................................................... 11
1.7 8-Chloroadenosine (8-Cl-Ado) ............................................................................ 14
1.8 Seahorse assay .................................................................................................. 14
1.9 Mitochondrial stress test ..................................................................................... 15
1.10 Glycolysis stress test ........................................................................................ 16
CHAPTER 2: MATERIALS AND METHODS .............................................................. 17
2.1 Cell Culture and viral infection ............................................................................ 18
2.2 Cell Proliferation Assay ....................................................................................... 18
2.3 Western blot and anti-bodies .............................................................................. 19
2.4 Seahorse analysis ............................................................................................... 20
2.5 ROS measurement ............................................................................................. 20
2.6 ATP measurement .............................................................................................. 21
2.7 Mitochondrial function ......................................................................................... 21
v
2.8 Statistical analysis............................................................................................... 22
CHAPTER 3: RESULTS .............................................................................................. 23
3.1 8-Cl-Ado selectively targets LKB1 deficient NSCLC cells ................................... 24
.................................................................................................................................. 27
3.2 LKB1 regulates the cellular response to 8-Cl-Ado .............................................. 28
3.3 LKB1 protects cell from 8-Cl-Ado induced ATP depletion .................................. 31
3.4 8-Cl-Ado induces oxidative stress in cells with LKB1 deficiency ........................ 38
3.5 LKB1 deficiency cells have enhanced the Warburg effect and impaired
mitochondrial membrane potential ............................................................................ 43
CHAPTER 4: DISCUSSION ......................................................................................... 48
4.1 Overview ............................................................................................................. 49
4.2 8-Cl-Ado and ATP depletion ............................................................................... 50
4.3 LKB1 and metabolic reprograming ..................................................................... 51
4.4 8-Cl-Ado and oxidative stress ............................................................................. 52
4.5 LKB1 and oxidative stress .................................................................................. 53
4.6 8-Cl-Ado and cell death ...................................................................................... 54
4.7 Future direction ................................................................................................... 57
CHAPTER 5: REFERENCES ...................................................................................... 59
Vita ............................................................................................................................... 77
vi
List of Figures
Figure 1.1 Illustration of cell metabolism……………………………………………8
Figure 1.2 Illustration of the production of ROS in mitochondria………………..10
Figure 1.3 AMPK-regulated proteins mediate cell
metabolism……………………………………………………………………………13
Figure 3.1 NSCLC cells with LKB1 deficiency are associated with increased
sensitivity
(A) ……………………………………………………………………..25
(B) And(C) …………...……………………………….…..…………..26
Figure 3.2 LKB1 regulates the sensitivity to 8-Cl-Ado
(A) And (B) ………………………………………………..………....29
(C) And (D) ……………………...……………………………...……30
Figure 3.3 8-Cl-Ado decreases cellular ATP level and induces AMPK activation
in a LKB1 dependent manner
(A) And (B) ……………...……………………………………..……..34
(C) And (D) …………………………………………………………...35
(E) ……………………………………………………..………………36
(F) And (G) …………………………………………………….……..37
Figure 3.4 LKB1 attenuates 8-Cl-Ado induced oxidative stress
(A) And (B) ………………………...…………………………..……..40
(C), (D) And (E) ………………………………...………………..…..41
(F) …………………………………………….……………………….42
Figure 3.5 LKB1 deficiency shifts cells from aerobic respiration to anaerobic
respiration
(A) And (B) …………………………...…………………………..…..45
(C) And (D) ………………………………………………….………..46
vii
(E), (F) And (G) ………………………………………...……..……..47
Figure 4.1 Cell cycle analysis…………………………………….…………………56
Table 1 IC50 to 8-Cl-Ado and KRAS and LKB1 mutation status of screened
cell lines……………………………………………………………………………….27
viii
CHAPTER 1: INTRODUCTION
1
1.1 Overview
Lung cancer is the second most frequent cancer in United States, which
represents about 13.5% of new cancer cases every year (1). It is also the
leading cause of cancer related death in the U.S., which accounts for about
27.2% of all cancer related deaths (1). In 2014, it is estimated that 220,000
people will be diagnosed with this disease and more than 159,000 people will
die from it (2). On average, only about 16% of lung cancer patients survive
beyond 5 years (1). The development of targeted therapies has greatly
improved the prognosis of a small subtype of lung cancer. However, for most
patients, less effective chemotherapy is still being used as the first line
treatment. Therefore, there is an urgent need to better understand the
molecular biology of lung cancer and develop more effective treatment
strategies.
1.2 Non-small cell lung cancer
Lung cancer is classified into two major groups: small cell lung cancer (SCLC)
and non-small cell lung cancer (NSCLC). NSCLC accounts for about 85% to 90%
of lung cancers. It is furthered grouped into adenocarcinoma (AD), squamous
cell carcinoma (SQ), and large cell carcinoma (LC) (3).
Various genetic alternations have been discovered in NSCLC, and these dictate
the heterogeneity of lung cancer and the difficulties for treatment. Among all the
mutated genes in lung cancer, TP53, KRAS, LKB1, EGFR, KEAP1 and NFE2L2
2
are the most frequently mutated (4). Detailed subtype studies have revealed
some distinct patterns between subtypes and genetic alteration (4). For
instance, MYCN amplifications often happen in squamous cell carcinoma;
KRAS, EGFR, and LKB1 mutations mainly occur in adenocarcinoma (4). Due to
these differences in mutations, the weakness of general chemotherapy as the
first line of treatment is apparent.
1.3 LKB1 in lung cancer
LKB1 is a serine/threonine protein kinase that functions as a tumor suppressor.
It was first identified in Peutz-Jeghers syndrome (PJS), which is characterized
by the development of hamartomatous polyps in the gastrointerstinal tract and
melanin pigmentation on the lips (5-7). PJS patients with germ-line LKB1
mutation are associated with increased risk for developing cancer at multiple
sites (8). When compared to the cancer risk for general population by the age of
60, the risk of PSJ patients carrying an LKB1 mutation is elevated by eight-fold
(9).
The human LKB1 gene is located on the short arm of chromosome 19 (10,11) .
This region is one of the most frequently altered regions in lung
adenocarcinomas (11,12). Adenocarcinoma is one of the most abundant
subtypes of lung cancer and accounts for about 40% of lung cancer cases
worldwide (3). Recent studies have shown that LKB1 is mutated in at least 15 to
30% of NSCLC (11,13-16). LKB1 mutations commonly overlap with activating
3
KRAS mutation, accounting for about 7%-10% of all NSCLC (17,18). These two
mutations result in a very aggressive phenotype. Mouse model studies have
shown that mice with KrasG12D and LKB1 deletion in lung have dramatically
increased tumor burden and metastasis (14,19). It has also been demonstrated
that LKB1/KRAS mutant tumors have a worse response to chemotherapy
compared to tumors with KRAS mutations alone (20).
1.4 LKB1 signaling
LKB1 is a master kinase that phosphorylates and activates 14 other kinases
(21,22). LKB1 was first discovered to play a role in regulating G1 cell cycle
arrest as demonstrated by ectopic expression of LKB1 in HeLa and G361 cells,
which do not normally express LKB1 (23). Transgenic mouse studies have
demonstrated a role for LKB1 in early development by showing that LKB1
knockout is associated with embryonic lethality (24-26). LKB1 functions in a
complex with two other proteins, MO25 and STRAD (27,28). Several studies
have suggested that binding to MO25 and STRAD could re-localize LKB1 from
the nucleus into the cytoplasm and thereby enhance its catalytic activity
(27,29) .
The AMP-activated protein kinase (AMPK) is the best characterized substrate
of LKB1 (30-33). AMPK is known for its function in regulating cellular energy
metabolism (34). In response to an increasing AMP/ATP ratio, LKB1, when
bound to MO25 and STRAD, has been shown to phosphorylate AMPK at
4
Thr172. Subsequently, phosphorylated AMPK regulates the activity of multiple
downstream signaling pathways to restore ATP levels in the cell.
Research has shown a conserved role of LKB1 in regulating cell polarity
(35,36). LKB1-AMPK modulates the phosphorylation of myosin regulatory light
chain and microtubule-associated proteins, which is important for the
establishment and maintenance of cell polarity (37,38). LKB1 loss is also
associated with epithelial-mesenchymal transition (EMT) (39). Given the role of
cell polarity in the maintenance of EMT (40), it suggests that disrupted cell
polarity due to LKB1 loss may contribute to the increased migration and
invasive characters of lung cancer cells.
Mammalian target of rapamycin (mTOR) is known for its role in regulating cell
growth and proliferation. Its downstream proteins are involved in variety of
cellular processes, such as cell cycle, protein synthesis and autophagy (41).
Under energy stress, LKB1 phosphorylates AMPK which modulates the
Tuberous sclerosis proteins 1 and 2 (TSC1-TSC2) complex through direct
phosphorylation on TSC2 T1227 and S1345. The phosphorylated complex then
inhibits the mTOR signaling by inactivating Rheb (42). This inhibition of mTOR
stalls cell growth and proliferation to conserve energy, thus protects cells from
this unfavorable condition.
Prostate cancer studies have shown that AMPK activation offers a survival
advantage to prostate cancer cells during energy stress and AMPK inhibition
could lead to increasing in apoptosis (43). Furthermore, it has been shown that
5
AMPK phosphorylation at Thr-172 was nearly completely lost in embryonic
fibroblasts lacking LKB1 expression (33). Those fibroblasts were also sensitive
to apoptosis induced by energy stress (33). Given the role of AMPK in
regulating the energy metabolism, these findings suggest that LKB1 deficient
lung cancer may be associated with altered energy metabolism and sensitivity
to metabolic interruption.
1.5 Metabolism and cancer
Cell metabolism refers to a series of chemical reactions that converts nutrients
to energy and building blocks (proteins, nucleic acid, and lipids) for cell growth
and proliferation. Adenosine triphosphate (ATP) is the principal energy supplier
for energy-dependent biological processes, however the cycle of cellular
metabolism relies on the compound nicotinamide adenine dinucleotide
phosphate (NADPH). ATP can be produced through both glycolysis and
oxidative phosphorylation. Glycolysis, as shown in Figure 1.1, is comprised of a
ten step reaction that takes place in the cytoplasm by metabolizing glucose to
pyruvate.
These reactions yield two molecules of ATP, two molecules of
pyruvate and two electron carrying molecules of NADH per molecule of glucose.
The pyruvate can further be converted to acetyl-coA, which will enter the
mitochondria to generate citrate. Citrate will then pass through the TCA cycle to
generate electron carriers for the electron transport chain. During oxidative
phosphorylation, the electrons from the carriers produced by TCA cycle will be
donated and passed though mitochondrial complexes to generate a proton
6
gradient. Finally, the protons will flux back into the mitochondria through ATP
synthase, which converts ADP to ATP. Overall, oxidative phosphorylation
generates 38 molecules of ATP per molecule of glucose (44).
Warburg first proposed that cancer cells display enhanced aerobic glycolysis in
the
presence
of
high
oxygen
tension
(45).
