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Targeting Antioxidant Systems to Treat Pancreatic Cancer
by
Francesco Gabriele Tatangelo Fazzari
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Laboratory Medicine & Pathobiology University of Toronto
© Copyright by Francesco Gabriele Tatangelo Fazzari 2018
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Targeting Antioxidant Systems to Treat Pancreatic Cancer
Francesco Fazzari
Master of Science
Department of Laboratory Medicine & Pathobiology
University of Toronto
2018
Abstract
Pancreatic cancer has the worst prognosis of all cancers. Pancreatic tumours are characterized by
excessive oxidative stress that may provide a unique vulnerability to target in the development of
new therapeutic strategies. I investigated a novel combination therapy targeting the two main
intracellular antioxidant systems, glutathione and thioredoxin. Buthionine sulfoximine (BSO)
targets glutathione synthesis and auranofin targets thioredoxin recycling. These drugs have been
tested in human clinical trials and are strong candidates for repurposing in cancer therapy. I found
that BSO potentiated auranofin’s cytotoxic effects in vitro. Furthermore, the combination
augmented oxidative stress in cells. Auranofin engaged its target TrxR in vitro at concentrations
causing cell death. I also assessed the biodistribution of auranofin in vivo and observed a
preferential accumulation in the stroma 24 hours after treatment. Overall, I conclude that this
treatment strategy can be effective against pancreatic cancer and deserves attention for future
development.
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Acknowledgments
First and foremost, I would like to thank my supervisor, Dr. David Hedley, for providing an
excellent opportunity to research in his lab. His guidance was instrumental in helping me to
develop a successful project and for supporting me as I take the next steps in my career.
I would also like the thank all the members of the Hedley lab past and present for their assistance
throughout my tenure. They provided an enjoyable work environment and were always willing to
lend a helping hand to teach me different techniques.
Next, I would like to thank my committee members, Drs. Richard Hill and Marianne Koritzisnky.
Their critical assessment of my work helped to shape my project to what it has become.
Lastly, I would like to thank my family and friends for providing support throughout my studies,
and for helping me to enjoy my time outside of the lab.
This work was made possible by support through funding from the Ontario Centres of Excellence.
My stipend was partially supported by the University of Toronto Fellowship Award.
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Table of Contents
ACKNOWLEDGMENTS ........................................................................................................... III
TABLE OF CONTENTS ............................................................................................................ IV
LIST OF TABLES .................................................................................................................... VI
LIST OF FIGURES .................................................................................................................. VII
LIST OF ABBREVIATIONS .................................................................................................... VIII
CHAPTER 1 INTRODUCTION .................................................................................................... 1
1.1 PANCREATIC CANCER ............................................................................................................ 1
1.1.1 Pancreatic Cancer Therapy Development ................................................................... 2
1.1.2 Pancreatic Carcinogenesis .......................................................................................... 5
1.2 OXIDATIVE STRESS ................................................................................................................ 6
1.2.1 Hypoxia ....................................................................................................................... 7
1.2.2 The Impact of High ROS Levels .................................................................................... 7
1.2.3 Adaptations to High ROS Levels ................................................................................ 10
1.2.4 The Impact of Low ROS Levels ................................................................................... 13
1.3 THE ROLE OF ANTIOXIDANTS ................................................................................................ 15
1.3.1 The Glutathione Antioxidant System ........................................................................ 16
1.3.2 The Thioredoxin Antioxidant System ........................................................................ 22
1.4 A NEW THERAPEUTIC STRATEGY FOR PANCREATIC CANCER ........................................................ 28
CHAPTER 2 ........................................................................................................................... 30
2.1 INTRODUCTION .................................................................................................................. 31
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2.2 MATERIALS & METHODS ..................................................................................................... 33
2.3 RESULTS ........................................................................................................................... 38
2.4 DISCUSSION ...................................................................................................................... 49
CHAPTER 3 ........................................................................................................................... 53
3.1 OVERALL DISCUSSION ......................................................................................................... 54
3.1.1 BSO potentiates the acute cytotoxicity of auranofin ................................................ 54
3.1.2 Apoptosis contributes to cell death .......................................................................... 55
3.1.3 The drugs engage their expected targets ................................................................. 56
3.1.4 Oxidative stress follows combination therapy .......................................................... 56
3.1.5 IMC is an effective tool to examine auranofin in vivo............................................... 57
3.2 FUTURE DIRECTIONS ........................................................................................................... 58
3.3 CONCLUSIONS ................................................................................................................... 60
REFERENCES ........................................................................................................................ 61
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List of Tables
Table 2.1. IC50 values of auranofin monotherapy and in combination with BSO in primary pancreatic cancer cell lines. ............................................................. 39
Table 2.2. Panel of markers measured by IMC using subcutaneous PDX tumours.
...................................................................................................................... 47
Table 2.3. Estimation of the mean ions/pixel in the stroma and epithelium of
subcutaneous tumours from auranofin treated PDX mice. ........................... 49
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List of Figures
Figure 2.1. Dose-response of antioxidant pathway inhibitors, TrxR isozyme levels, and colony formation potential. .................................................................... 40
Figure 2.2. Representative two parameter plots measuring cell death and apoptosis in OCIP23. ..................................................................................... 42
Figure 2.3. Representative two parameter plots assessing the GSH content of apoptotic cells in OCIP23. .............................................................................. 43
Figure 2.4. Effect of treatment on thiol and ROS levels in 9A and OCIP23 cell lines. ...................................................................................................................... 44
Figure 2.5. Auranofin-TrxR interaction in vitro. .................................................... 46
Figure 2.6. Distribution of auranofin in subcutaneous PDX tumours. ................... 48
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List of Abbreviations
5-FU 5-fluorouracil
ARE antioxidant response element
ASK1 apoptosis signaling kinase 1
Bax bcl-2-like protein 4
BCNU carmustine
BSA bovine serum albumin
BSO buthionine sulfoximine
c-FLIP cellular FLICE-inhibitory protein
CETSA cellular thermal shift assay
DCF 2’,7’-dichlorofluorescein
DMEM Dulbecco’s Modified Eagle Medium
DMSO dimethyl sulfoxide
ECM extracellular matrix
EGFR epidermal growth factor receptor
ERK extracellular signal-regulated kinase
FBS fetal bovine serum
FFPE formalin-fixed, paraffin-embedded
G6PD glucose 6-phosphate dehydrogenase
GCL glutamate-cysteine ligase
GCLC glutamate-cysteine ligase catalytic subunit
GCLM glutamate-cysteine ligase modifier subunit
GGT -glutamyltranspeptidase
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GLUD1 glutamate dehydrogenase
GOT1 aspartate transaminase
GPx glutathione peroxidase
GR glutathione reductase
Grx glutaredoxin
GS glutathione synthetase
GSH glutathione
GSSG oxidized glutathione
GST glutathione S-transferase
HNE 4-hydroxynonenal
IKK- inhibitor of nuclear factor kappa-B kinase subunit beta
IMC imaging mass cytometry
Keap1 kelch-like ECH-associated protein 1
mBBr monobromobimane
MDH1 malate dehydrogenase
ME1 malic enzyme 1
MMP mitochondrial membrane potential
Msr methionine sulfoxide reductase
NADPH nicotinamide adenine dinucleotide phosphate
Nrf2 nuclear factor, erythroid derived 2, like 2
P-gp multidrug resistance protein 1
PanIN pancreatic intraepithelial neoplasia
PBS phosphate-buffered saline
PD-L1 programmed death ligand 1
x
PDX patient-derived xenograft
PEGPH20 pegvorhyaluronidase alfa
PHGDH phosphoglycerate dehydrogenase
PI propidium iodide
PPP pentose phosphate pathway
Prx peroxiredoxin
PTEN phosphatase and tensin homolog
RNR ribonucleotide reductase
ROS reactive oxygen species
RPMI Roswell Park Memorial Institute
RT room temperature
SCID severe combined immunodeficiency
System XC– cystine/glutamate antiporter system
TRAIL TNF-related apoptosis-inducing ligand
Trx thioredoxin
TrxR thioredoxin reductase
VEGF-A vascular endothelial growth factor A
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Chapter 1 Introduction
1.1 Pancreatic Cancer
Pancreatic cancer has the worst prognosis amongst all cancers; 5 year survival rates for the disease
range from less than 5 to 8%1–3. Although it is only the twelfth most commonly diagnosed, it is
the fourth most common cause of cancer death in Canada2. Unfortunately, mortality from
pancreatic cancer has not improved during the era of modern medicine4,5. Estimates predict that
pancreatic cancer will become the second most common cause of cancer death behind non-small-
cell lung cancer by 20306. The dismal outlook for patients with pancreatic cancer can be partially
attributed to the lack of progress made in the development of diagnostics and therapeutics, which
have remained stagnant over the last 50 years7. During this time vast improvements have been
made for the diagnosis and treatment of other tumour types. For example, the development of the
Pap smear can reduce cervical cancer death by up to 80%8, and the development of trastuzumab
has increased overall survival by 33% in HER2+ breast cancers9,10. The identification of new
biomarkers and treatment strategies is required to overcome pancreatic cancer.
Early stage pancreatic cancer is generally asymptomatic, leading to difficulties in detection. The
disease may be identified secondary to non-specific symptoms, or as an incidental finding. In
addition to this, screening tests have not been developed. Currently the only treatment that offers
the potential for long-term survival is surgery in early-stage disease, however the 5 year survival
rate in patients with resectable tumours remains only 15-20%11. For patients with advanced
metastatic disease there are few systemic options that offer little benefit in survival.