Compared
to
oxidative
phosphorylation, glycolysis is a rapid but less efficient way of producing energy.
In order to support the rapid growth of cell division, tumor cells have to keep an
abnormally high rate of glycolysis. This transformation consumes a huge
amount of glucose, but also provides a biosynthetic advantage for tumor cells.
The increased glucose flux allows for effective diversion of carbon for the
increased energy, biosynthesis and redox needs (46).
The fast growing tumor requires a sustained supply of nutrients and oxygen.
However, the lagging growth of new blood vessels decreases nutrients and
results in hypoxic stress (47-49). In this regard, high glycolysis permits tumor
cells to live under low oxygen tension. Also, the production of lactate by
glycolysis creates an acidic microenvironment, which facilitates tumor invasion
and suppresses immune effectors (50-52) .
The pentose phosphate pathway (PPP) is the main source of NADPH synthesis.
This pathway uses the intermediate products of glycolysis to generate NADPH
(53). NADPH is a critical substrate for fatty acid synthesis. It is also a major
antioxidant agent to protect cells from the harsh tumor microenvironment and
chemotherapy (53).
7
Figure 1.1 Illustration of cell metabolism.
Glucose is taken up by cells and converted to pyruvate. In normal cells,
pyruvate will be converted to acetyl-coA to undergo oxidative
phosphorylation (OXPHOS), through TCA cycle. Glucose-6-phosphate can
also be taken up by pentose phosphate pathway (PPP) to produce NADPH.
Citrate from TCA cycle can be converted back to Acetyl-CoA and undergoes
fatty acid synthesis. Under energy stress, fatty acid oxidation breaks down
lipids to acetyl-CoA and produce FADH2 and NADH to fuel TCA cycle and
OXPHOS. Citrate can also be converted to isocitrate and α-ketogluatarate to
restore NADPH level. In cancer cells, pyruvate is preferentially converted to
lactate in cytoplasm.
8
Reactive oxygen species (ROS) refers to a diverse class of radical species that
are byproducts of oxidative phosphorylation. The majority of ROS generation
takes place in the mitochondria, as shown in Figure 1.2. Excess electrons at
mitochondrial complex I and complex III will directly pass to oxygen and
generate superoxide anions (54,55). The superoxide anions will then be
dismutated into hydrogen peroxide by superoxide dismutase.
Low levels of ROS are thought to promote tumorigenesis by modulating kinase
and phosphatase activities (56-58). At moderate levels, ROS stabilizes HIF-1,
which in turn regulates the transcription of genes that control metabolic
reprograming, such as GLUT1 (glucose transporter), PKM2 (increase glucose
shunting to PPP), and PDK1 (prevents pyruvate to be used by mitochondrial
respiration) (59-61). At high levels, however, ROS damages the cellular
membrane and macromolecules, triggering senescence and causing apoptosis
(62-65).
9
Figure 1.2 The production of ROS in mitochondria.
The NADH and FADH2 generated by TCA cycle will denote electrons to
electron transport chain to reduce oxygen to water and produce ATP at
complex V. However, electron leaked from complex I and III will generate
superoxide (O2.-). Then, superoxide will be dismutated to hydrogen peroxide
(H2O2) by superoxide dismutases (SOD1 in the intermembrane space and
SOD2 in the matrix). Reprinted from Journal of Hematology & Oncology
(permission is not required by journal) 2013, 6:19 doi:10.1186/1756-8722-619 (66).
10
1.6 LKB1-AMPK in cell metabolism
LKB1 modulates cell metabolism by mediating the activation of AMPK. AMPK is
the master sensor of cellular energy status and has a crucial role in the
response to metabolic stress. Tissue specific LKB1 knockout mouse models
have shown that LKB1 is the principal upstream kinase that activates AMPK
(67). As mentioned above, AMPK inhibits mTOR signaling by phosphorylating
TSC complex in response to energy stress. In addition to the role of mTOR in
regulating protein translation by 4EBP1 and S6K1, mTOR also modulates the
translation of Hypoxia-inducible factor 1-alpha (HIF1-α) and MYC (68). HIF1-α
is a master regulator of cellular response to oxygen fluctuation (69). In normal
cells, the HIF1-α protein is degraded by unbiquitination under normoxic
condition but stabilized under hypoxic condition (70). In tumor cells, however,
HIF1-α is activated by oncogenic signaling, amplifying the transcription of
various glycolytic enzymes, promoting the cell to carry out glycolysis.
In
addition, MYC collaborates with HIF1-α in terms of enhancing glycolysis. It also
activates the transcription of genes that involve in the mitochondrial biogenesis
(71,72) .
The LKB1-AMPK signaling pathway has also been shown to modulate other
key metabolic signaling pathways other than mTOR signaling. AMPK inhibits
acetyl-CoA carboxylase (ACC) by phosphorylation (73). ACC is the major
enzyme in fatty acid synthesis. Fatty acid is an important energy source in
addition to glucose and glutamine. The synthesis is an anabolic process that
requires acetyl-CoA and NADPH. Its oxidation however is a catabolic process
11
that generates NADPH and acetyl-CoA for the electron transport chain and TCA
cycle to generate ATP. Under energy stress, activation of AMPK by LKB1
inhibits fatty acid synthesis through ACC phosphorylation, and in turn stalls
energy consumption and promotes its production (74).
As mentioned previously, NADPH is a key product of the pentose phosphate
pathway. It plays a role as cofactor and reduces many enzymatic reactions that
are important to macromolecular biosynthesis. In addition, NADPH is a crucial
anti-oxidant, which reduces both glutathione (GSH) and thioredoxin (TRX) to
scavenge ROS (75). It has been shown that LKB1 decreases ROS levels by
promoting the NADPH production through fatty acid synthesis (74).
12
Figure 1.3 AMPK-regulated proteins mediate cell metabolism. The left panel
shows the anabolic processes that consume ATP. Whereas, the right panel
demonstrates the catabolic processes. Under energy stress, increased AMP
promotes AMPK phosphorylation by LKB1. Subsequently, AMPK will
suppress the ATP consuming processes and promote ATP production
processes.
13
1.7 8-Chloroadenosine (8-Cl-Ado)
8-Cl-Ado is a RNA-directed nucleoside analogue that has been investigated
under a phase I clinical trial for the treatment of chronic lymphocytic leukemia
(CLL) (76). Preclinical studies have shown that 8-Cl-Ado could target various
cancer cell lines, such as primary CLL cells, glioma, myeloid leukemia, colon
and lung (76-82). The cytotoxicity of 8-Cl-Ado depends on its phosphorylation to
8-Cl-ADP and 8-Cl-ATP by adenosine kinase (83). The increased 8-Cl-ATP
inhibits mRNA transcript synthesis by incorporating into the RNA as a chain
terminator or by inhibiting polyadenylation (84,85). In addition, 8-Cl-ADP may
compete with ADP for binding to the active site of ATP synthase to inhibit ATP
production (84,86).
1.8 Seahorse assay
The seahorse assay (Seahorse Bioscience) provides a comprehensive method
to assess two key parameters of cellular metabolism: oxygen consumption rate
(OCR) and extracellular acidification rate (ECAR). Oxygen molecules are used
at the final step of the electron transport chain to accept electrons and protons
to form water. Its consumption is proportional to mitochondrial respiration.
Therefore, the OCR is used as an indicator of mitochondrial respiration. As
mentioned previously, glucose is metabolized to pyruvate, which is then
converted to lactate during glycolysis. The production of lactate releases proton
into the extracellular medium, which then acidifies the medium surrounding the
14
cells. The extracellular acidification rate is therefore used as an indicator of
glycolysis.
1.9 Mitochondrial stress test
This assay studies the function of the mitochondria by assessing its response to
various parameters. The cells are metabolically perturbed by sequentially
adding four different compounds. The first injected compound is Oligomycin,
which is a known inhibitor of ATP synthase (Complex V). Upon adding it to the
medium, it blocks the proton channel of ATP synthase, thus causing a decrease
in oxygen consumption. It is used to evaluate the percentage of oxygen
consumption that is devoted to ATP synthase. The second compound is FCCP
(carbonilcyanide p-triflouromethoxyphenylhydrazone), which depolarizes the
mitochondrial membrane potential by transporting protons across the
mitochondrial membrane rather than ATP synthase. The collapse of the
mitochondria membrane potential causes a rapid increase in energy and
oxygen consumption, but with no ATP production. Overall, FCCP treatment is
used to measure the maximum respiration and determine the spare respiratory
capacity of cells. The third and fourth compounds are Rotenone and Antimycin
A, both of which are injected into cells together. Rotenone is a complex I
inhibitor, which prevents the electron transport. Antimycin A is a complex III
inhibitor, which stalls the generation of proton gradient. These two compounds
can shut down mitochondrial respiration, causing a decrease in oxygen
consumption. They are used to calculate the non-mitochondrial respiration (87).
15
1.10 Glycolysis stress test
Similar to the mitochondria stress test, the glycolysis stress test studies the
glycolytic potential of cells by exposing them to chemical insults. Initially, the
ECAR of cells are measured in the medium without glucose. Glucose is then
injected into the cell medium, which will be taken up and metabolized to lactate.
The product of lactate leads to the acidification of medium surrounding the cells,
and in turn it causes a rapid increase in ECAR. The difference of ECAR with
and without glucose is the rate of glycolysis under basal condition. The second
injection is Oligomycin, as mentioned previously, this shifts the energy
production to glycolysis by inhibiting ATP synthase. Subsequently, Oligomycin
causes a sharp increase in ECAR, which is the maximum glycolytic capacity of
the cells. The final injection is 2-DG, which prevents glucose from being
catabolized by glucose hexokinase which results in a subsequent sharp
reduction in ECAR. This decrease in ECAR confirms that the ECAR produced
in experiment is due to glycolysis (88).
16
CHAPTER 2: MATERIALS AND METHODS
17
2.1 Cell Culture and viral infection
All cells were cultured in RPMI-1640 media (Sigma) supplemented with 10%
fetal bovine serum, and 1% penicillin-streptomycin. pLenti-GIII empty vector or
construct expressing wild type LKB1 (LV323938, Applied Biological Materials
Inc) was used for expressing LKB1 in A549 and H460 cells. Two independent
shRNAs (V3LHS_348649 and V3LHS_639000) and scramble control vectors
were purchased from Open Biosystems for stably knocking down LKB1.
Vectors were transiently transfected with 2nd Generation Packaging System
(psPAX2 and pMD2.G) into 293T cells by using Lipofectamine 2000 and Plus
reagents (Life Technologies). Viral supernatant was collected 3 days after
transfection and mixed with PEG-it Virus Precipitation Solution (LV810A-1,
System Biosciences) overnight at 4°C. Virus pellets were then collected and resuspended in HBSS solution containing HEPES. NSCLC cells were infected
and incubated with the viral particles supplemented with polybrene (AL-118,
Sigma) overnight in the CO2 incubator at 37°C. Infected cells were selected by
2 μg/mL puromycin.
2.2 Cell Proliferation Assay
Cells were seeded in 96 well plates at 2,000 cells per well. Various doses of 8Cl-Ado were added to each well on second day after plating. The effect of each
dose of 8-Cl-Ado was measured in triplicate. Sevety-two hours later, 10 μl/well
of CellTiter-Glo® Luminescent Cell Viability Assay (Promega) was added to the
18
treated cells. The plates were read using the FLUOstar Optima luminescence
reader (BMG Labtech). IC50 values were calculated by Calcusyn (Biosoft).
2.3 Western blot and anti-bodies
Protein lysates were collected by lysing cells with cell lysis buffer (1% Triton X100, 50mM HEPES, 150mM NaCl, 1.5mM MgCl2, 1mM EGTA, 100mM NaF,
10mM Na pyrophosphate, 1mM Na3VO4, 10% glycerol, and freshly added
protease
and
phosphatase
inhibitors
from
Roche
Applied
Science
#04693116001 and 049006845001, respectively. pH was adjusted to 7.4. The
protein concentration was determined by DC Protein Assay kit (Bio-Rad). 30 μg
of protein was mixed with 5x sample buffer (60 mM Tris-Cl pH 6.8, 2% SDS, 10%
glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue), and boiled at 95°C
for 5 minutes. Samples were resolved on polyacrylamide gel (Criterion™
TGX™, Bio-Rad) and transferred to nitrocellulose membrane.
Membranes
were blocked with 5% milk at room temperature for 1 hour, and then incubated
overnight at 4°C with primary antibodies: LKB1(#3050, Cell Signaling), pACC
Ser79 (#11818, Cell Signaling), pAMPK Thr172 (#4188, Cell Signaling), NRF2
(2178-1,
Epitomics),
and
p-p70S6K
Thr389
(#9205,
Cell
Signaling).
Immunodetection was performed using Goat anti-Rabbit IgG(H+L)-HRP
conjugated secondary antibody (Bio-Rad, #170-6515) and SuperSignal West
Pico Chemiluminescent Substrate(Thermo Scientific,#34080).
19
2.4 Seahorse analysis
The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)
was measured by a Seahorse Bioscience XF96 extracellular flux analyzer. The
Mito Stress test kit and Glycolysis Stress test kit were purchased from
Seahorse Biosciences. Briefly, cells were plated at 15,000 cells/well the night
before the experiment. The following day, the cells were washed 3 times with
XF Assay Medium (#102365-100) and incubated in a CO2 free incubator for 1
hour. Following incubation, the plates and Cartridges loaded with test
compounds were loaded into the analyzer and analyzed according to
manufacturer’s protocol (Seahorse Bioscience).
2.5 ROS measurement
ROS was measured by using either DCFDA or CellRox DeepRed (C10422, Life
Technologies). Briefly, the cells were plated into 6-well plates at 100,000
cells/well. On following day, 8-Cl-Ado (5µM) was added into medium. After 48
hours, the cells were washed with PBS once and incubated in fresh medium
containing probe (DCFDA: 20µM, DeepRed 2.5µM) for 30 mins. The cells were
then trypsinized into a single cell suspension and washed with PBS again,
suspended in medium and analyzed by FACS.
20
2.6 ATP measurement
Luminescence based measurement: cells were initially plated into a 384-well
plate at 1,000 cells/well. The following day, 8-Cl-Ado was added into cell
medium. After 6 h, cells were processed according to the manufacture’s
protocol (ATPlite, PerkinElmer). All the plates were screened by the BMG
reader for luminescence signal.
LC-MS based ATP measurement was carried out according to the procedure
outlined before (86).
2.7 Mitochondrial function
The mitochondrial mass and potential were measured by MitoTracker Deep
Red FM (M22426, Life Technologies) and MitoTracker Orange CMTMRos
(M7510, Life Technologies) respectively. Briefly, the cells were plated into 6well plates at 200,000 cells/well. The following day, the cells were washed with
PBS once and incubated in fresh medium containing 25nM MitroTracker probe
for 30 minutes. The cells were then trypsinized into a single cell suspension and
washed with PBS again. Finally, the cells were suspended in medium and
analyzed by FACS.
21
2.8 Statistical analysis
Unless indicated in the figure legends, all the statistical analysis was done by
using GraphPad Prism 6 (GraphPad Software, Inc). Results are reported as
mean ± SE. Statistical significance was calculated by a two-sided student t test
or one-way ANOVA and determined to be significant as p < 0.05.
22
CHAPTER 3: RESULTS
23
3.1 8-Cl-Ado selectively targets LKB1 deficient NSCLC cells
A panel of NSCLC cell lines was initially screened with various commonly used
chemotherapy agents to study the correlation between LKB1 status and drug
sensitivity (in collaboration with Dr. John Minna at UTSW). The results were
consistent with reported mouse studies which showed that KRAS driven mice
with LKB1 conditional knockout in lung had a worse response rate to
chemotherapy (20). As shown in Figure 3.1 (A) and (B), LKB1 deficient cells
did not show any significant increase in the sensitivity to either chemotherapies
or their combinations which have been commonly used in the clinic for the
treatment of lung cancer. However, cells with LKB1 deficiency showed a
selective vulnerability to 8-Choloradenosine (8-Cl-Ado). Further, we correlated
the sensitivity to 8-Cl-Ado of NSCLC cell lines with their LKB1 protein
expression determined by RPPA. As shown in Figure 3.1 (C), each cell line’s
LKB1 protein level was correlated to its IC50 value to 8-Cl-Ado. On average, cell
lines with high LKB1 protein levels were associated with higher IC50 values than
those with low LKB1 protein levels. The result confirms the finding that LKB1
deficient cells are selectively vulnerable to 8-Cl-Ado. Overall, these findings
suggest that LKB1 plays a significant role in regulating the response to 8-ClAdo in NSCLC cells.
24
A
LKB1 deficient cells LKB1 deficient cells
more sensitive
More resistant
Pemetrexed
Erlotinib
Pemetrexed +
Cisplatin
Paclitaxel
Docetaxel
Paclitaxel +
Carboplatin
Cisplatin
Carboplatin
Gemcitabine
Gemcitabine +
Cisplatin
8-Cl-Ado
-3
-2
-1
0
1
2
3
Fold change
Figure 3.1 NSCLC cells with LKB1 deficiency are associated with increased
sensitivity to 8-Cl-Ao. (A) Response of LKB1 deficient cells to standard agents
used for NSCLC treatment in clinic. Cells were grouped according to their
LKB1 status (in collaboration with Dr. John Minna at UTSW). The fold change
refers to the percentage difference of average IC50 between LKB1 deficient
and LKB1 wild type groups. The P value was determined by Wilcox test. The
significant difference was marked with asterisk sign.
25
Log10 IC50 M
B
C
Figure 3.1 NSCLC cells with LKB1 deficiency are associated with
increased sensitivity to 8-Cl-Ao (in collaboration with Dr. John Minna at
UTSW). (B) Comparison of average IC50 (Log10) according to LKB1 status
(**p=0.0005). (C) Comparison of average IC50 (Log10) between cells with low
LKB1 and high LKB1 protein expression (**p=0.007). Cells with LKB1 protein
level lower than the median value are considered LKB1 low, and vice versa.
26
Cell
Line
KRAS
Status
LKB1
Status
WT
IC50 to 8Cl-Ado
(µM)
0.47
HCC95
WT
WT
IC50 to 8Cl-Ado
(µM)
0.17
WT
WT
1.55
H1155
Mutated
WT
0.47
Calu-3
WT
WT
1.58
H1648
WT
WT
0.56
HCC1171
Mutated
WT
1.58
H2347
Mutated
WT
0.74
SK-LU-1
Mutated
WT
1.66
H358
Mutated
WT
0.76
Cell Line
KRAS
Status
LKB1
Status
H441
Mutated
HCC2279
H1299
WT
WT
1.86
Calu-6
Mutated
WT
0.95
HCC827
WT
WT
2.00
H292
Mutated
WT
0.95
HCC1359
WT
WT
2.00
WT
WT
1.05
PC-9
WT
WT
2.40
WT
WT
1.17
H1819
WT
WT
2.57
H1975
HCC40
11
HCC78
WT
WT
1.55
HCC4006
WT
WT
3.39
H2126
Mutated
Mutated
0.49
H820
WT
WT
3.47
A549
Mutated
Mutated
0.87
H1792
Mutated
WT
3.80
H460
Mutated
Mutated
0.30
H226
WT
WT
3.89
H1355
Mutated
Mutated
2.75
H1650
Mutated
WT
3.89
H1395
WT
Mutated
4.17
H2882
WT
WT
4.07
H157
Mutated
Mutated
0.16
H2087
WT
WT
4.47
H2073
WT
Mutated
1.17
H3255
WT
WT
4.57
H1993
WT
Mutated
1.29
H1693
WT
WT
5.13
WT
Mutated
2.75
H322
WT
WT
6.03
Mutated
Mutated
4.37
HCC2935
WT
WT
6.46
H1666
HCC51
5
H2122
Mutated
Mutated
0.50
HCC4017
Mutated
WT
7.24
H23
Mutated
Mutated
0.52
HCC193
WT
WT
7.94
H1437
WT
Mutated
0.85
Calu-1
Mutated
WT
9.55
H2009
Mutated
Mutated
1.07
H596
WT
WT
10.00
Mutated
Mutated
0.85
H661
WT
WT
16.98
WT
Mutated
0.37
H2887
Mutated
WT
19.95
Mutated
Mutated
0.93
H522
WT
WT
32.36
HCC44
HCC11
95
HCC36
6
HCC15
WT
Mutated
0.95
Table 1 IC50 to 8-Cl-Ado and KRAS and LKB1 mutation status of screened cell
lines (in collaboration with Dr. John Minna at UTSW).
27
3.2 LKB1 regulates the cellular response to 8-Cl-Ado
In order to study the role of LKB1 in the increased sensitivity toward 8-Cl-Ado
treatment, two LKB1 deficient NSCLC cell lines were chosen: A549 and H460.
These two cell lines were re-constituted with LKB1 by infecting with lentivirus
carrying wild type LKB1 cDNA. The LKB1 expression was confirmed by western
blot as shown in Figure 3.2 (A).
Cells carrying either wild-type LKB1 or empty control vectors were treated with
various doses of 8-Cl-Ado ranging from 0.1 µM to 20 µM for 72 hours. The
comparison between the IC50 values shows that both A549-LKB1 and H460LKB1 cells are more resistance to 8-Cl-Ado than those with empty control
vector (CTR), as shown in Figure 3.2 (B). In addition, the results were
confirmed by using lenti-virus carrying shRNAs to stably knockdown LKB1 in
KRAS mutant cell line, Calu6, as shown in Figure 3.2 (C). The proliferation
assay was then repeated as mentioned previously on LKB1 isogenic Calu6
cells, as shown in Figure 3.2 (D). The IC50 value revealed a consistent pattern
that cells with LKB1 are more resistant to 8-Cl-Ado than cells without LKB1.