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1.1.1 Pancreatic Cancer Therapy Development
The first systemic therapy used in the treatment of pancreatic cancer was 5-fluorouracil (5-FU). 5-
FU is a non-specific agent that inhibits the synthesis of the nucleoside thymidine. 5-FU provided
a 4 month survival benefit when used in combination with radiotherapy, compared to radiotherapy
alone12. Since the indroduction of 5-FU in pancreatic cancer clinics some small advancements
have been made. Gemcitabine became the new standard in the late 1990s when a clinical trial
showed it offered a significant improvement in median overall survival when compared to 5-FU
(5.6 months vs. 4.4 months)13. However, gemcitabine is another non-specific agent. It is a
nucleoside analog that replaces cytidine during DNA replication. Since the introduction of
gemcitabine in the treatment of pancreatic cancer, two therapies have been developed for clinical
use. The combination therapy FOLFIRINOX (folinic acid, 5-FU, irinotecan, and oxaliplatin)
improved median overall survival compared to gemcitabine in a clinical trial (11.1 months vs. 6.8
months)14, and the combination of nab-paclitaxel with gemcitabine improved median overall
survival compared to gemcitabine alone (8.5 months vs. 6.7 months)15. Although there appears to
be a survival difference between FOLFIRINOX and gemcitabine/nab-paclitaxel, the
FOLFIRINOX regimen is more toxic and the patient population was skewed slightly in favour of
the FOLFIRINOX cohort, leading many investigators to consider the two regimens to be similar16–
18. Although these therapies are an improvement on past therapies, median survival falls short of
one year with either regimen.
Great interest has centred on the development of targeted therapies to provide effective non-toxic
treatment for pancreatic cancer. There have been many proposed targets and clinical trials19,
however no such therapy has entered clinical practice in Canada to date. Trials have examined
agents such as bevacizumab, inhibiting angiogenesis by targeting vascular endothelial growth
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factor A (VEGF-A), or cetuximab, inhibiting epidermal growth factor receptor (EGFR), in
combination with gemcitabine to no benefit20,21, among many other attempts. From the trials, only
erlotinib in combination with gemcitabine has shown a significant effect on overall survival (6.2
months vs. 5.9 months), although this is not clinically relevant22. In summary, current attempts at
targeted therapy have proven ineffective in the management of pancreatic cancer. This may change
in the future, however current prospects seem bleak and new biomarkers may be required to
develop these therapies further23.
Beyond targeted therapy there have been attempts to develop immunotherapies for pancreatic
cancer. Immunotherapy is a treatment approach that relies on augmenting the host’s immune
response against cancer24. This method derives from the observation that the immune system can
eliminate malignant cells during the early stages of transformation25. While there have been many
unequivocal successes in other tumour types, particularly in advanced melanoma26–28, pancreatic
cancer has proven resistant to these approaches. Clinical trials have observed a lack of response to
immune checkpoint blockade with ipilimumab29 and programmed death ligand 1 (PD-L1) directed
monoclonal antibodies30. These failures have resulted from the poorly immunogenic and
immunosuppressive nature of the pancreatic cancer microenvironment7,31. A small number of
patients enrolled in these trials have received some therapeutic benefit, leading to continued
interest in developing new immunotherapy strategies. One such strategy includes attempting to
achieve a synergistic response through the use of multiple agents. This may include combining
immune checkpoint blockade, inhibition of immunosuppressive signalling, stimulation of T cells,
and/or conventional cytotoxic therapies32.
In relation to immunotherapy, other strategies attempt to target additional features of the tumour
microenvironment. Attention has focused on the role of the stroma in determining therapy
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response. These tumours are characterized by a dense desmoplastic reaction comprised of fibrotic
tissue, extracellular matrix proteins, pancreatic stellate cells, immune cells, and blood vessels7.
The extensiveness of desmoplasia correlates with worse disease outcome33. Furthermore,
desmoplasia limits tumour vascularization, creating a barrier to drug uptake by tumours34. A
clinical trial using the modified hyaluronidase molecule pegvorhyaluronidase alfa (PEGPH20) to
degrade stromal hyaluronan in combination with nab-paclitaxel and gemcitabine has shown
improvement in progression free survival by the perceived mechanism of increasing tumour
vasculature to aid drug delivery35. However, there is evidence to suggest that the stroma may have
a role in protection against tumour progression and an increased tumour vasculature may
contribute to accelerated growth if not targeted properly7. Therefore, there is a difficult balance to
achieve via this strategy. Clinical trials have also given attention to hypoxia, which is a feature of
solid tumours that can result from the lack of vasculature in the stroma.
Hypoxia is a tumour microenvironment state where oxygen utilization exceeds its supply.
Traditionally, pancreatic tumours have been considered to be characterized by extensive hypoxia,
which may be caused by the lack of blood supply resulting from the desmoplastic reaction36. In
patient-derived xenograft (PDX) mouse models, hypoxia correlates with aggressive growth and
metastasis37, making it a logical choice to target. The prodrug evofosfamide is activated under
hypoxic conditions to release a DNA alkylating agent that inhibits cancer cell replication38.
However, a phase III clinical trial observed no significant improvement in median overall survival
using evofosfamide/gemcitabine compared to gemcitabine alone (8.7 months vs. 7.6 months)38. It
is possible that hypoxia in patient tumours is more variable than previously believed, which would
limit the effectiveness of therapy across the entire patient population39. This can be addressed by
non-invasive imaging using positron-emitting hypoxia tracers in order to stratify patients towards
hypoxia-targeting clinical trials40.
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In summary, the most effective systemic therapies available for metastatic pancreatic cancer
provide only a small benefit to patients. Although there are promising avenues to explore further,
attempts to develop new strategies against pancreatic cancer have fallen short of expectations.
Further studies are required to identify and examine novel, exploitable targets in pancreatic cancer.
1.1.2 Pancreatic Carcinogenesis
Four driver genes are frequently mutated in pancreatic cancer. These are the oncogene KRAS and
the tumour suppressors CDKN2A, SMAD4, and TP5341–44. These mutations are shared by
pancreatic intraepithelial neoplasia (PanIN), which is a precursor lesion to pancreatic cancer.
Sequencing of these genes in PanINs suggests a stepwise progression of pancreatic cancer.
Activation of KRAS is present in the earliest PanIN-1 lesions, making it the initiating event in
pancreatic cancer tumorigenesis. Inactivation of CDKN2A is found in PanIN-2, followed by
inactivation of SMAD4 and TP53 in PanIN-3 lesions45. However, more recent whole-genome
sequencing of tumour cell-enriched primaries showed cases that displayed either simultaneous
inactivation of multiple drivers or tumours that did not follow the predicted mutation sequence.
These data are consistent with punctuated rather than gradual evolution46. Regardless, KRAS
activation occurs in more than 90% of human pancreatic cancer and its effects likely have the
greatest impact on pancreatic cancer41. Interestingly, KRAS has been implicated in mediating
pancreatic carcinogenesis in part through its impact on oxidative stress. Its most important effect
on oxidative stress is the promotion of low intracellular reactive oxygen species (ROS) levels by
inducing nuclear factor, erythroid derived 2, like 2 (Nrf2) activation47. This aspect will be
discussed further in the next section.
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1.2 Oxidative Stress
Pancreatic cancer, among other cancers, is characterized by excessive oxidative stress48. Oxidative
stress is caused by the overproduction of ROS, leading to damage of lipids, proteins, and DNA49.
It occurs when there is an imbalance between intracellular ROS and antioxidants in favour of the
oxidants. ROS are produced at an extreme rate due to the high metabolism inherent in transformed
cells, and may promote aggressive tumour features50,51. Oxidative stress and ROS represent an
interesting target in pancreatic cancer because ROS exhibit dual roles; lower concentrations of
ROS can stimulate cancer cell proliferation, whereas higher concentrations of ROS are damaging
to cells49,52. Cancer cells adapt during oxidative stress conditions to maintain low ROS levels to
avoid cell death and promote proliferation by enhancing antioxidant defences53.
ROS exist as multiple species including superoxide (O2•–) , hydroxyl radical (HO•), and hydrogen
peroxide (H2O2)54. They are produced in various subcellular compartments: the cytoplasm, the
mitochondria, peroxisomes, and the endoplasmic reticulum. The major source of cytoplasmic ROS
in pancreatic cancer is NADPH oxidase, which produces superoxide anion by facilitating the
reduction of molecular oxygen by NADPH55. Mitochondrial ROS are produced in dysfunctional
mitochondria that result from inefficient hypermetabolism in rapidly proliferating cancer cells.
This can cause leakage of electrons at complex I and complex III of the mitochondrial electron
transport chain, leading to the production of superoxide and its dismutation to hydrogen peroxide
by superoxide dismutases56,57. Peroxisomal ROS are produced during the -oxidation of fatty
acids58. Lastly, endoplasmic reticulum can contribute to ROS production through its role in
providing an oxidizing environment to promote disulfide bonding and protein folding59. Although
ROS themselves are short-lived molecules, they can generate longer lasting lipid peroxides such
as 4-hydroxynonenal (HNE)60. HNE is capable of reacting with cellular macromolecules such as
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proteins, lipids, and DNA. In addition, HNE can stimulate signaling cascades to affect cell
proliferation and differentiation61. As such, it has been implicated in different cancers.
1.2.1 Hypoxia
Hypoxic tumours experience both chronic and acute hypoxia. Chronic hypoxia exists in regions
located away from blood vessels and is limited by the diffusion of oxygen through the dense
tumour stroma. Acute or cycling hypoxia exists in regions proximal to blood vessels and is
perfusion-limited in that there are temporal instabilities in blood flow through a tumour. Cycling
hypoxia generates greater oxidative stress because the acute influx of oxygen can contribute to an
increase in ROS62. In pancreatic xenografts hypoxia is associated with aggressive growth and
metastasis37. However, human pancreatic tumours may not possess extensive hypoxia as
previously suggested40. Although hypoxia stems from a lack of oxygen, hypoxic tumours have
been stated to overproduce ROS54. However, direct evidence suggesting this phenomenon is
lacking. Hypoxia can contribute to resistance to chemo and radiotherapy, which rely on ROS to
mediate their effects63. This evidence contradicts the idea hypoxia of greater oxidative stress
during hypoxia.
1.2.2 The Impact of High ROS Levels
At high levels ROS disrupt cellular processes by non-specifically damaging proteins, lipids, and
DNA. Furthermore, ROS are implicated in both the extrinsic (death receptor-dependent) and
intrinsic (mitochondrial) pathways of apoptosis. The extrinsic pathway is mediated by death-
inducing ligands, such as TNF-related apoptosis-inducing ligand (TRAIL)64. Following ligand-
receptor interaction the caspase cascade is activated, leading to apoptosis65. ROS negatively
regulate cellular FLICE-inhibitory protein (c-FLIP) by a post-transcriptional method to target it
for degradation and activate the extrinsic apoptosis pathway. The intrinsic pathway is mediated by
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a permeability transition in the mitochondria. Mitochondrial stress, including oxidative stress,
stimulates pore formation in the inner mitochondrial membrane which causes cytochrome-c
mobilization and mitochondrial swelling66. This can lead to rupture of the outer membrane, and
with the help of pro or anti-apoptotic Bcl-2 family member proteins can stimulate or prevent
apoptosis. Bcl-2-like protein 4 (Bax) in particular mediates the formation of the mitochondrial
apoptosis-induced channel (MAC) to release mitochondrial pro-apoptotic factors, such as
cytochrome-c, which leads to activation of the executioner caspase proteins67–69. As mitochondria
are a significant source of ROS production, the intrinsic pathway is the largest contributor to ROS-
mediated apoptosis. In this pathway, electron transport chain-produced superoxide triggers
eventual cytochrome-c release, leading to the induction of apoptosis70. Apoptosis following
oxidative stress displays several distinct steps that can be measured by flow cytometry methods.
Following glutathione (GSH) depletion in cells, ROS production causes the mitochondrial
permeability transition and the subsequent loss of mitochondrial membrane potential (MMP), due
to cellular damage. Finally, cells lose viability after perforation of the cell membrane71.
In addition to apoptosis, ROS contribute to ferroptosis, a form of cell death that depends on
intracellular iron72. NADPH oxidase-induced accumulation of ROS was identified to contribute to
ferroptosis. Furthermore, the compounds erastin and sulfasalazine, which inhibit the
cystine/glutamate antiporter system (system XC–), lead to toxic accumulation of ROS and
ferroptosis72,73. The lethal effect can be reversed by addition of the lipophilic antioxidants
ferrostatin-1 and vitamin E, suggesting an important role for lipid radicals in mediating
ferroptosis72,74. KRAS driven cancer cells have been shown to be susceptible to ROS-mediated
ferroptosis following treatment with lanperisone, an FDA-approved muscle relaxant75, suggesting
that ROS can be utilized to induce ferroptosis in a KRAS driven tumour such as pancreatic cancer.
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High ROS also have indirect effects on cell death by sensitizing cancer cells to chemo and
radiotherapy. The multidrug resistance protein 1 (P-gp) facilitates the removal of several
anticancer agents from cells. High ROS concentrations have been associated with decreased P-gp
expression, which allows for cytotoxic drugs to accumulate within a cell76. Furthermore, ROS have
been identified to mediate the effects of some cytotoxic chemotherapies. For example, gemcitabine
acts through a ROS-dependent mechanism that may promote metastasis due to the action of
antioxidants77. This feature may contribute to gemcitabine’s limited effectiveness in pancreatic
cancer. Moreover, both 5-FU and oxaliplatin have been observed to augment oxidative stress in
patients with colon cancer78. These drugs are two of the components of the combination
FOLFIRINOX, which is one of the most effective systemic therapies against metastatic pancreatic
cancer. Paclitaxel, which is the main component of the nab-paclitaxel/gemcitabine therapy for
pancreatic cancer, and doxorubicin also enhance oxidative stress79,80. While augmented oxidative
stress is not the primary mechanism of action of these therapies, it is interesting to note that the
most effective therapies in clinical use for pancreatic cancer elicit an effect on the redox
environment of the tumour.
In addition to chemosensitization, high ROS levels are involved in mediating the cytotoxic effects
of radiotherapy. Radiotherapy utilizes ionizing radiation to generate ROS, particularly hydroxyl
radical. Hydroxyl radicals enact the therapeutic and side effects of radiotherapy. It propagates by
reacting with other molecules including DNA, leading to abundant DNA lesions and subsequent
cell death81. It has been observed that radical scavengers are effective at attenuating the cytotoxic
effects of radiation, indicating that ROS are required82–84. The evidence presenting the role of ROS
in chemo and radiosensitization suggests that exploiting an increase in ROS can be effective in the
development of new therapies.
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Cancer cells require multiple adaptations to protect from inherently high ROS levels allowing them
to survive and proliferate. Signaling through the oxidative stress response mediates this adaptation.
The master regulator of this response is the transcription factor Nrf2. Nrf2’s activity is modulated
by its repressor Kelch-like ECH-associated protein 1 (Keap1). In a normal redox state Nrf2 is
constitutively degraded. Keap1 promotes its ubiquitination by Cullin E3 ubiquitin ligase to label
Nrf2 for proteasome degradation85. During oxidative stress conditions, redox-sensitive cysteine
residues on Keap1 are oxidized by ROS. In this state Keap1 is unable to bind Nrf2 to recruit
ubiquitin ligases to target it for degradation. Nrf2 subsequently translocates to the nucleus where
it heterodimerizes with small Maf proteins to stimulate transcription of genes associated with
antioxidant response element (ARE) promoters86. The lipid peroxide HNE is a potent inducer of
Nrf2 by covalent binding to Keap1 redox-sensitive cysteines and by activating upstream kinases
such as protein kinase C87,88 It has been observed that nuclear Nrf2 expression associates with poor
survival in pancreatic cancer89, whereas cytoplasmic Keap1 expression associates with reduced
metastasis and membrane-associated Keap1 displays a protective effect on survival90.
1.2.3 Adaptations to High ROS Levels
Nrf2 and ARE positively regulate the transcription of several cytoprotective genes including drug-
metabolizing enzymes, drug transporters, and antioxidants91. Nrf2 regulates the expression of
many phase I and II drug-metabolizing enzymes92–94, as well as members of the multi-drug
resistance-associated gene family of multidrug transporters95. It can indirectly modulate ROS
levels by regulating free Fe (II) homeostasis. This prevents the Fenton reaction, where Fe(II)
catalyses the oxidation of hydrogen peroxide to the more reactive hydroxyl radical96. This function
occurs by promoting the release of Fe (II) from heme, and its subsequent sequestration. However,
Nrf2’s most important role is functioning as the master regulator of antioxidants. The production
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and regeneration of the most abundant antioxidant cofactor, GSH, is controlled exclusively by
Nrf297. In addition, Nrf2 supports the expression of enzymes involved in GSH utilization98, as well
as enzymes involved in the production, regeneration, and utilization of thioredoxin (Trx)99. These
actions of Nrf2 are responsible for direct modulation of oxidative stress. These antioxidants will
be discussed in further detail in section 1.3. In parallel to antioxidant production, cancer cells
require the redirection of cellular metabolism to support antioxidant function.
Cancer cell metabolism differs from that of normal cells and requires abundant ATP production to
support rapid proliferation. Cancer cells experience a metabolic switch to rely on aerobic
glycolysis, rather than oxidative phosphorylation, to supplement their needs. This phenomenon is
termed the Warburg effect100,101. Constitutively active KRAS in pancreatic cancer plays a key role
in activating the metabolic switch towards supporting anabolic pathways102. In addition to this
switch, Nrf2 reprograms metabolism in oxidative stress conditions to support the antioxidants it
promotes. It reprograms metabolism by increasing transcription of NADPH-generating enzymes
to modulate glucose, glutamine, and serine metabolism103,104. NADPH is an important cofactor
that is required by antioxidant enzymes to supply reducing potential. Nrf2 promotes a proliferative
phenotype through the PI3K-Akt axis, which redirects glucose into anabolic pathways to increase
metabolism103. Three major pathways that produce NADPH are regulated by Nrf2: the pentose
phosphate pathway (PPP), the serine synthesis and folate pathway, and the malic enzyme pathway
of glutamine metabolism.
The PPP is a metabolic pathway that runs in parallel to glycolysis. It consists of oxidative and non-
oxidative phases. The oxidative arm is responsible for NADPH production, while the non-
oxidative arm is responsible for the generation of ribonucleotides105. Both phases are critical to
cancer cell survival. Glucose 6-phosphate dehydrogenase (G6PD) is the first enzyme in the
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oxidative arm of the PPP and is positively regulated by Nrf293. G6PD commits glucose 6-
phosphate to the PPP, rather than glycolysis105. The PPP has been observed to promote cell survival
and proliferation during neoplastic transformation106. Although the oxidative phase has generally
been implicated to supply cancers with NADPH, constitutively active KRAS has been observed
to uncouple ribose biogenesis from NADPH production in pancreatic cancer107. Glycolytic
intermediates are utilized specifically in the non-oxidative arm of the PPP, but not the oxidative
arm. Inhibition of the PPP by knockdown of G6PD, as well as glucose deprivation, in pancreatic
cancer cells led to minimal effect on ROS, indicating that the oxidative arm is not required to
maintain ROS levels in pancreatic cancer108. ROS could be affected, however, by modulating the
other two NADPH production pathways.
The serine synthesis pathway is another glycolytic shunt responsible for NADPH production. In
this pathway the glycolytic intermediate 3-phosphoglycerate is converted to serine via three
reactions. The first committed step is catalyzed by phosphoglycerate dehydrogenase (PHGDH).
After its production serine can be used in the folate cycle to generate glycine and NADPH109. The
transcription of the enzymes involved in this process is regulated by Nrf2104. Overexpression of
PHGDH has been reported in non-small cell lung, colorectal, breast, and pancreatic cancers104,110–
112. In pancreatic cancer, PHGDH has been described as an independent prognostic indicator for
patients; increased expression is associated with poor prognosis. Furthermore, PHGDH promotes
proliferation, migration, and invasion of pancreatic cancer cells in vitro112. Reduced levels of
serine hydroxymethyltransferase, the enzyme responsible for preparing serine for the folate cycle,
were responsible for decreasing NADPH while increasing ROS113. Therefore, Nrf2 promotes the
serine synthesis and folate pathway to produce NADPH, which allows the cancer cells to adapt to
oxidative stress and may contribute to worse outcomes in pancreatic cancer.