These results show that LKB1 plays a role in protecting cells from 8-Cl-Ado.
28
A
H460
Control LKB1
A549
Control
LKB1
LKB1
Actin
Actin
LKB1
B
Figure 3.2 LKB1 regulates the sensitivity to 8-Cl-Ado. (A) Western blot
analysis for LKB1 re-constitution in H460 and A549 cells. (B) Comparison of
IC50 determined by proliferation assay to show cells with LKB1 reconstitution became resistant to 8-Cl-Ado.
29
C
D
Figure 3.2 LKB1 regulates the sensitivity to 8-Cl-Ado. (B) Western blot
analysis for LKB1 knockdown in Calu6 cells. (D) Comparison of IC50
determined by proliferation assay to show cells with LKB1 knockdown
became sensitivity to 8-Cl-Ado.
30
3.3 LKB1 protects cell from 8-Cl-Ado induced ATP depletion
AMPK has been shown to be the main cellular energy sensor and LKB1 has
been shown to directly phosphorylate AMPK at the Thr172 site in response to
energetic stress (31-33). A549 and H460 with LKB1 stable overexpression cells
were then treated with 8-Cl-Ado for 24 hours. ACC is a direct downstream
target of AMPK and its phosphorylation at Ser79 has been widely used as an
indicator of AMPK activity (89). As shown in figure 3.3 (A) and (B), 8-Cl-Ado
increased AMPK phosphorylation at Thr172 site in cells with LKB1 but not with
empty vector control. Consistently, ACC phosphorylation was further increased
by 8-Cl-Ado in A549-LKB1 and H460-LKB1 cells compared with the A549-CTR
and H460-CTR cells. This finding indicates that 8-Cl-Ado has the potential to
activate AMPK in a LKB1-dependent manner.
In addition, 8-Cl-Ado has been shown to be able to decrease endogenous ATP
levels though targeting ATP synthase (90). Given, the finding that 8-Cl-Ado
activates AMPK signaling in a LKB1-dependent manner, we hypothesized that
8-Cl-Ado decreases the cellular ATP levels in NSCLC cells. However, LKB1
may compensate the ATP reduction induced by 8-Cl-Ado by activating
alternative pathways.
ATP level in cells with 6 hour 8-Cl-Ado treatment was measured. As shown in
Figure 3.3 (C), (D) and (E), 8-Cl-Ado decreased the ATP levels in all cell lines;
however, LKB1-expressing cells had less 8-Cl-Ado induced ATP reduction
compared to control cells. Treatment of cells with 8-Cl-Ado for 6 hours reduced
31
ATP levels in H661 cells with LKB1 knockdown to a greater extent than in cells
with scramble control.
The results were further confirmed by measuring 8-Cl-Ado induced ATP
depletion in isogenic H460 and A549 cells by an LC-MS based ATP assay
described previously (in collaboration with Dr. Varsha Gandhi) (76). As shown
in Figure 3.3 (F) and (G), H460-CTR cells had a lower ATP baseline levels
compared with H460-LKB1 cells. 8-Cl-Ado also decreased endogenous ATP
levels in both H460-CTR and H460-LKB1 cells. However, H460-LKB1 cells had
a lesser reduction than H460-CTR cells and their ATP levels rebounded to the
baseline level after 24 hour treatment. Similar results were also found in
isogenic A549 cells treated with 8-Cl-Ado. 8-Cl-Ado decreased endogenous
ATP levels to a greater extent in A549-CTR than A549-LKB1 cells. These
findings confirm the role of LKB1-AMPK signaling in maintaining the
homeostasis of energy metabolism (74) and indicate the potential of targeting
LKB1 deficient NSCLC through ATP depletion.
It has been reported that AMPK can phosphorylate TSC2, which in turn inhibits
mTOR signaling (42). Therefore, we attempted to study the effect of 8-Cl-Ado in
the mTOR pathway. Phosphorylation of p70S6K was picked to be used as a
marker for mTOR signaling. As shown in Figure 3.3 (A) and (B), western blot
analysis demonstrates that 8-Cl-Ado causes de-phosphorylation of p70S6K in a
LKB1 dependent manner. This result suggests that 8-Cl-Ado has the potential
to inhibit mTOR signaling through LKB1. However, its effect on LKB1 deficient
cells is independent on mTOR signaling.
32
Overall, 8-Cl-Ado induces AMPK activation and mTOR inhibition in the
presence of LKB1. As mentioned previously, energy stress induces AMPK
phosphorylation by LKB1, and 8-Cl-Ado can decrease endogenous ATP.
Therefore, I hypothesize that 8-Cl-Ado might target cellular energy metabolism,
hence inhibit the growth of LKB1 deficient NSCLC.
33
A
B
Figure 3.3 8-Cl-Ado decreases cellular ATP level and induces AMPK
activation in a LKB1 dependent manner. (A) (B) Western blot analysis for
phos-ACC, phos-AMPK, phos-p70S6K and LKB1 showing that 8-Cl-Ado
induced AMPK activation is LKB1 dependent. Lysates were collected from
cells treated with 5µM 8-Cl-Ado for 24 hours
34
C
% to control in ATP
150
H460 LKB1
H460 CTR
*
*
100
*
50
0
0 µM
0.5 µM
1 µM
4 µM
8-Cl-Ado
D
% to control in ATP
150
A549 LKB1
A549 CTR
*
*
100
50
0
0 µM
0.5 µM
1 µM
4 µM
8-Cl-Ado
Figure 3.3 8-Cl-Ado decreases cellular ATP level and induces AMPK
activation in a LKB1 dependent manner. (C) (D) ATP level measurements of
cells treated with 8-Cl-Ado at 0.5 µM, 1 µM, and 4 µM for 6 hours by
luminescent method (ATPlite). Results were normalized with non-treated
control. Statistical significances were determined by two way ANOVA
multiple comparisons.
35
% to control in ATP
E
Figure 3.3 8-Cl-Ado decreases cellular ATP level and induces AMPK
activation in a LKB1 dependent manner. (E) ATP level measurements of
cells treated with 8-Cl-Ado at at 0.5 µM, 1 µM, and 4 µM for 6 hours
luminescent method (ATPlite). Result was normalized with non-treated
control. Statistical significances were determined by two way ANOVA
multiple comparisons.
36
F
200
***
% to control in ATP
H460 LKB1
H460 CTR
150
***
**
*
*
100
50
0
0 µM 0.2 µM 2 µM
6 hr
20 µM 0.2 µM 2 µM
20 µM
24 hr
G
150
A549 LKB1
A549 CTR
100
50
0
0 µM 0.2 µM 2 µM
6 hr
20 µM 0.2 µM 2 µM
20 µM
24 hr
Figure 3.3 8-Cl-Ado decreases cellular ATP level and induces AMPK
activation in a LKB1 dependent manner. (F) (G) ATP level measurements of
cells treated with 8-Cl-Ado at 0.2 µM, 2 µM, and 20 µM for 6 and 24 hours by
LC-MS method (in collaboration with Dr. Varsha Gandhi). Result was
normalized with cells carry empty control vector without treatment. Statistical
significances were determined by two way ANOVA multiple comparisons.
37
3.4 8-Cl-Ado induces oxidative stress in cells with LKB1 deficiency
ROS is a by-product of mitochondrial respiration (54). Low to moderate ROS
stimulates cell proliferation and differentiation (91). Excess ROS causes
oxidative stress and subsequent cell death by damaging cellular components,
such as DNA, proteins and lipids (54,72). Research has shown glucose
starvation/2-DG treatment reduces intracellular ATP and induce ROS
accumulation in lung cancer cells (74). Inside the cell, 8-Cl-Ado gets
metabolized into 8-Cl-ADP and 8-Cl-ATP (92). ATP synthase is the last enzyme
of the respiratory chain of oxidative phosphorylation. It catalyzes the synthesis
of ATP from ADP. It has been proposed that 8-Cl-ADP/8-Cl-ATP binds to ATP
synthase in a similar mode as ATP/ADP. 8-Cl-ADP and 8-Cl-ATP together may
be an inhibitor of ATP synthase (90). In addition, inhibiting ATP synthase with
the known inhibitor oligomycin could stall the electron transport chain and
subsequently cause accumulation of electrons. Then, the excess electrons will
convert oxygen to superoxide and to hydrogen peroxide. Based upon all the
above facts, I wanted to investigate whether 8-Cl-Ado induced ATP reduction is
associated with ROS accumulation.
H1299 (LKB1 wild-type) and A549 (LKB1 mutant) cells were treated with 8-ClAdo and 2-DG for 48 hours. As shown in Figure 3.4 (A) and (B), both 8-Cl-Ado
and 2-DG treated H1299 cells did not show any accumulation of ROS. However,
A549 cells showed an increase in ROS level for both treatments, with 8-Cl-Ado
treatment causing the most increase in the accumulation of ROS. These results
38
suggest that LKB1 plays a role in protecting cells from 8-Cl-Ado induced ROS
accumulation.
To further investigate the role of LKB1 in 8-Cl-Ado induced ROS accumulation,
isogenic A549, H661 and Calu6 cells were treated with 8-Cl-Ado for 48 hours.
As expected, LKB1 reduces 8-Cl-Ado induced ROS accumulation in both A549LKB1 and Calu-6-SCR cells; however, A549-CTR and Calu-6 shLKB1 cells had
greater accumulation of ROS, as shown in Figure 3.4 (C), (D) and (E). The
NRF2 protein level in H460 and A549 isogenic pairs after 8-Cl-Ado treatment
was also measured. As expected, 8-Cl-Ado increased NRF2 protein level in
H460-CTR and A549-CTR cells, but not in H460-LKB1 and A549-LKB1 cells, as
shown in Figure 3.4 (F). These findings suggest that 8-Cl-Ado induces ROS
accumulation. However, LKB1 could protect cells from 8-Cl-Ado induced ROS.
39
A
B
Figure 3.4 LKB1 attenuates 8-Cl-Ado induced oxidative stress. (A) (B)
FACS analysis of cellular ROS level of H1299 and A549 cells after 48 hour 5
µM 8-Cl-Ado treatment.
40
C
100
80
60
40
20
0
A549 CTR
A549 LKB1
8-Cl-Ado
D
E
50
40
40
30
30
20
20
10
10
0
Calu6
SCR
Calu6
Calu6
shLKB1#1 shLKB1#2
8-Cl-Ado
0
H661
SCR
H661
shLKB1
8-Cl-Ado
Figure 3.4 LKB1 attenuates 8-Cl-Ado induced oxidative stress. (C) (D) (E)
FACS analysis of cellular ROS level A549 cells with LKB1 re-constitution and
Calu6 and H661 cells with LKB1 knockdown after 48 hour 5 µM 8-Cl-Ado
treatment. The experiment was performed twice. The bar graph represents
the percentage difference in mean value determined by FACS based upon
measurement of 10,000 cells.
41
F
Figure 4. LKB1 attenuates 8-Cl-Ado induced oxidative stress. (F) Western
blot analysis for LKB1 and NRF2.