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Finally, glutamine metabolism through the malic enzyme (ME1) pathway is another source of
NADPH production that may be the most important in pancreatic cancer108. ME1 is positively
regulated by Nrf299. Cancer cells exhibit glutamine addiction because of its essential canonical
roles in supplying intermediates for the Kreb’s cycle through anaplerosis, and its roles in
nucleotide, amino acid, and lipid biosynthesis114–116. Most cells utilize the enzyme glutamate
dehydrogenase (GLUD1) to convert glutamine-derived glutamate into -ketoglutarate to fuel the
Krebs cycle, however constitutively active KRAS in pancreatic cancer downregulates GLUD1 and
upregulates aspartate transaminase (GOT1) to shunt glutamine into the non-canonical ME1
pathway for NADPH production108. In this pathway glutamine-derived aspartate is converted to
oxaloacetate via GOT1 in the cytoplasm, and then oxaloacetate is metabolized by malate
dehydrogenase (MDH1) into malate. The final step involves the oxidation of malate to pyruvate
by ME1. This reaction is responsible for NADPH regeneration and assists in protecting from
damage during oxidative stress117. Confirming this, glutamine deprivation and ME1 knockdown
led to elevated ROS and decreased clonogenicity in pancreatic cancer cells. The cells could be
rescued with supplemented oxaloacetate, malate, or GSH, indicating that glutamine is required for
redox homeostasis in pancreatic cancer108. Further demonstrating the importance of glutamine
metabolism in pancreatic cancer, ME1 pathway inhibition diminished tumour xenograft growth108.
1.2.4 The Impact of Low ROS Levels
The adaptations controlled by Nrf2 maintain ROS at low to moderate levels (ie. above background
levels but below cytotoxic levels) to prevent cell death. These low ROS levels are implicated in
stem cell phenotype and in tumour cell proliferation and act as important signaling molecules
capable of regulating various functions including proliferation, apoptosis, and tumour cell
invasion48. Low ROS levels are associated with maintaining cancer stem cell-like properties in
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cells that confers therapy resistance118. For example, human breast cancer stem cells possess robust
ROS scavenging systems that maintain low ROS levels84. This feature prevents ROS-induced
DNA damage following ionizing radiation, protecting the cells from death. Furthermore,
pharmacologic depletion of antioxidants results in loss of stem cell properties84.
Low ROS are involved in signal transduction pathways to regulate various cellular responses. This
function is made possible because of ROS-mediated inactivation of protein-tyrosine phosphatases,
which allows for increased signaling through receptor tyrosine kinases119. As such, ROS activate
signaling of the MAPK pathway by activating p38 MAPK to promote tumour cell differentiation
and proliferation120. In addition, ROS can inactivate PI3K/Akt phosphatases like PTEN. This
stimulates signaling of the PI3K pathway to promote proliferation and apoptosis evasion55,121.
Proliferation promotion by ROS has been observed in various cancer cell types122,123. These
functions implicate ROS in carcinogenesis. Moreover, ROS may contribute to the promotion of
invasion and metastasis. Tumour cell invasion and metastasis requires cell mobility through the
extracellular matrix (ECM). Cells degrade glycosaminoglycan in the ECM to allow for motility.
ROS-dependent degradation of the ECM occurs due to the stimulation of matrix metalloproteinase
activity, expression, and secretion124. ROS-activated MAPK pathways contribute to the expression
of matrix metalloproteinases. Specifically, extracellular signal-regulated kinase (ERK) activation
by ROS promotes invasion and metastasis125. The metastatic phenotype is enabled and sustained
by concomitant ROS production and the ability to protect from the cytotoxicity of oxidative stress
with antioxidants to maintain low ROS levels126. These functions of low ROS levels allow for
tumour growth and spread that may contribute to worse prognosis in pancreatic cancer.
Taken together this information indicates that low ROS levels contribute to many of the
tumorigenic properties of cancer cells. Cancer cells utilize antioxidants to protect themselves from
15
ROS and maintain these low ROS levels. These antioxidants are regulated by the master regulator
Nrf2 during the oxidative stress response. ROS scavenging prevents ROS-induced toxicity and
assists in ROS-mediated proliferation. Strategies designed to exacerbate ROS through targeted
inhibition of antioxidants may provide a relatively non-toxic therapy, as cancer cells are reliant on
antioxidants whereas normal cells can survive change. This strategy has been employed using
various drugs against a number of targets to great success75,127,128.
1.3 The Role of Antioxidants
The most important factor in the cell that moderates ROS levels are the antioxidants regulated by
Nrf2129. The role of antioxidants and Nrf2 in cancer is controversial as studies suggest them to be
either tumour suppressive or oncogenic. The answer to this controversy is likely to be that the role
of antioxidants is context specific91,130. Nrf2-knockout mice are more susceptible to carcinogenesis
and show diminished efficacy of chemopreventive enzymes131. Furthermore, the natural
compound sulforaphane activates Nrf2 and was found in a phase II clinical trial to protect against
hepatocellular carcinoma by preventing DNA adduct formation132. Although these studies suggest
that the antioxidant response mediated by Nrf2 is required for chemoprevention, most clinical trials
suggest that dietary antioxidants do not provide this effect133,134, and may even increase cancer
risk135,136.
Nrf2 may promote tumorigenesis by protecting cells from oxidative stress and maintaining low
ROS. Oncogenes such as KRAS in pancreatic cancer increase Nrf2 to provide cytoprotection and
decrease ROS levels47. Loss-of-function mutations in KEAP1, resulting in constitutive Nrf2
activation, have been identified in various solid tumours137. Lastly, high Nrf2 expression has been
correlated with resistance to platinum-based chemotherapy in ovarian cancer138.
16
These contrasting sets of data suggest that the role of Nrf2 may depend on the stage of
tumorigenesis. Before the cancer development, Nrf2 and antioxidants may help to suppress cancer
by the prevention of mutations and oxidative stress. Once the cancer develops, Nrf2 and the
antioxidants it regulates may promote cancer progression91,130. Therefore, once a tumour has been
developed, antioxidants may prove to be an important target for cancer therapy. As previously
discussed, the GSH and Trx systems are both coordinated by Nrf2. These are likely two of the
main factors contributing to tumour cytoprotection. Not only is the production of GSH and Trx
controlled by Nrf2, but also the production of the enzymes that recycle and utilize them to evoke
their effects.
1.3.1 The Glutathione Antioxidant System
GSH is the most abundant non-protein thiol in cells and is critical to providing protection from
oxidative stress139. GSH exists in two forms: reduced GSH, or oxidized glutathione (GSSG) which
is a dimer. A number of enzymes responsible for GSH homeostasis are regulated by Nrf2 during
periods of oxidative stress. GSH synthesis is performed in a two-step process from the amino acids
cysteine, glutamate, and glycine. The first reaction in GSH biosynthesis is the rate-limiting step
catalyzed by glutamate-cysteine ligase (GCL), which has both a catalytic (GCLC) and modifier
(GCLM) subunit140. GCLC performs the catalytic activity of the enzyme141, while GCLM is an
important regulatory subunit that lowers the Km for glutamate142. In this reaction glutamate and
cysteine are ligated to form -glutamylcysteine. This dimer contains a non-conventional peptide
bond between the -carboxyl group of glutamate and the amine of cysteine, rather than the -
carboxyl group. Hydrolysis of this bond can only be performed by -glutamyltranspeptidase
(GGT) which is found on the external surface of certain cell types and supplies cells with GSH
precursors143. As such GSH resists intracellular degradation. The second reaction to add glycine
17
to -glutamylcysteine is catalyzed by GSH synthetase (GS). Overexpression of GS fails to increase
GSH levels, whereas GCL overexpression leads to an increase in GSH144, supporting the notion
that GCL is more important in the regulation of GSH synthesis. Since -glutamylcysteine is found
at very low intracellular concentrations, GCL is considered to be rate-limiting140. Cystine uptake
through the system XC– transporter can also be a limiting step in GSH synthesis, as intracellular
cysteine concentrations are low145. Cysteine auto-oxidizes extracellularly to cystine, which can
then be taken up by the system XC– transporter before rapid intracellular reduction145. The
expression of both GCL subunits and the critical xCT subunit of the system XC– are regulated by
Nrf2146,147.
1.3.1.1 Functions of the Glutathione System
As a redox sensitive thiol the primary function of GSH is to scavenge ROS148. Supporting the
antioxidant role of GSH, depletion of GSH leads to increases in ROS and subsequent ROS-induced
toxicity149. GSH is particularly required in the mitochondria to protect from pathologically
generated oxidative stress150. This antioxidant function is performed in reduction reactions
catalyzed by glutathione peroxidase (GPx). GPx catalyzes the reduction of hydrogen peroxide and
lipid peroxides, which in turn oxidizes GSH to GSSG151. Members of the GPx enzyme family,
including GPx2 and GPx4, are regulated by Nrf294,152. GSH recycling is performed when cells
reduce GSSG using the enzyme glutathione reductase (GR) and NADPH to maintain a reducing
environment. This key enzyme for maintaining GSH homeostasis is regulated by Nrf2153. Since
the GSH to GSSG ratio is a significant contributor to the redox equilibrium, cells require
substantially more GSH than GSSG to maintain a reducing environment. During periods of
oxidative stress when GSSG accumulates, cells can export GSSG when the capacity of GR to
recycle GSH is outmatched154. Another mechanism to prevent toxic GSSG accumulation during
18
oxidative stress is the reaction of GSSG with a protein thiol catalyzed by protein disulfide
isomerase to form a protein mixed disulfide155.
GSH can contribute to redox signaling in addition to its antioxidant function. This role is executed
by modifying the oxidation state of critical modifying cysteine residues in proteins156.
Mechanistically this is performed by the reversible binding of GSH to protein thiols to alter the
protein structure and function. This process is called glutathionylation and can activate or
inactivate proteins157. Glutathionylation can happen enzymatically with the assistance of GSH S-
transferase (GST) or non-enzymatically158,159. This mechanism exists to protect sensitive proteins
from irreversible oxidation. Deglutathionylation is catalyzed by the oxidoreductase glutaredoxin
(Grx) which is itself reduced by GSH160. A significant number of proteins critical in cell signaling
have been identified to be regulated by glutathionylation. These include GAPDH, inhibitor of
nuclear factor kappa-B kinase subunit beta (IKK-), and p53161–164. This process may affect ROS
generation, as glutathionylation of electron transport chain proteins can alter their function165. GST
has an additional role in phase II detoxification of xenobiotics, including chemotherapeutic drugs.
In this process GSH is conjugated to xenobiotics during metabolism to prepare for excretion166.
GST-mediated detoxification can contribute to chemoresistance167.
GSH contributes to the regulation of growth in both normal and transformed cells. Increased GSH
levels are essential for cell cycle progression and are associated with proliferation168–170.
Hepatocytes increase GSH in response to injury, as has been observed during liver regeneration
following partial hepatectomy171. Blocking this GSH generation causes a reduction in the total
amount of DNA synthesis associated with cell division170. GSH levels in various cancer types
including hepatocellular carcinoma, melanoma, and pancreatic cancer have been correlated with
cell and tumour growth170,172,173. Interestingly, the redox environment of the nucleus has been
19
observed to be critical during the cell cycle. During proliferation GSH localizes to the nucleus and
can affect gene expression of multiple proteins through influence on histones174. Cell cycle
progression requires a reducing environment moderated by GSH to occur. In addition to cell
growth regulation, GSH is also involved in cell death regulation. This regulation is carried out
through effects on the mechanism of apoptosis. GSH levels impact the expression and activity of
caspases that are involved in effecting apoptosis175. As previously mentioned, GSH levels fall as
ROS propagate during the apoptotic cascade149. This can occur because of decreased GCL activity
following GCLC cleavage by caspase 3 during apoptosis176.
1.3.1.2 Targeting the Glutathione System in Cancer
GSH is critical for malignant behaviour of cancer cells. GSH is required during tumour initiation,
as its depletion impairs tumorigenesis127. This is because GSH prevents oxidative stress from
enacting its cytotoxic effects on transformed cells. Following tumour formation GSH becomes
important to maintain high growth rates, as GSH levels are correlated to growth177. These levels
are elevated in cancer due to increased expression of GCL and the system XC–178,179. In addition,
GGT is an early marker of neoplastic transformation and assists with the GSH cycle to supply
cancer cells with GSH metabolic precursors180. GGT overexpression has been observed in
melanoma and hepatocellular carcinoma181,182, among other cancers. Lastly, GSH is required for
establishing distant metastases because metastatic cells experience higher oxidative stress. In
response they elevate GSH levels to combat the microenvironment and maintain redox
homeostasis126. To highlight the importance of GSH in cancer, many studies have attempted
pharmacologic GSH depletion and observed sensitization to various therapies. Cancer cells are
sensitized to radiation upon GSH depletion84. In addition, chemoresistance has been associated
with high GSH levels in multiple myeloma and ovarian cancer165,166. This chemoresistance can be
eliminated following depletion of GSH184
20
As GSH is critical to cancer progression and survival there is reason to believe that it can be
exploited in cancer therapy. Various key nodes in GSH metabolism can be targeted as an anticancer
strategy. First, substrate supply for GSH biosynthesis can be targeted. Glutamine analogs targeting
GGT have been evaluated in humans but have been found too toxic for clinical use185. Newer
uncompetitive inhibitors are in development but have yet to prove effective and will require time
before clinical application186. The system XC– transporter may be a critical target of an anti-GSH
cancer therapy as it is required to regulate intracellular cysteine and has been observed to be
necessary for cancer cell survival187. Multiple inhibitors against this system have been described.
Sulfasalazine is a drug that has been used in clinics for rheumatoid arthritis, ulcerative colitis, and
Crohn’s disease, and inhibits the system XC–188. However, the mechanism of action is poorly
understood, it is metabolically unstable, and lacks potency73. Erastin is a newer molecule that is
more potent than sulfasalazine and more selective for system XC–74. However, it has yet to be tested
in humans.
A second potential target critical to GSH metabolism is GSH recycling. The nitrogen mustard
compound carmustine (BCNU) is primarily an alkylating agent used in chemotherapy for brain
cancer and lymphomas189,190, however its off-target effect is GR inhibition191. Unfortunately,
BCNU is in short supply, costly, and displays prolonged myelosuppression and potentially lethal
pulmonary toxicity190,192,193. A newer molecule 2-AAPA has been described as a GR inhibitor that
increases GSSG and stimulates protein glutathionylation194. However, 2-AAPA has yet to be tested
in humans and possesses off-target effects including Grx and thioredoxin reductase (TrxR)
inhibition195,196. These off-target effects and potentially others that have yet to be uncovered make
2-AAPA too unpredictable for use in cancer therapy.
21
Lastly, a third strategy involves direct targeting of GSH synthesis. GCL, the rate-limiting enzyme,
is more important for GSH levels than GS, the enzyme catalyzing the second step of GSH
synthesis144. Therefore, GCL would be the ideal target. Fortunately, an irreversible inhibitor of
GCL, buthionine sulfoximine (BSO), has been studied extensively in vitro, in vivo, and in human
clinical trials127,197,198. BSO has proven to be minimally toxic in humans when used on its own199.
Although it is rapidly metabolized following intravenous bolus, concentrations of ~500M
following continuous infusion are achievable in humans while maintaining safe toxicity profiles200.
BSO has garnered great interest for use in drug combinations against cancers that have been
observed to require GSH for chemoresistance197. Mechanistically, it contributes to oxidative stress
in the cells by acute GSH depletion and long-term ROS exacerbation149. This can lead to both
apoptosis and ferroptosis149,201. BSO’s safety profile, specificity, low cost, previous clinical trials,
and mechanistic understanding make it an excellent drug to develop further for cancer therapy that
modulates oxidative stress.
BSO on its own is not very cytotoxic with short-term treatments and many cell lines are resistant
to acute GSH depletion197. It is likely that therapy involving BSO would require a second agent to
enhance toxicity. Adaptation to GSH depletion involves Nrf2 activation to upregulate other
antioxidants, including Trx202. Since antioxidants have similar, sometimes overlapping functions,
compensation from alternative antioxidant systems is a plausible mechanism for resistance to GSH
depletion. Compensation between antioxidants has been observed in other contexts including
between GSH and Trx, and between Trx and heme oxygenase127,203,204. Therefore, therapy tailored
to inhibit more than one antioxidant system may be able to overcome compensation and produce
strong anti-cancer effects.
22
1.3.2 The Thioredoxin Antioxidant System
The Trx system is a reductase system that is required for DNA synthesis and protection from
oxidative stress203. The three major components of the system are Trx, TrxR, and NADPH. As
with other antioxidant genes, Trx and TrxR are transcriptionally regulated during oxidative stress
by Nrf299,205. Trx is a small (~12 kDa) reductase that is the effector of the Trx system. It catalyzes
the reduction of protein disulfides. The active site dithiol in reduced Trx interacts with various
protein substrates via a thiol-disulfide exchange reaction that forms a disulfide in Trx (oxidized
Trx). This oxidized form is reduced by electron transfer from NADPH via the reaction catalyzed
by TrxR. There is a mitochondrial Trx2 with two active site cysteines, and cytosolic and nuclear
Trx1 with 3 additional cysteines that contribute to enzymatic regulation206. TrxR is a homodimeric
oxidoreductase that recycles Trx, and belongs to the same protein family as GR207. Mammalian
cells contain three TrxRs: cytosolic TrxR1, mitochondrial TrxR2, and testis-specific TrxR3208. Of
these, TrxR1 has been identified as a fitness gene209. Although structurally and functionally
similar, the TrxR enzymes have different amino acid sequences210. These enzymes contain a
unique C-terminal selenocysteine residue that contributes to its reaction mechanism.
In the mechanism of TrxR reduction electrons from NADPH are first transferred to enzyme-bound
FAD, then FAD transfers reducing equivalents to the N-terminal disulfide in the redox centre of
the same subunit. This reduces the disulfide to a dithiol, which then transfers the electrons to the
C-terminal selenenylsulfide of the other subunit. Addition of another NADPH molecule reduces
these residues to a selenol-thiol pair211. The reduced enzyme can transfer these electrons to its
substrates, including proteins such as oxidized Trx and Grx2, and small molecules such as
hydrogen peroxide and cytochrome c212. When transferring electrons to Trx the selenol of TrxR is
deprotonated to a selenolate anion. This anion attacks the disulfide of oxidized Trx, resulting in a
23
TrxR-Trx mixed selenenylsulfide. The free thiol on the C-terminal TrxR reduces this bond, causing
the reformation of the selenenylsulfide in TrxR. This allows the now reduced Trx to leave with
reduced dithiols in its active site211. This mechanism is made possible because of the
selenocysteine residue. Selenocysteine is a cysteine analog that replaces sulfur with selenium. This
maintains many chemical properties of the amino acid, however the pKa of the selenol group in
selenocysteine is significantly lower than the thiol group in cysteine (cysteine pKa = ~5.8 vs.
selenocysteine pKa = ~8.3). This property allows the selenol to exist primarily as the ionized
selenolate at physiological conditions, whereas the thiol exists in the protonated form213. The
ionized molecule is more reactive than the protonated version. In fact, replacement of
selenocysteine with cysteine dramatically decreases activity of TrxR1214.
1.3.2.1 Functions of the Thioredoxin System
As one of the major thiol-dependent electron donor systems in the cell, the Trx system carries out
multiple important functions. The Trx system can perform mechanisms of both direct and indirect
ROS scavenging. First, Trx can directly neutralize ROS. This action involves quenching hydroxyl
radical by providing electrons, and requires Trx’s catalytic cysteines, as shown by loss of ROS
scavenging following mutation of these cysteines to alanine215. Second, TrxR can indirectly
scavenge ROS by providing electrons to other endogenous antioxidants, including ubiquinone.
The reduced form of ubiquinone, ubiquinol, can act as a ROS scavenger by reducing radicals to
protect from lipid peroxidation216. The reduction of ubiquinone is catalyzed by TrxR under
physiologic conditions, and may contribute to protection from oxidative stress217.
A critical function of the Trx system involves the regulation of protein disulfide formation. During
oxidative stress ROS enact damage to cellular macromolecules. As proteins are the most highly
concentrated macromolecule in cells, they are likely the most significant target of ROS218. Not all
24
proteins are equally susceptible to oxidative damage, as subcellular localization and content of
oxidation-sensitive amino acids may contribute219. The most oxidation-sensitive amino acid
residue is cysteine, that readily forms disulfide bridges with other cysteine residues upon
oxidation220. These aberrant disulfides can cause structural changes that impact protein function.