42
3.5 LKB1 deficiency cells have enhanced the Warburg effect and impaired
mitochondrial membrane potential
In order to investigate the role of LKB1 in cellular metabolism, the seahorse
assay was used to study the oxygen consumption rate (OCR) of isogenic A549
and H661 cells at four different concentrations. According to manufacturer’s
protocol, the OCR is a direct indicator of mitochondrial respiration. As show in
Figure 3.5 (A) and (B), A549 cells with LKB1 expression showed an increase in
OCR. However, H661 cells with LKB1 knockdown presented decreased OCR.
This finding indicates a potential role of LKB1 in regulating cellular respiration.
In addition, the Warburg effect suggests that cancer cells preferentially undergo
aerobic glycolysis (45). This led to the hypothesis that LKB1 deficient cells are
highly dependent on glycolysis for energy production. The glycolytic potential of
isogenic A549 cells was then investigated using the seahorse assay. As
expected, A549-LKB1 cells showed a decrease in the glycolysis compared to
A549-CTR cells, as shown on Figure 3.5 (C). Overall, these findings suggest a
novel role of LKB1 in regulating cellular metabolism by reversing the Warburg
effect.
The mitochondrial stress test was then performed on isogenic A549 cells. As
shown in figure 3.5 (D), both A549-LKB1 and A549-CTR cells showed a
reduction in OCR upon oligomycin treatment. However, A549-CTR cells failed
to response to mitochondrial uncoupler FCCP, which collapses mitochondrial
potential by depolarizing the mitochondrial member. FCCP causes a rapid
consumption of energy and oxygen, but without generation of ATP (87).
43
This observation suggests that A549 cells with LKB1 deficiency may have a
defective mitochondrial membrane potential. It further suggests that A549 CTR
cells associated with high glycolysis may be due to the defective mitochondria.
Finally, the mitochondrial membrane potential and mass in A549-CTR and
A549 LKB1 cells was investigated. As is shown in Figure 3.5 (E) (F) and (G),
A549-LKB1 cells had slightly higher membrane potential than A549 CTR cells.
However, the mitochondrial mass of A549-CTR cells was much higher than in
A549-LKB1 cells. The average mitochondrial membrane potential per unit of
mitochondrial mass of A549-CTR cells was however much lower than in A549LKB1 cells. This finding confirms the results obtained earlier which showed that
A549-CTR cells have a defective mitochondrial potential and subsequently
depend more on glycolysis for energy production.
44
OCR (pMoles/min)
A
OCR (pMoles/min)
B
Figure 3.5 LKB1 deficiency shifts cells from aerobic respiration to anaerobic
respiration. (A) (B) seahorse analysis of baseline OCR of A549 with LKB1
re-constitution and H661 with LKB1 knockdown cells at three different cell
concentrations. Data are presented as mean+/- SE. The experiment was
performed once. Each cell line at each concentration had 4 replicates. The
final OCR value is the average of 14 independent measurements of all 4
replicates.
45
C
D
Figure 3.5 LKB1 deficiency shifts cells from aerobic respiration to anaerobic
respiration. (C) Seahorse glycolytic analysis of A549 with LKB1 reconstitution. The experiment was performed twice with 5 replicates for each
cell line. (D) Seahorse mitochondrial stress test of A549 with LKB1 reconstitution. The experiment was performed with 4 different concentrations
of FCCP. Each cell line had 5 replicates. Data are presented as mean+/- SE.
46
E
MitoTracker Orange CMXros 576nm
F
150
Mitochondrial
mass (MM)
100
50
0
A549
CTR
A549
LKB1
G
1.5
MMP/MM
1.0
0.5
0.0
A549
CTR
A549
LKB1
Figure 3.5 LKb1 deficiency shifts cells from aerobic respiration to anaerobic
respiration. (E) FACS analysis of mitochondrial potential of A549 cells with
LKB1 re-constitution (F) FACS analysis of mitochondrial mass of A549 cells
with LKB1 re-constitution. (G) Mitochondrial potential per unit mitochondrial
mass of A549 cells with LKB1 re-constitution. The experiment was
performed once. The bar graph represents the mean value determined by
FACS based upon measurement of 10,000 cells.
47
CHAPTER 4: DISCUSSION
48
4.1 Overview
Recent studies have shown a promising potential of targeting cell metabolism in
LKB1 deficient NSCLC, but very little is known about the underlying mechanism
involved. Since LKB1 is the third most mutated gene (after P53 and KRAS) in
lung cancer (4), this study specifically investigated the vulnerability of LKB1
deficient NSCLC to metabolic disruption. It supports the ability of disrupting
cellular energy metabolism and redox homeostasis to stall the growth of LKB1
deficient NSCLC. To investigate the hypothesis, we screened a panel of
NSCLC cell lines and identified 8-Cl-Ado selectively targets LKB1 deficient cells.
The role of LKB1 in regulating cellular response to 8-Cl-Ado was further
confirmed by stably re-constituting and knocking-down LKB1 in NSCLC cells. It
has been previously shown that 8-Cl-Ado decreases endogenous ATP levels
(76,90). In this study, we show that 8-Cl-Ado depletes endogenous ATP levels
to a greater extent in LKB1 deficient cells compared to LKB1 expressing cells.
We also demonstrate that 8-Cl-Ado causes the accumulation of reactive oxygen
species in cells lacking LKB1 expression and again confirmed the protective
role asserted by LKB1. Additionally, this study reveals that LKB1 regulates
cellular metabolic reprogramming. It was supported by the observation that
expression of LKB1 is associated with increased mitochondrial potential and
oxygen consumption rate. Research has shown that defects in mitochondrial
oxidative phosphorylation have been linked to Warburg effect (93). In addition
to producing energy, the mitochondria also play an important role in regulating
the redox state, cellular Ca2+ and apoptosis (55). Therefore, defective
49
mitochondrion may render the sensitivity of LKB1 deficient cells to energetic
and oxidative stresses induced by 8-Cl-Ado.
4.2 8-Cl-Ado and ATP depletion
8-Cl-Ado has been shown to deplete ATP and was first reported in a study of
mantle cell lymphoma (MCL) cell lines (94). In that study, it was evident that 8Cl-Ado gets metabolized to 8-Cl-ATP. The accumulation of 8-Cl-ATP was
associated with a reduction in cellular ATP level. Later, studies have also
shown that 8-Cl-ADP/8-Cl-ATP, metabolites of 8-Cl-Ado, could inhibit the ATP
synthase on mitochondrial membrane by occupying the tight and loose binding
sites of ATP synthase in a similar way as ADP/ATP (95). Oliogmycin used in
the seahorse assay in this study is a known ATP synthase inhibitor. It causes
immediate ATP synthase inhibition upon adding to the cell. However,
substitution of Oliogmycin with 8-Cl-Ado on the seahorse assay did not show
any acute inhibition of ATP synthase. This observation may be due to the fact
that a longer incubation time is needed for 8-Cl-Ado to inhibit ATP synthase
given that 8-Cl-Ado needs to be metabolized to 8-Cl-ATP and 8-Cl-ADP. It also
suggests that 8-Cl-Ado may decrease cellular ATP level through alternative
mechanisms. The data also show that LKB1 deficiency is associated with
decreased oxygen consumption rate, increased extracellular acidification rate,
and decreased mitochondrial membrane potential. It suggests that LKB1
deficient cells are shifting towards glycolysis for energy production. Thus, it
50
raises a question of whether 8-Cl-Ado affects glycolysis and hence causes ATP
depletion.
4.3 LKB1 and metabolic reprograming
This study revealed a novel role of LKB1 in reprograming the balance between
oxidative phosphorylation and glycolysis in NSCLC. The “Warburg effect” states
that cancer cells have increased glycolytic flux even in the presence of high
oxygen tension (45,96,97). It has been shown that high glycolysis could offer
cancer cells with several survival advantages. First, high glycolysis in cancer
cells could enable them to survive under condition with limited oxygen supply.
Second, production of lactic acid could make an acidic environment that favors
cancer cell invasion. Third, NADPH plays an anti-oxidant role in protecting cells
from chemotherapeutic agents (97). Mice studies have shown that KRASG12D
driven mice with LKB1 conditionally knockout in lung are resistant to
chemotherapy and chemotherapy plus MEK inhibitor (20). In addition, lung
tumors from those mice displayed an increased distant metastasis and
expression of angiogenic factors (13,14). However, the role of LKB1 in
increased resistance and metastasis has not yet been elucidated. Given the
advantages of high glycolysis to tumor cells, the observation of LKB1 deficiency
induced high glycolysis in this study would potentially fill this gap of knowledge.
AMPK directly regulates the autophagy kinase ULK1 to regulate mitophagy and
survival under conditions of cellular stress. In response to energy stress, AMPK
51
can activate mitophagy through activating ULK1(100). Mitophagy then can
remove damaged or dysfunctional mitochondria to prevent cell death. In this
case, the difference in mitochondrial mass may be due to the failure of
mitophagy activation in LKB1 deficient cells. Alternatively, cells may increase
mitochondrial mass to compensate for low mitochondrial activity. Thus, further
studies are needed to investigate this mechanism.
4.4 8-Cl-Ado and oxidative stress
ROS is a byproduct of oxidative phosphorylation in proliferating cells. Electrons
from NAPH and succinate of TCA cycle pass through the electron transport
chain and reduce oxygen to water. However, excess electrons at complex I and
complex III contribute to a partial reduction of oxygen to superoxide anions.
Immediately, these superoxide anions get dismutated to hydrogen peroxide by
dismutases. For the first time, this study demonstrates that 8-Cl-Ado could
cause ROS accumulation, especially in those cells without LKB1 expression.
Inhibition of ATP synthase by oligomycin could stall the electron transport chain
and reduce the electron carriers in complexes I and III and CoQ, which causes
an accumulation of free electrons. Those excess electrons can then be
converted to superoxide and hydrogen peroxide (55). As mentioned above, 8Cl-Ado could inhibit ATP synthase, which is the last complex on the electron
transport chain. Therefore, 8-Cl-Ado may function in a similar way as
52
oligomycin by raising the mitochondrial inner membrane polarization and
subsequently resulting in an accumulation of electrons.
4.5 LKB1 and oxidative stress
Recently a study has shown that LKB1 regulates fatty acid oxidation through
AMPK-ACC axis and thereby protecting cells from energetic and oxidative
stress (18). Fatty acid are an alternative energy source in addition to glucose
and glutamine. Proliferating cells require sustained supply of fatty acids for their
cellular membrane synthesis. The synthesis of fatty acids is an anabolic
process, which consumes ATP. On the other hand, fatty acid oxidation is a
catabolic process that fuels the TCA cycle and electron transport chain with
acetyl CoA, NADPH and FADH2. In addition, acetyl CoA from fatty acid
oxidation could enter a series reaction to give rise to cytosolic NADPH, which is
a key anti-oxidant molecule in cells.