Trx maintains protein redox status via its reductase activity which can reduce disulfides in an
extensive number of target proteins to dithiols221. Trx targets regulate a vast array of cellular
processes including proliferation, metabolism, and apoptosis. In general, the role of Trx is to
provide a reducing environment to protect from oxidative stress to prevent cell death and promote
cell growth. The most important targets of Trx are reductive enzymes including Prx, ribonucleotide
reductase, and methionine sulfoxide reductase. This highlights the importance of Trx in the
maintenance of other reductive pathways222.
Prxs are a peroxidase family that act primarily as cellular antioxidants. Three subgroups of Prx
exist: 2-cysteine Prx isoforms (Prx1-4), atypical 2-cysteine Prx isoform (Prx5), and 1-cysteine Prx
isoform (Prx6)223. Reduced Trx is the electron donor for Prx1-5 which can then remove ROS such
as hydrogen peroxide, lipid hydroperoxides, and peroxynitrite. Hyperoxidation of Prx occurs from
ROS overexposure. The catalytic cysteine residues oxidize to form disulfide bonds, which must
be reversed by Trx224. This establishes a redox cascade where the flow of electrons travels from
NADPH through the Trx system to Prx and finally to neutralize toxic ROS225. The speed of
hydrogen peroxide scavenging by Prx is comparable to other significant ROS scavenging enzymes
like glutathione peroxidase, on the magnitude of 107 – 108 M-1s-1, indicating that it is a major
contributor to peroxide scavenging in cells226,227. Prxs can inhibit cancer development or promote
tumorigenesis depending on the specific Prx member and cancer context228. However, high levels
of Prxs are often associated with radioresistance and chemoresistance. For example, high Prx2
levels correlates to radioresistance in breast cancer cells, as well as with cisplatin resistance in
25
gastric cancer cells229,230. Silencing of Prx2 sensitizes colorectal cancer cells to both radiation and
oxaliplatin treatment231. These data indicate that Trx may have an indirect role in promoting
therapy resistance through Prx.
Another significant reductase target of Trx is ribonucleotide reductase (RNR). RNR reduces all
four ribonucleotides to deoxyribonucleotides for use in DNA synthesis. During ribonucleotide
reduction the two active site cysteines of RNR form a double bond which must be reduced by a C-
terminal dithiol through a thiol-disulfide exchange reaction232. The original conformation of the
C-terminal cysteines is restored by an external reductase system, either Trx or Grx which both
have similar catalytic efficiency with mammalian RNR233–235. This redundancy allows for cells to
withstand the loss of one reductase system. This is supported by studies showing that TrxR
downregulation causes no change in deoxyribonucleotide triphosphate pools in mouse cancer
cells236. Some differences may exist as Trx is believed to support S phase electron donation,
whereas Grx is believed to support DNA repair and mitochondrial DNA synthesis235. Interestingly,
Grx is an important effector of the GSH system, supporting compensatory mechanisms between
Trx and GSH.
Methionine is another oxidation-sensitive amino acid. However, protein reduction by Trx is
limited to disulfides. Methionine sulfoxide reductase (Msr) is capable of reducing oxidized
methionine in proteins and acquires its reducing potential from Trx237. Although there are multiple
Msr isoforms with different numbers of catalytic cysteines and mechanisms, they all require Trx
to maintain their reductive capacity222. This is yet another pathway by which Trx is required to
protect cells from damage caused by oxidative stress.
Beyond reductase systems, Trx is responsible for post-translational modifications to regulate
numerous signaling cascades and transcription factors. Apoptosis signaling kinase 1 (ASK1) is
26
part of the MAPKKK family that activates the JNK and p38 MAP kinase pathway238. Reduced
Trx can bind ASK1 to inhibit its activation, directing it to ubiquitin-mediated degradation, which
prevents ASK1 from activating proapoptotic gene expression239. Inactivation of PTEN, a negative
regulator of the PI3K-Akt pathway, results in the stimulation of proliferation. Trx1 binds PTEN
and inhibits its activity, which may stimulate cancer cell proliferation240. These data indicate a
potential role for Trx in cancer proliferation and highlight the importance of Trx inhibition in
cancer therapy.
1.3.2.2 Targeting the Thioredoxin System in Cancer
A novel strategy for pancreatic cancer therapy may involve Trx system inhibition, as the Trx
system is involved in protection from oxidative stress that may promote cancer. Excluding
downstream targets of Trx, three major targets can be identified within the Trx system: NADPH,
Trx, and TrxR. Since NADPH is ubiquitous and required in a vast array of major biosynthetic
pathways, complete inhibition of NADPH production would not be feasible. The individual
pathways of NADPH production could potentially be targeted. The molecule dichloroacetate
inhibits the oxidative phase of the PPP and has been clinically tested241,242. However, this likely
may not be effective in a KRAS driven tumour like pancreatic cancer since the oxidative phase of
the PPP is less involved in NADPH production than other pathways108. When targeting the serine
biosynthesis pathway PHGDH knockdown in overexpressing cancers inhibits proliferation111.
However, inhibitors of PHGDH, including PKUMDL-WQ-2101 and PKUMDL-WQ-2201, have
only recently been identified and require further development before use in humans243. Targeting
glutamine metabolism through inhibition of ME1 has shown to be promising by inducing
senescence and apoptosis in vitro244. However, an inhibitor of ME1 has yet to be characterized.
An inhibitor of the mitochondrial isoform ME2, NPD389, has been described but not developed245.
27
As the effector of most functions of the Trx system, Trx would seem to be an ideal target. The
synthetic molecule PX-12 is the most developed Trx inhibitor that covalently binds to Trx cysteine
residues, forming mixed disulfides. PX-12 inhibits breast cancer cell proliferation in vitro and
reduced tumour volume in vivo. Interestingly, PX-12 can be a substrate for TrxR causing
competitive inhibition of TrxR for its normal substrate, oxidized Trx246. PX-12 has been tested in
clinical trials, and was shown to decrease plasma Trx in a phase I trial247. However, a phase II trial
of PX-12 in patients with advanced pancreatic cancer refractory to gemcitabine therapy showed
no antitumour activity248. These results may stem from the inability to achieve adequate plasma
concentrations of PX-12 without significant toxicities because of rapid binding to plasma
components. Combined results of clinical trials suggest that development of PX-12 for intravenous
infusion is not feasible249.
The third and last potential target, TrxR, may contain the most potential for anticancer therapy.
High TrxR1 expression correlates to poor prognosis in hepatocellular carcinoma, breast, and
pancreatic cancer, among others250,251. TrxR1 inhibition following siRNA knockdown
dramatically reduced tumour progression in xenograft models of lung cancer252. In contrast,
normal adult tissues can survive TrxR1 loss253. Recent development has centered around attempts
to develop specific inhibitors of TrxR1 with some success254. However, these molecules require
extensive development and testing before potential clinical use. The pan-TrxR inhibitor auranofin
is an FDA approved drug for the treatment of rheumatoid arthritis that has recently garnered
interest for repurposing in cancer therapy255. Auranofin is an orally available gold complex that
irreversibly inhibits all TrxR isoforms, and is inexpensive, well-characterized, and has relatively
minor toxicities256. Although it has been reported to inhibit proteasome deubiquitinases in addition
to TrxR257, evidence to support this is sparse and seems to be secondary to cell death. Auranofin
is capable of inducing apoptotic cell death following the proliferation of ROS258,259. However, it
28
appears to work best in combination with other drugs after being testing as a combination therapy
against various cancers260–262. In addition, auranofin has been used in phase I and II clinical trials
for chronic lymphocytic leukemia therapy (see www.clinicaltrials.gov NCT01419691). Therefore,
auranofin is likely the most ideal candidate to develop for pancreatic cancer therapy that targets
the Trx system to augment oxidative stress.
1.4 A New Therapeutic Strategy for Pancreatic Cancer
The hallmarks of cancer describe essential alterations involved in tumour development. They
include the sustainment of proliferative signaling, evasion of growth suppressors, activation of
invasion and metastasis, enabling replicative immortality, induction of angiogenesis, resisting cell
death, deregulation of cellular energetics, and avoidance of immune destruction263. Redox
signaling and the responses to oxidative stress are involved in every hallmark of cancer264.
Therefore, consideration for cancer promotion by antioxidants should be made when developing
novel anti-cancer therapies. Normal tissues are resistant to antioxidant loss, which may prevent
harmful toxicities of antioxidant inhibition.
As previously discussed, BSO and auranofin appear to be useful drug candidates for combined
targeting of the GSH and Trx systems. Evidence suggests that these systems overlap to compensate
and protect against inefficiencies or inhibition of either system. The Trx system can be an
alternative route for cells to reduce oxidized GSH in vivo265. In addition, physiologic
concentrations of GSH and Grx are capable of reducing oxidized Trx in vitro, suggesting GSH as
a backup to TrxR function266. However, this observation was made in a cell-free system which
may limit its applicability in the complex cell environment.
29
Combination therapy using both BSO and auranofin can help to overcome compensation. Ideally
the combination of the two drugs will produce improved effectiveness compared to monotherapy
with either compound. This result can either be stated as synergy or potentiation. Synergy is
observed when the effect of two or more drugs is greater than the sum of effects when the drugs
are administered as monotherapies. The effect is mutual in that each drug enhances the effect of
the other267. In contrast, a scenario by which the enhancement of effect is one-sided can be deemed
a potentiation or augmentation. Evaluation of a potentiation can be stated as a fold change in the
effect of one drug, caused by the effect of the other drug267.
Previous studies have shown that dual-inhibition of the GSH and Trx systems can lead to greater
anti-cancer responses127. Use of BSO and auranofin in combination sensitized mouse tumours to
radiotherapy83, and mesothelioma cells show extensive oxidative stress and cell death following
combination of the drugs268. These studies do not evaluate potential synergism of the combination
of BSO and auranofin. Nor do any studies evaluate this combination in pancreatic cancer, which
seems to be an exceptional target of pro-oxidant therapy.
Considering that the Trx and GSH systems comprise the main antioxidant systems in the cell, I
hypothesize that combination therapy using auranofin and BSO will cause primary pancreatic
cancer cells to undergo apoptosis by exacerbating oxidative stress. Auranofin will be identifiable
in pancreatic PDX tumours using IMC. I have three aims to test this hypothesis:
Aim 1: Evaluate the effect of combination therapy on cell viability and death.
Aim 2: Evaluate the effect of combination therapy on the redox environment of the cell.
Aim 3: Evaluate the drug-target interaction of auranofin-TrxR in vitro and biodistribution of
auranofin in vivo.
30
Chapter 2
Targeting the Thioredoxin and Glutathione Antioxidant Systems to Treat Pancreatic Cancer
Francesco Fazzari1, Samir Barghout2, Qing Chang3, David Hedley1,2,4
1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada 2Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada 3Fluidigm Canada Inc, Markham, Ontario, Canada 4Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, Ontario, Canada
The work presented in this chapter is presented as a manuscript for future submission.
31
2.1 Introduction
Pancreatic cancer is the 4th leading cause of cancer death in Canada, however it is only the 12th
most commonly diagnosed2. There is poor prognosis with an overall 5-year survival rate of 5%4.
Significant advancements in the development of new treatment options for the disease have not
been made. Many patients with advanced disease are given standard chemotherapy drugs that
provide little benefit, leading to an urgent need for new strategies to combat this disease.
Pancreatic cancer, among other cancers, is characterized by excessive oxidative stress48, which
occurs when production of intracellular reactive oxygen species (ROS) outpaces the capacity of
antioxidants to protect from ROS. These ROS are produced in mitochondria at an extreme rate in
transformed cells due to inherently high metabolism, and may promote aggressive tumour
features50. Adaptations to protect from ROS are required for cancer cells to survive. These
adaptations primarily involve oxidative stress signaling through Nrf2, which mediates the
oxidative stress response to promote the production of the antioxidants glutathione (GSH) and
thioredoxin (Trx)54. The main oncogenic driver of pancreatic cancer, KRAS, induces Nrf2
activation to maintain antioxidant defenses47. In conjunction to stimulating antioxidant production,
pancreatic cancer relies on glutamine metabolism for the production of NADPH108. This adaptation
occurs because of the need to protect against high ROS during oxidative stress, as NADPH
provides reducing power to antioxidant systems in the cell to prevent cytotoxic damage caused by
high ROS levels.
ROS exhibit dual roles; lower concentrations of ROS that are promoted by antioxidants can
stimulate cancer cell proliferation, whereas higher concentrations of ROS are damaging to
cells49,52. Therefore, antioxidants may contribute to aggressive growth and metastasis in cancer
because of their functions in ROS scavenging, preventing ROS-mediated cellular damage, and
32
promoting ROS-mediated proliferative signalling148,269. The reliance of pancreatic cancer on
antioxidants to protect from oxidative stress may provide a unique vulnerability in the tumour.
Targeting antioxidants may promote cytotoxic damage specifically in tumours by augmenting
oxidative stress.
Both the GSH and Trx systems have been extensively studied and tested inhibitors exist. GSH is
the most abundant antioxidant cofactor in cells and is responsible for ROS scavenging148. The rate-
limiting step of GSH synthesis is catalyzed by glutamate-cysteine ligase (GCL)148. An inhibitor of
GCL, buthionine sulfoximine (BSO), has been well characterized and previously tested in human
subjects with minimal toxicity197,198. Trx reduces aberrant protein disulfides caused by oxidative
stress269. Oxidized Trx is recycled by thioredoxin reductase (TrxR) using NADPH to replenish its
reducing power256. Three TrxR isozymes exist: cytosolic TrxR1 that has been identified as a fitness
gene209, mitochondrial TrxR2, and testes-specific TrxR3. The gold complex auranofin is an
inhibitor of all three TrxR proteins and has previously been used in the treatment of rheumatoid
arthritis255. It has garnered interest for repurposing as a therapy in various cancers260,261,270.
Many recent studies have identified oxidative stress as an effective target in cancer therapy,
including in KRAS driven tumours75,128,271–273. Targeting one antioxidant pathway may allow for
compensation from another to continue providing protection to the cell204. This may possibly be
overcome by a double hit. To our knowledge no studies have attempted a double hit in pancreatic
cancer. Considering that Trx and GSH comprise the main antioxidant systems in the cell,
combination therapy using auranofin and BSO may cause cell death by depleting antioxidant
capacity and increasing ROS in primary pancreatic cancer cells.
33
2.2 Materials & Methods
Cell Culture and Reagents
Primary cell lines (9A and OCIP23) were derived from human pancreatic cancer tumours. 9A was
maintained in Roswell Park Memorial Institute (RPMI) 1640 (Wisent) medium supplemented with
10% fetal bovine serum (FBS) (VWR Seradigm Life Science) and 1x penicillin/streptomycin
(Wisent). OCIP23 was maintained in Dulbecco’s Modified Eagle Medium (DMEM)/F-12
(Wisent) supplemented with 2.5% FBS and 1x penicillin/streptomycin. Cells were cultured at 37C
in a humidified incubator with 5% CO2.
Auranofin (Sigma-Aldrich) was diluted in DMSO (Sigma-Aldrich) at 100mM, aliquoted and
stored at -10C to be used once without refreezing. BSO (Sigma-Aldrich) was prepared fresh in
sterilized MilliQ water at 100mM.
MTS Assay
Cell viability was assayed by MTS assay and performed according to the manufacturer’s protocol
(Promega). In summary, 96 well plates were seeded with 5000 cells/100L/well and incubated for
24 hours. Medium was replaced with medium containing DMSO (Sigma-Aldrich) [0.003% for
OCIP23, 0.004% for 9A], auranofin, BSO, or combination of auranofin and BSO at the indicated
concentrations. Each treatment was performed in triplicate. Cells were incubated for 24 hours, then
cell viability was determined by adding 20L of MTS reagent followed by incubation for 3 hours
at 37C and absorbance reading at 492nm using the Multiskan EX microplate spectrophotometer
(Thermo Fisher).
Clonogenic Assay
34
Clonogenic assays evaluated drug treatment effects on colony formation and was performed as
previously described274. Briefly, cells were grown to 70-80% confluence in 10cm plates then
treated with vehicle (1L/mL DMSO), auranofin, BSO, or a combination at the indicated
concentrations for 24 hours. Cells were harvested and reseeded in 6-well plates at 105, 104, 103,
and 102 cells/well in triplicate, then allowed to incubate for 12 days before fixing and staining with
0.2% methylene blue in 50% ethanol/water. Colonies were counted using the Gel Count (Oxford
Optronix) counter. Plating efficiency was calculated as the average number of colonies divided by
the number of cells plated. Surviving fraction was expressed as the plating efficiency of the sample
divided by the plating efficiency on vehicle control plates.
Multiparametric Flow Cytometry
Apoptosis, ROS levels, and GSH levels were assayed by flow cytometry. 7x105 cells/plate were
seeded in 6cm plate. After 24 (OCIP23) or 48 hours (9A), media was replaced with either normal
medium or BSO-supplemented medium. Auranofin was diluted in DMSO to 1000x the indicated
concentrations and added at 1L/mL. After 24 hour treatment the cells were trypsinized and
resuspended in medium. Cells were centrifuged at 1100rpm for 5 minutes, then the pellets were
resuspended in 0.5mL serum free media. Cell suspensions were then incubated in a water bath at
37C with 40nM DiIC1(5) (ThermoFisher Scientific) and 5M 2’,7’-dichlorofluorescein (DCF)
(ThermoFisher Scientific) for 30 minutes. 5 minutes before the end of the incubation, 40M
monobromobimane (mBBr) (ThermoFisher Scientific) and 1g/mL propidium iodide (PI)
(ThermoFisher Scientific) were added. Following incubation, samples were cooled on ice for 10
minutes then analyzed with the CytoFLEX flow cytometer (Beckman Coulter). During the time
course experiment samples were prepared in the same manner, however the drug treatments were
35
for 2 and 5 hours, and mBBr was excluded from the panel of fluorescent probes. Data analysis was
performed using FlowJo Software (BD).
Tumour sample preparation
Animal experiments were carried out according to protocols approved by the University Health
Network Animal Care Committee. Patient-derived xenograft (PDX) mice were established
subcutaneously in 5 week old severe combined immunodeficiency (SCID) mice. Two models,
OCIP83 and 9A were used. Mice were given an intraperitoneal injection of 10mg/kg auranofin for
a 24 hour treatment. Mice were later given 120mg/kg pimonidazole (Hypoxyprobe, Inc.), followed
by 30mg/kg EF5 (obtained from Dr. Cameron J. Koch, University of Pennsylvania) 5 and 3 hours
prior to sacrifice, respectively. Tumours were excised, fixed, and processed for paraffin
embedding.
Immunohistochemistry (IHC)
Formalin-fixed, paraffin-embedded (FFPE) sections of subcutaneous PDX tumours were
incubated with mouse pimonidazole antibody (Hypoxyprobe, Inc.) at 1:400, overnight at 4C.
Biotinylated horse anti-mouse IgG was added at 1:200 concentration and incubated for 1 hour.
Detection was performed using the HRP labeling (Vector Labs) and DAB chromogen (Dako).
Imaging Mass Cytometry (IMC) Staining, Data Acquisition, and Visualization
Imaging mass cytometry was used to directly image the distribution of atomic gold in tissue
sections following treatment with auranofin. Tissue staining for IMC was performed as previously
described275. In summary, FFPE sections were dewaxed and rehydrated as routine. Antigen
retrieval and blocking with BSA was performed, followed by incubation with primary antibody
36
against pimonidazole for 1 hour at room temperature (RT). Samples were then incubated overnight
at 4C with a metal-conjugated antibody cocktail diluted in PBS/0.5% BSA (Table 1). Following
this, samples were washed and exposed to 25M Ir-Intercalator (Fluidigm) for 30 minutes at RT.
Lastly, the samples were rinsed then dried before IMC ablation.
Regions of interest were determined by assessing IHC samples stained for pimonidazole. During
data acquisition, the IMC immunostained samples are inserted into a laser ablation chamber where
the tissue is ablated in a 1m diameter spot. The ablation spot is vaporized, and the plume is carried
with high time-fidelity into the inductively coupled plasma ion source for simultaneous analysis
by the mass cytometer (Fluidigm). The metal isotopes located on each spot are simultaneously
measured. This process continues for the entire area, eventually yielding an intensity map of the
targets throughout the tissue region of interest.