Under energetic stress, LKB1 will phosphorylate AMPK in response to the
stress. Phosphorylated AMPK will then inhibit ACC through phosphorylation.
Inhibition of ACC could stop the synthesis of fatty acid (98), which conserves
the ATP. At the same time, inhibition of ACC turns on the fatty acid oxidation
process. As mentioned above, it produces acetyl CoA, NADPH and FADH2 for
the TCA cycle and electron transport chain thus generating more ATP. It also
gives rise to NADPH, which detoxifies ROS, and hence protects the cell from
both energetic and oxidative stress. In this study, we have shown that 8-Cl-Ado
53
could induce ACC phosphorylation in a LKB1 dependent manner. Also, LKB1
could attenuate 8-Cl-Ado induced ATP reduction and ROS accumulation. Given
the role of fatty acid oxidation and its connection with LKB1-AMPK signaling,
dysregulation of fatty acid synthesis and oxidation may render the sensitivity of
LKB1 deficient cells to 8-Cl-Ado. Overall, this study revealed 8-Cl-Ado as a
potential drug that could target LKB1 deficient NSCLC. It also suggests that
disrupted metabolic and redox controls in LKB1 deficient cells can be a
potential vulnerability for targeting.
4.6 8-Cl-Ado and cell death
Cell cycle anaylsis, as shown on Figure 4.1, did not show any change in cell
cycle due to LKB1. Calu6 cells with LKB1 knockdown displayed an increase in
sub G1 phase after 8-Cl-Ado treatment. Sub G1 phase includes dead cells with
low DNA content. This result indictates that 8-Cl-Ado may induce cell death in
LKB1 deficient cells. However, similar results are not seen on H441 cells with
LKB1 knockdown. Therefore, further studies are needed to investigate 8-Cl-Ado
induced cell death in LKB1 deficient cells.
The initial cell line screening was performed using an MTT assay (in
collaboration with Dr. John Minna at UTSW). The MTT assay is based upon
cellular NADH, which is considered as an indicator of cellular viability. CellTiterGlo assay is an ATP based assay. It is believed that the the amount of ATP is
proportional to the amount of live cells. Given by the role of LKB1 and 8-Cl-Ado
54
in cell metabolism, both methods used in this study may not be ideal
approaches. However, the acute effect of 8-Cl-Ado is not expected to last
beyond 72 hours. Thus, the end point differences determined by these two
methods are believed to be mainly dominated by the actual differences in cell
number. Future approaches, such as counting cell number by DAPI staining
and DNA content based assays should be deployed.
55
Figure 4.1 Cell cycle analysis. Cell cycle analysis by FACS on H441 and
Calu6 cells with LKB1 knockdown after 72 hours 8-Cl-Ado treatment.
56
4.7 Future direction
Although this work has demonstrated that LKB1 deficient NSCLC cells are
associated with a metabolic alternation and a potential sensitivity to energy
stress induced by 8-Cl-Ado, there still remains some unanswered questions. It
is important to investigate the mechanism of how LKB1 regulates the switch
between glycolysis and mitochondrial respiration. Global metabolomics
screening could provide information on the metabolites affected by LKB1. This
metabolic alteration may bring survival advantages to LKB1 deficient NSCLC,
but it also can be a potential target. Given the reported findings that NSCLC
with Kras mutation more depends on glutamine for energy production but not
glycolysis (99), it is also interesting to study the contribution of increased
glycolysis to the tumorigenesis of LKB1 deficient NSCLC. Furthermore, it would
be critical to investigate the mechanism of action of 8-Cl-Ado in ATP depletion
and ROS accumulation. It is necessary to test if 8-Cl-Ado targets ATP synthase
to decrease ATP level in LKB1 deficient NSCLC cells, or it may actually target
glycolysis. ROS accumulation may be due to either excess production or
defective anti-oxidant response. It is important to determine if 8-Cl-Ado could
affect any of these two mechanisms.
It would also be important to determine the pathways that LKB1 regulated that
contribute to the protection of cells from 8-Cl-Ado. A gene expression analysis
could provide a complete understanding of pathways that regulated by LKB1
and affected by 8-Cl-Ado. The result can also be correlated with metabolomics
57
analysis, which will give a comprehensive understanding of how LKB1 regulates
cellular metabolism and the mechanism of action of 8-Cl-Ado.
58
CHAPTER 5: REFERENCES
59
1. Cancer of the lung and bronchus - SEER stat fact sheets; Available from:
http://seer.cancer.gov/statfacts/html/lungb.html. Accessed 16 July 2014.
2. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin
2014;64(1):9-29.
3.
What
is
non-small
cell
lung
cancer?;
Available
from:
http://www.cancer.org/cancer/lungcancer-non-smallcell/detailedguide/nonsmall-cell-lung-cancer-what-is-non-small-cell-lung-cancer. Accessed 16 July
2014.
4. Network Genomic Medicine (NGM). A genomics-based classification of human
lung tumors. Sci Transl Med 2013, Oct 30;5(209):209ra153.
5. Hemminki A, Tomlinson I, Markie D, Järvinen H, Sistonen P, Björkqvist AM,
Knuutila S, Salovaara R, Bodmer W, Shibata D, de la Chapelle A, Aaltonen LA.
Localization of a susceptibility locus for peutz-jeghers syndrome to 19p using
comparative genomic hybridization and targeted linkage analysis. Nat Genet
1997, Jan;15(1):87-90.
6. Hemminki A. The molecular basis and clinical aspects of peutz-jeghers
syndrome. Cell Mol Life Sci 1999, May;55(5):735-50.
7. Jenne DE, Reimann H, Nezu J, Friedel W, Loff S, Jeschke R, Müller O, Back W,
Zimmer M. Peutz-Jeghers syndrome is caused by mutations in a novel serine
threonine kinase. Nat Genet 1998, Jan;18(1):38-43.
60
8. Lim W, Hearle N, Shah B, Murday V, Hodgson SV, Lucassen A, Eccles D,
Talbot I, Neale K, Lim AG, O'Donohue J, Donaldson A, Macdonald RC, Young
ID, Robinson MH, Lee PW, Stoodley BJ, Tomlinson I, Alderson D, Holbrook AG,
Vyas S, Swarbrick ET, Lewis AA, Phillips RK, Houlston RS. Further
observations on LKB1/STK11 status and cancer risk in peutz-jeghers syndrome.
Br J Cancer 2003, Jul 21;89(2):308-13.
9. Launonen V. Mutations in the human LKB1/STK11 gene. Hum Mutat 2005,
Oct;26(4):291-7.
10. Hemminki A, Markie D, Tomlinson I, Avizienyte E, Roth S, Loukola A, Bignell
G, Warren W, Aminoff M, Höglund P. A serine/threonine kinase gene defective
in peutz--jeghers syndrome. Nature 1998;391(6663):184-7.
11. Sanchez-Cespedes M, Parrella P, Esteller M, Nomoto S, Trink B, Engles JM,
Westra WH, Herman JG, Sidransky D. Inactivation of LKB1/STK11 is a
common event in adenocarcinomas of the lung. Cancer Res 2002, Jul
1;62(13):3659-62.
12. Virmani AK, Fong KM, Kodagoda D, McIntire D, Hung J, Tonk V, Minna JD,
Gazdar AF. Allelotyping demonstrates common and distinct patterns of
chromosomal loss in human lung cancer types. Genes Chromosomes Cancer
1998, Apr;21(4):308-19.
13. Carretero J, Medina PP, Pio R, Montuenga LM, Sanchez-Cespedes M. Novel
and natural knockout lung cancer cell lines for the LKB1/STK11 tumor
suppressor gene. Oncogene 2004, May 13;23(22):4037-40.
61
14. Ji H, Ramsey MR, Hayes DN, Fan C, McNamara K, Kozlowski P, Torrice C,
Wu MC, Shimamura T, Perera SA, Liang MC, Cai D, Naumov GN, Bao L,
Contreras CM, Li D, Chen L, Krishnamurthy J, Koivunen J, Chirieac LR, Padera
RF, Bronson RT, Lindeman NI, Christiani DC, Lin X, Shapiro GI, Jänne PA,
Johnson BE, Meyerson M, Kwiatkowski DJ, Castrillon DH, Bardeesy N,
Sharpless NE, Wong KK. LKB1 modulates lung cancer differentiation and
metastasis. Nature 2007, Aug 16;448(7155):807-10.
15. Matsumoto K, Szajek L, Krishna MC, Cook JA, Seidel J, Grimes K, Carson J,
Sowers AL, English S, Green MV, Bacharach SL, Eckelman WC, Mitchell JB.
The influence of tumor oxygenation on hypoxia imaging in murine squamous
cell carcinoma using [64cu]cu-atsm or [18F]fluoromisonidazole positron
emission tomography. Int J Oncol 2007, Apr;30(4):873-81.
16. Koivunen JP, Mermel C, Zejnullahu K, Murphy C, Lifshits E, Holmes AJ, Choi
HG, Kim J, Chiang D, Thomas R. EML4-ALK fusion gene and efficacy of an
ALK
kinase
inhibitor
in
lung
cancer.
Clinical
Cancer
Research
2008;14(13):4275-83.
17. Ding L, Getz G, Wheeler DA, Mardis ER, McLellan MD, Cibulskis K, Sougnez
C, Greulich H, Muzny DM, Morgan MB, Fulton L, Fulton RS, Zhang Q, Wendl
MC, Lawrence MS, Larson DE, Chen K, Dooling DJ, Sabo A, Hawes AC, Shen
H, Jhangiani SN, Lewis LR, Hall O, Zhu Y, Mathew T, Ren Y, Yao J, Scherer
SE, Clerc K, Metcalf GA, Ng B, Milosavljevic A, Gonzalez-Garay ML, Osborne
JR, Meyer R, Shi X, Tang Y, Koboldt DC, Lin L, Abbott R, Miner TL, Pohl C,
62
Fewell G, Haipek C, Schmidt H, Dunford-Shore BH, Kraja A, Crosby SD,
Sawyer CS, Vickery T, Sander S, Robinson J, Winckler W, Baldwin J, Chirieac
LR, Dutt A, Fennell T, Hanna M, Johnson BE, Onofrio RC, Thomas RK, Tonon
G, Weir BA, Zhao X, Ziaugra L, Zody MC, Giordano T, Orringer MB, Roth JA,
Spitz MR, Wistuba II, Ozenberger B, Good PJ, Chang AC, Beer DG, Watson
MA, Ladanyi M, Broderick S, Yoshizawa A, Travis WD, Pao W, Province MA,
Weinstock GM, Varmus HE, Gabriel SB, Lander ES, Gibbs RA, Meyerson M,
Wilson RK. Somatic mutations affect key pathways in lung adenocarcinoma.
Nature 2008, Oct 23;455(7216):1069-75.
18. Weir BA, Woo MS, Getz G, Perner S, Ding L, Beroukhim R, Lin WM, Province
MA, Kraja A, Johnson LA, Shah K, Sato M, Thomas RK, Barletta JA, Borecki IB,
Broderick S, Chang AC, Chiang DY, Chirieac LR, Cho J, Fujii Y, Gazdar AF,
Giordano T, Greulich H, Hanna M, Johnson BE, Kris MG, Lash A, Lin L,
Lindeman N, Mardis ER, McPherson JD, Minna JD, Morgan MB, Nadel M,
Orringer MB, Osborne JR, Ozenberger B, Ramos AH, Robinson J, Roth JA,
Rusch V, Sasaki H, Shepherd F, Sougnez C, Spitz MR, Tsao MS, Twomey D,
Verhaak RG, Weinstock GM, Wheeler DA, Winckler W, Yoshizawa A, Yu S,
Zakowski MF, Zhang Q, Beer DG, Wistuba II, Watson MA, Garraway LA,
Ladanyi M, Travis WD, Pao W, Rubin MA, Gabriel SB, Gibbs RA, Varmus HE,
Wilson RK, Lander ES, Meyerson M. Characterizing the cancer genome in lung
adenocarcinoma. Nature 2007, Dec 6;450(7171):893-8.
19. Carretero J, Shimamura T, Rikova K, Jackson AL, Wilkerson MD, Borgman CL,
Buttarazzi MS, Sanofsky BA, McNamara KL, Brandstetter KA, Walton ZE, Gu
63
TL, Silva JC, Crosby K, Shapiro GI, Maira SM, Ji H, Castrillon DH, Kim CF,
García-Echeverría C, Bardeesy N, Sharpless NE, Hayes ND, Kim WY,
Engelman JA, Wong KK. Integrative genomic and proteomic analyses identify
targets for lkb1-deficient metastatic lung tumors. Cancer Cell 2010, Jun
15;17(6):547-59.
20. Chen Z, Cheng K, Walton Z, Wang Y, Ebi H, Shimamura T, Liu Y, Tupper T,
Ouyang J, Li J, Gao P, Woo MS, Xu C, Yanagita M, Altabef A, Wang S, Lee C,
Nakada Y, Peña CG, Sun Y, Franchetti Y, Yao C, Saur A, Cameron MD,
Nishino M, Hayes DN, Wilkerson MD, Roberts PJ, Lee CB, Bardeesy N,
Butaney M, Chirieac LR, Costa DB, Jackman D, Sharpless NE, Castrillon DH,
Demetri GD, Jänne PA, Pandolfi PP, Cantley LC, Kung AL, Engelman JA,
Wong KK. A murine lung cancer co-clinical trial identifies genetic modifiers of
therapeutic response. Nature 2012, Mar 29;483(7391):613-7.
21. Lizcano JM, Göransson O, Toth R, Deak M, Morrice NA, Boudeau J, Hawley
SA, Udd L, Mäkelä TP, Hardie DG, Alessi DR. LKB1 is a master kinase that
activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J
2004, Feb 25;23(4):833-43.
22. Jaleel M, McBride A, Lizcano JM, Deak M, Toth R, Morrice NA, Alessi DR.
Identification of the sucrose non-fermenting related kinase SNRK, as a novel
LKB1 substrate. FEBS Lett 2005, Feb 28;579(6):1417-23.
64
23. Tiainen M, Ylikorkala A, Mäkelä TP. Growth suppression by lkb1 is mediated
by a G(1) cell cycle arrest. Proc Natl Acad Sci U S A 1999, Aug 3;96(16):924851.
24. Jishage K, Nezu J, Kawase Y, Iwata T, Watanabe M, Miyoshi A, Ose A, Habu
K, Kake T, Kamada N, Ueda O, Kinoshita M, Jenne DE, Shimane M, Suzuki H.
Role of lkb1, the causative gene of peutz-jegher's syndrome, in embryogenesis
and polyposis. Proc Natl Acad Sci U S A 2002, Jun 25;99(13):8903-8.
25. Miyoshi H, Nakau M, Ishikawa TO, Seldin MF, Oshima M, Taketo MM.
Gastrointestinal hamartomatous polyposis in lkb1 heterozygous knockout mice.
Cancer Res 2002, Apr 15;62(8):2261-6.
26. Ylikorkala A, Rossi DJ, Korsisaari N, Luukko K, Alitalo K, Henkemeyer M,
Mäkelä TP. Vascular abnormalities and deregulation of VEGF in lkb1-deficient
mice. Science 2001, Aug 17;293(5533):1323-6.
27. Baas AF, Boudeau J, Sapkota GP, Smit L, Medema R, Morrice NA, Alessi DR,
Clevers HC. Activation of the tumour suppressor kinase LKB1 by the ste20-like
pseudokinase STRAD. EMBO J 2003, Jun 16;22(12):3062-72.
28. Boudeau J, Baas AF, Deak M, Morrice NA, Kieloch A, Schutkowski M,
Prescott
AR,
Clevers
HC,
Alessi
DR.
MO25alpha/beta
interact
with
stradalpha/beta enhancing their ability to bind, activate and localize LKB1 in the
cytoplasm. EMBO J 2003, Oct 1;22(19):5102-14.
65
29. Boudeau J, Scott JW, Resta N, Deak M, Kieloch A, Komander D, Hardie DG,
Prescott AR, van Aalten DM, Alessi DR. Analysis of the LKB1-STRAD-MO25
complex. J Cell Sci 2004, Dec 15;117(Pt 26):6365-75.
30. Hong SP, Leiper FC, Woods A, Carling D, Carlson M. Activation of yeast snf1
and mammalian amp-activated protein kinase by upstream kinases. Proc Natl
Acad Sci U S A 2003, Jul 22;100(15):8839-43.
31. Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Mäkelä TP, Alessi DR,
Hardie DG. Complexes between the LKB1 tumor suppressor, STRAD
alpha/beta and MO25 alpha/beta are upstream kinases in the amp-activated
protein kinase cascade. J Biol 2003;2(4):28.
32. Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D,
Schlattner U, Wallimann T, Carlson M, Carling D. LKB1 is the upstream kinase
in
the
amp-activated
protein
kinase
cascade.
Curr
Biol
2003,
Nov
11;13(22):2004-8.
33. Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA,
Cantley LC. The tumor suppressor LKB1 kinase directly activates ampactivated kinase and regulates apoptosis in response to energy stress. Proc
Natl Acad Sci U S A 2004, Mar 9;101(10):3329-35.
34. Hardie D, Scott JW, Pan DA, Hudson ER. Management of cellular energy by
the amp-activated protein kinase system. FEBS Lett 2003, Jul;546(1):113-20.
66
35. Baas AF, Smit L, Clevers H. LKB1 tumor suppressor protein: PARtaker in cell
polarity. Trends Cell Biol 2004, Jun;14(6):312-9.
36. Forcet C, Etienne-Manneville S, Gaude H, Fournier L, Debilly S, Salmi M,
Baas A, Olschwang S, Clevers H, Billaud M. Functional analysis of peutzjeghers mutations reveals that the LKB1 c-terminal region exerts a crucial role
in regulating both the AMPK pathway and the cell polarity. Hum Mol Genet
2005, May 15;14(10):1283-92.
37. Lee JH, Koh H, Kim M, Kim Y, Lee SY, Karess RE, Lee SH, Shong M, Kim JM,
Kim J, Chung J. Energy-dependent regulation of cell structure by amp-activated
protein kinase. Nature 2007, Jun 21;447(7147):1017-20.
38. Zhang S, Schafer-Hales K, Khuri FR, Zhou W, Vertino PM, Marcus AI. The
tumor suppressor LKB1 regulates lung cancer cell polarity by mediating cdc42
recruitment and activity. Cancer Res 2008, Feb 1;68(3):740-8.
39. Roy BC, Kohno T, Iwakawa R, Moriguchi T, Kiyono T, Morishita K, SanchezCespedes M, Akiyama T, Yokota J. Involvement of LKB1 in epithelialmesenchymal transition (EMT) of human lung cancer cells. Lung Cancer 2010,
Nov;70(2):136-45.
40. Wu Y, Zhou BP. New insights of epithelial-mesenchymal transition in cancer
metastasis. Acta Biochimica Et Biophysica Sinica 2008, Jul;40(7):643-50.
41. Inoki K, Kim J, Guan K-L. AMPK and mtor in cellular energy homeostasis and
drug targets. Annual Review of Pharmacology and Toxicology 2012;52:381-400.
67
42. Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control
cell growth and survival. Cell 2003, Nov 26;115(5):577-90.
43. Chhipa RR, Wu Y, Mohler JL, Ip C. Survival advantage of AMPK activation to
androgen-independent prostate cancer cells during energy stress. Cell Signal
2010, Oct;22(10):1554-61.
44. Alberts B. Molecular biology of the cell. New York: Garland Science; 2008.
45. Warburg OH. Über den stoffwechsel der tumoren. J. Springer; 1926.
46. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the warburg
effect: The metabolic requirements of cell proliferation. Science 2009, May
22;324(5930):1029-33.
47. Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K, Dewerchin M,
Neeman M, Bono F, Abramovitch R, Maxwell P, Koch CJ, Ratcliffe P, Moons L,
Jain RK, Collen D, Keshert E, Keshet E. Role of hif-1alpha in hypoxia-mediated
apoptosis, cell proliferation and tumour angiogenesis. Nature 1998, Jul
30;394(6692):485-90.
48. Bertout JA, Patel SA, Simon MC. The impact of O2 availability on human
cancer. Nat Rev Cancer 2008, Dec;8(12):967-75.
49. Semenza GL. HIF-1: Upstream and downstream of cancer metabolism. Curr
Opin Genet Dev 2010, Feb;20(1):51-6.
50. Koukourakis MI, Giatromanolaki A, Harris AL, Sivridis E. Comparison of
metabolic pathways between cancer cells and stromal cells in colorectal
68
carcinomas: A metabolic survival role for tumor-associated stroma. Cancer Res
2006, Jan 15;66(2):632-7.
51. Swietach P, Vaughan-Jones RD, Harris AL. Regulation of tumor ph and the
role of carbonic anhydrase 9. Cancer Metastasis Rev 2007, Jun;26(2):299-310.
52. Fischer K, Hoffmann P, Voelkl S, Meidenbauer N, Ammer J, Edinger M,
Gottfried E, Schwarz S, Rothe G, Hoves S, Renner K, Timischl B, Mackensen A,
Kunz-Schughart L, Andreesen R, Krause SW, Kreutz M. Inhibitory effect of
tumor cell-derived lactic acid on human T cells. Blood 2007, May
1;109(9):3812-9.
53. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat
Rev Cancer 2004, Nov;4(11):891-9.
54. Fleury C, Mignotte B, Vayssière J-L. Mitochondrial reactive oxygen species in
cell death signaling. Biochimie 2002;84(2):131-41.
55.
Wallace
DC.
Mitochondria
and
cancer.
Nature
Reviews
Cancer
2012;12(10):685-98.
56. Giannoni E, Buricchi F, Raugei G, Ramponi G, Chiarugi P. Intracellular
reactive oxygen species activate src tyrosine kinase during cell adhesion and
anchorage-dependent cell growth. Mol Cell Biol 2005, Aug;25(15):6391-403.
57. Lee SR, Yang KS, Kwon J, Lee C, Jeong W, Rhee SG. Reversible inactivation
of the tumor suppressor PTEN by H2O2. J Biol Chem 2002, Jun
7;277(23):20336-42.
69
58. Cao J, Schulte J, Knight A, Leslie NR, Zagozdzon A, Bronson R, Manevich Y,
Beeson C, Neumann CA. Prdx1 inhibits tumorigenesis via regulating
PTEN/AKT activity. EMBO J 2009, May 20;28(10):1505-17.
59. Gao P, Zhang H, Dinavahi R, Li F, Xiang Y, Raman V, Bhujwalla ZM, Felsher
DW, Cheng L, Pevsner J, Lee LA, Semenza GL, Dang CV. HIF-dependent
antitumorigenic effect of antioxidants in vivo. Cancer Cell 2007, Sep;12(3):2308.
60. Keith B, Johnson RS, Simon MC. HIF1α and hif2α: Sibling rivalry in hypoxic
tumour growth and progression. Nat Rev Cancer 2012, Jan;12(1):9-22.
61. Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell 2012,
Feb 3;148(3):399-408.
62. Ramsey MR, Sharpless NE. ROS as a tumour suppressor? Nat Cell Biol
2006;8(11):1213-5.
63. Takahashi A, Ohtani N, Yamakoshi K, Iida S, Tahara H, Nakayama K,
Nakayama KI, Ide T, Saya H, Hara E. Mitogenic signalling and the p16ink4a-rb
pathway cooperate to enforce irreversible cellular senescence. Nat Cell Biol
2006, Nov;8(11):1291-7.
64. Garrido C, Galluzzi L, Brunet M, Puig PE, Didelot C, Kroemer G. Mechanisms
of cytochrome c release from mitochondria. Cell Death Differ 2006,
Sep;13(9):1423-33.
70
65. Han D, Antunes F, Canali R, Rettori D, Cadenas E. Voltage-dependent anion
channels control the release of the superoxide anion from mitochondria to
cytosol. J Biol Chem 2003, Feb 21;278(8):5557-63.
66. Li X, Fang P, Mai J, Choi ET, Wang H, Yang XF. Targeting mitochondrial
reactive oxygen species as novel therapy for inflammatory diseases and
cancers. J Hematol Oncol 2013;6:19.
67. Shackelford DB, Shaw RJ. The LKB1--AMPK pathway: Metabolism and
growth control in tumour suppression. Nature Reviews Cancer 2009;9(8):56375.
68. Guertin DA, Sabatini DM. Defining the role of mtor in cancer. Cancer Cell 2007,
Jul;12(1):9-22.
69. Semenza GL. Hypoxia-inducible factor 1: Master regulator of O< sub> 2</sub>
homeostasis. Curr Opin Genet Dev 1998;8(5):588-94.
70. Bertout JA, Patel SA, Simon MC. The impact of O2 availability on human
cancer. Nat Rev Cancer 2008, Dec;8(12):967-75.
71. Li F, Wang Y, Zeller KI, Potter JJ, Wonsey DR, O'Donnell KA, Kim JW,
Yustein JT, Lee LA, Dang CV. Myc stimulates nuclearly encoded mitochondrial
genes and mitochondrial biogenesis. Mol Cell Biol 2005, Jul;25(14):6225-34.
72. Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nature
Reviews Cancer 2011;11(2):85-95.
71
73. Davies SP, Carling D, Munday MR, Hardie DG. Diurnal rhythm of
phosphorylation of rat liver acetyl-coa carboxylase by the amp-activated protein
kinase, demonstrated using freeze-clamping. Effects of high fat diets. Eur J
Biochem 1992, Feb 1;203(3):615-23.
74. Jeon SM, Chandel NS, Hay N. AMPK regulates NADPH homeostasis to
promote tumour cell survival during energy stress. Nature 2012, May
31;485(7400):661-5.
75. Nathan C, Ding A. SnapShot: Reactive oxygen intermediates (ROI). Cell 2010,
Mar 19;140(6):951-951.e2.
76. Gandhi V, Ayres M, Halgren RG, Krett NL, Newman RA, Rosen ST. 8-chlorocAMP and 8-chloro-adenosine act by the same mechanism in multiple myeloma
cells. Cancer Res 2001;61(14):5474-9.
77. Balakrishnan K, Stellrecht CM, Genini D, Ayres M, Wierda WG, Keating MJ,
Leoni LM, Gandhi V. Cell death of bioenergetically compromised and
transcriptionally challenged CLL lymphocytes by chlorinated ATP. Blood 2005,
Jun 1;105(11):4455-62.
78. Langeveld CH, Jongenelen CA, Theeuwes JW, Baak JP, Heimans JJ, Stoof
JC, Peters GJ. The antiproliferative effect of 8-chloro-adenosine, an active
metabolite of 8-chloro-cyclic adenosine monophosphate, and disturbances in
nucleic acid synthesis and cell cycle kinetics. Biochem Pharmacol 1997, Jan
24;53(2):141-8.
72
79. Halgren RG, Traynor AE, Pillay S, Zell JL, Heller KF, Krett NL, Rosen ST. 8ClcAMP cytotoxicity in both steroid sensitive and insensitive multiple myeloma cell
lines is mediated by 8cl-adenosine. Blood 1998, Oct 15;92(8):2893-8.
80. Zhu B, Zhang LH, Zhao YM, Cui JR, Strada SJ. 8-chloroadenosine induced
HL-60 cell growth inhibition, differentiation, and G(0)/G(1) arrest involves
attenuated cyclin D1 and telomerase and up-regulated p21(WAF1/CIP1). J Cell
Biochem 2006, Jan 1;97(1):166-77.
81. Carlson CC, Chinery R, Burnham LL, Dransfield DT. 8-Cl-adenosine-induced
inhibition of colorectal cancer growth in vitro and in vivo. Neoplasia
2000;2(5):441-8.
82. Zhang HY, Gu YY, Li ZG, Jia YH, Yuan L, Li SY, An GS, Ni JH, Jia HT.
Exposure of human lung cancer cells to 8-chloro-adenosine induces G2/M
arrest and mitotic catastrophe. Neoplasia 2004;6(6):802-12.
83. Gandhi V, Ayres M, Halgren RG, Krett NL, Newman RA, Rosen ST. 8-chlorocAMP and 8-chloro-adenosine act by the same mechanism in multiple myeloma
cells. Cancer Res 2001;61(14):5474-9.
84. Stellrecht CM, Rodriguez CO, Ayres M, Gandhi V. RNA-directed actions of 8chloro-adenosine in multiple myeloma cells. Cancer Res 2003, Nov
15;63(22):7968-74.
73
85. Chen LS, Sheppard TL. Chain termination and inhibition of saccharomyces
cerevisiae poly(A) polymerase by c-8-modified ATP analogs. J Biol Chem 2004,
Sep 24;279(39):40405-11.
86. Dennison JB, Balakrishnan K, Gandhi V. Preclinical activity of 8chloroadenosine with mantle cell lymphoma: Roles of energy depletion and
inhibition of DNA and RNA synthesis. Br J Haematol 2009, Nov;147(3):297-307.
87. XF cell mito stress test kit user manual X96 instructions. .
88. XF glycolysis stress test kit user manual (XF 96). .
89. Davies SP, Carling D, Munday MR, Hardie DG. Diurnal rhythm of
phosphorylation of rat liver acetyl-coa carboxylase by the amp-activated protein
kinase, demonstrated using freeze-clamping. Effects of high fat diets. Eur J
Biochem 1992, Feb 1;203(3):615-23.
90. Chen LS, Nowak BJ, Ayres ML, Krett NL, Rosen ST, Zhang S, Gandhi V.
Inhibition of ATP synthase by chlorinated adenosine analogue. Biochem
Pharmacol 2009, Sep 15;78(6):583-91.
91. Gorrini C, Baniasadi PS, Harris IS, Silvester J, Inoue S, Snow B, Joshi PA,
Wakeham A, Molyneux SD, Martin B, Bouwman P, Cescon DW, Elia AJ,
Winterton-Perks Z, Cruickshank J, Brenner D, Tseng A, Musgrave M, Berman
HK, Khokha R, Jonkers J, Mak TW, Gauthier ML. BRCA1 interacts with nrf2 to
regulate antioxidant signaling and cell survival. J Exp Med 2013, Jul
29;210(8):1529-44.
74
92. Dennison JB, Ayres ML, Kaluarachchi K, Plunkett W, Gandhi V. Intracellular
succinylation of 8-chloroadenosine and its effect on fumarate levels. J Biol
Chem 2010, Mar 12;285(11):8022-30.
93. López-Ríos F, Sánchez-Aragó M, García-García E, Ortega AD, Berrendero JR,
Pozo-Rodríguez F, López-Encuentra A, Ballestín C, Cuezva JM. Loss of the
mitochondrial bioenergetic capacity underlies the glucose avidity of carcinomas.
Cancer Res 2007, Oct 1;67(19):9013-7.
94. Dennison JB, Ayres ML, Kaluarachchi K, Plunkett W, Gandhi V. Intracellular
succinylation of 8-chloroadenosine and its effect on fumarate levels. J Biol
Chem 2010, Mar 12;285(11):8022-30.
95. Chen LS, Nowak BJ, Ayres ML, Krett NL, Rosen ST, Zhang S, Gandhi V.
Inhibition of ATP synthase by chlorinated adenosine analogue. Biochem
Pharmacol 2009, Sep 15;78(6):583-91.
96. Brahimi-Horn MC, Chiche J, Pouysségur J. Hypoxia signalling controls
metabolic demand. Curr Opin Cell Biol 2007, Apr;19(2):223-9.
97. Kroemer G, Pouyssegur J. Tumor cell metabolism: Cancer's achilles' heel.
Cancer Cell 2008, Jun;13(6):472-82.
98. Currie E, Schulze A, Zechner R, Walther TC, Farese RV. Cellular fatty acid
metabolism and cancer. Cell Metab 2013, Aug 6;18(2):153-61.
99. White E. Exploiting the bad eating habits of ras-driven cancers. Genes Dev
2013, Oct 1;27(19):2065-71.
75
100.Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, Mair W,
Vasquez DS, Joshi A, Gwinn DM, Taylor R, Asara JM, Fitzpatrick J, Dillin A,
Viollet B, Kundu M, Hansen M, Shaw RJ. Phosphorylation of ULK1 (hatg1) by
amp-activated protein kinase connects energy sensing to mitophagy. Science
2011, Jan 28;331(6016):456-61.
76
Vita
Chao Yang was born on August 5th 1987 in Baoji, Shaanxi, China. After
graduating from Baoji High School, he came to United States for college. Chao
obtained his bachelor’s degree in Molecular and Cellular Biology with minor in
Chemistry from Towson University in Maryland in May 2011. The summer of
2011, he entered The University of Texas Health Science Center Graduate
School of Biomedical Sciences to pursue a degree in Cancer Biology.
77