Data-processing and image visualization was performed using the in-house-developed MCD
Viewer (Fluidigm). The signal data from the mass cytometer was exported in text format, then
reconstructed into images. The epithelial cells were defined by E-cadherin and pan-keratin
staining, and the stromal cells were defined by collagen and SMA. Areas within these regions
were selected in the MCD viewer to semi-quantitatively determine the average number of gold
ions within them
Cell Lysis and Protein Determination
Lysis was performed using a lab-made lysis buffer containing 50mM Hepes (pH 8.00), 10%
glycerol, 1% Triton-X, 150mM NaCl, 1mM EDTA, 1.5mM MgCl2, 100mM NaF, 10mM
Na4P2O710H2O, 1mM Na3VO4, and 1 tablet/7mL buffer cOmplete protease inhibitor cocktail
(Sigma-Aldrich). Cell cultures were incubated with 700L lysis buffer for 1 hour at 4C, then
37
collected and centrifuged at 14,000rpm for 15 minutes at 4C. Supernatants were collected and
stored at -80C until use. Protein determination was performed using the BCA Assay kit
(ThermoFisher Scientific) as per the manufacturer’s protocol.
Gel Electrophoresis and Immunoblot
Samples were resolved using 10% SDS-PAGE. Following transfer to polyvinylidene difluoride
membranes, proteins were detected with antibodies to TrxR1 1:1000 (Cell Signaling Technology),
TrxR2 1:1000 (Cell Signaling Technology), -actin 1:7000 (Abcam), GAPDH 1:4000 (Cell
Signaling Technology). Horseradish peroxidase (HRP) conjugated secondary sheep anti-mouse
(Abcam) and donkey anti-rabbit (Abcam) were used for detection at 1:5000. Quantification was
performed by densitometry using ImageJ (NIH).
Cellular Thermal Shift Assay
Cellular Thermal Shift Assay (CETSA) was performed as previously described276 to assess
auranofin-TrxR engagement. Briefly, to determine the optimal temperature at which maximal
thermal shifts were observed, cells were treated with vehicle or 10M auranofin for 1 h, then
divided into equal portions, centrifuged, and resuspended in PBS with protease inhibitors. Cells
were then heated at temperatures ranging from 46-70C for 3 min in a thermal cycler (SimpliAmp,
Applied Biosystems), cooled at RT for 3 min, snap-frozen in liquid nitrogen, thawed at RT, and
then lysed by 3 freeze/thaw cycles with vortexing in between. Samples were then centrifuged at
20,000 x g for 20 min at 4C and supernatants were collected. Lysates were analyzed by
immunoblotting. When evaluating changes with different auranofin concentrations the same
procedure was followed, however cells were treated with increasing auranofin concentrations at
the determined optimal temperature of 56C.
38
Statistical Analysis
Data are presented as the mean value S.E.M. GraphPad Prism version 7.0 (GraphPad Software,
Inc., San Diego, CA, USA) was used for statistical analysis. A value of P<0.05 was considered to
indicate statistically significant differences.
2.3 Results
Auranofin, but not BSO, inhibits cell growth and viability of primary pancreatic cancer cell
lines.
To evaluate the effect of Auranofin and BSO on cell viability, human primary pancreatic cancer
cells were treated with different concentrations of either drug for 24 hours. MTS assay measured
cell metabolic activity. BSO alone displayed no significant effect on the viability of the primary
cells (Fig. 2.1A). In contrast, auranofin reduced viability in a concentration dependent manner with
IC50 values 1.40μM and 2.35μM in OCIP23 and 9A, respectively (Table 2.1; Fig. 2.1B). These
data suggest that after 24 hours, these two primary pancreatic cancer cell lines are susceptible to
auranofin, but not BSO. Since BSO does not reduce viability at this time point, any interaction
between the two drugs cannot be deemed synergy.
BSO potentiates auranofin’s inhibitory effect on cell viability of primary pancreatic cancer cells.
Using the MTS assay, we evaluated cell viability following treatment of cells with a combination
of auranofin and BSO. Co-treatment with 10μM BSO caused a 61.1-fold decrease in the IC50 of
auranofin in OCIP23 (IC50 = 22.9nM), and a 4.07-fold decrease in 9A (IC50 = 578nM) (Fig.
2.1B). The addition of 50μM BSO caused a 238-fold decrease in the IC50 of auranofin in OCIP23
(IC50 = 5.88nM), and a 11.0-fold decrease in the IC50 in 9A (IC50 = 213nM) (Fig. 2.1B). These
39
data support a strong BSO-mediated potentiation of auranofin cytotoxicity. The higher IC50 and
lower potentiation observed with 9A cells suggest they possess a higher reserve capacity of
TrxR1/2 activity that makes them more resistant to drug-induced oxidative stress. To test this
hypothesis, we assessed expression levels of Trx1/2 in 9A and OCIP23 cells. 9A cells showed a
significantly higher expression level of TrxR1, but not TrxR2, compared to OCIP23 cells
(p=0.0007) (Fig. 2.1C and D). This may explain, at least in part, the differential response of
OCIP23 and 9A to auranofin and combination with BSO.
Combination of BSO and auranofin inhibits clonogenicity of primary pancreatic cancer cells.
We then conducted clonogenic assay to determine whether the BSO-auranofin combination
inhibited long-term viability and if low auranofin concentrations on their own improved viability
as observed by MTS assay. There was a significant decrease in clonogenicity following
combination treatment in 9A (p=0.0155) with 43.1% surviving fraction (Fig. 2.1E) and OCIP23
(p=0.0088) with 43.8% surviving fraction (Fig. 2.1F) when compared to vehicle controls. Single
treatment with auranofin (100nM or 400nM) showed no increase in clonogenicity, which contrasts
the improved viability of low auranofin concentrations observed via MTS assay (Fig. 2.1B). The
results of these two assays may differ because of the different endpoints measured, metabolism
versus colony growth. These data support the potentiation of auranofin by BSO at low auranofin
concentrations that otherwise have no effect on cell death and viability when used as monotherapy.
Table 2.1. IC50 values of auranofin monotherapy and in combination with BSO in primary
pancreatic cancer cell lines.
Cell Line BSO Concentration (M) Auranofin IC50 (nM)
OCIP23 - 1400
OCIP23 10 22.9
OCIP23 50 5.88
9A - 2350
9A 10 578
9A 50 213
40
Figure 2.1. Dose-response of antioxidant pathway inhibitors, TrxR isozyme levels, and
colony formation potential. (A and B) Viability of primary pancreatic cancer cells was
determined after 24 hour treatment using an MTS assay. Shown are the toxicity profiles of A) BSO
monotherapy (n=3), and B) auranofin monotherapy or combination (n=3). (C and D) Relative
amounts of C) TrxR1 and TrxR2 in untreated cell lines (n=3) and their corresponding D)
immunoblots. Levels were normalized to the first OCIP23 sample. (E and F) Colony formation of
primary cell lines was determined after 24 hour treatment. Cells were reseeded and incubated for
12 days before colonies were assesed for E) 9A (n=3) (Combination of Au 400nM and BSO 10M)
and F) OCIP23 (n=3) (Combination of Au 100nM and BSO 10M). Combinations were compared
to vehicle controls. Surviving fraction is represented as a percentage of vehicle controls. Shown
are the S.E.M. Significant decrease from vehicle control, * = p<0.05, ** = p<0.01, *** = p<0.001.
0 20 40 60 80 100 1200
20
40
60
80
100
120
BSO concentration (uM)
% C
ell V
iabil
ity
BSO Dose-Response
OCIP239A
9A - 10uM BSO9A - 0 BSO
9A - 50uM BSO
OCIP23 - 50uM BSOOCIP23 - 10uM BSO
10-3 10-2 10-1 100 101 102 103 104 1050
20
40
60
80
100
120
140
160
180
200
220
Auranofin concentration (nM)
% C
ell V
iabil
ity
Auranofin Dose-Response with and without BSO
OCIP23 - 0 BSO
Vehic
le
BSO
10u
M
Au
400n
M
Au
100n
M
Com
bina
tion
0
20
40
60
80
100
120
140
Surv
ivin
g F
ract
ion (
%)
OCIP23
**
9A OCIP23
TrxR1
β-actin
TrxR2
β-actin
A) B)
C)
E) F)
D)
41
BSO-auranofin combination induces apoptosis in primary pancreatic cancer cells.
We conducted live cell flow cytometry to determine whether apoptosis contributed to cell death
after BSO-auranofin combination. The loss of mitochondrial membrane potential (MMP) has been
observed to be one of the first steps of apoptosis, and has previously been observed following
auranofin treatment258. In addition, the loss of membrane integrity, and subsequent uptake of
propidium iodide (PI), is a marker of cell death. Here we used DiIC1(5), a marker of MMP, and
PI to assess cell death after combination treatment. Auranofin or BSO alone did not increase the
MMP negative or PI positive populations. In contrast, combinations of these low concentrations
increased the proportion of cells observed to be starting apoptosis and those that have already died
in both OCIP23 (Fig. 2.2) and in 9A. These results indicate that combinations of low
concentrations of auranofin and BSO induces apoptosis in primary pancreatic cancer cells.
BSO-auranofin combination reduces thiol levels and induces an acute increase in ROS levels.
We also used live cell flow cytometry to assess the effect of combinatorial treatment on oxidative
stress. First, GSH was examined after 24 h treatment by using mBBr, which fluoresces after
binding to free thiols. GSH is the most prominent free thiol in cells, and contributes to
approximately 50% of the mBBr signal277. The treated cells were gated for PI negative populations
to restrict analysis to viable cells. Auranofin did not affect GSH levels when applied as a
monotherapy in OCIP23 but had a significant impact in 9A (p=0.0345). However, BSO had a
significant impact (OCIP23 p<0.0001; 9A p=0.0003) on GSH levels (Fig. 2.4A). The GSH status
of MMP negative populations, both PI negative and positive, was assessed during the same live
cell flow cytometry assay. Following 24 hour treatment both populations displayed depleted GSH
compared to MMP positive populations (Fig. 2.3). These data suggest that the combination therapy
may lead to cytotoxicity by depletion of GSH levels.
42
Figure 2.2. Representative two parameter plots measuring cell death and apoptosis in
OCIP23. Cells were treated with either monotherapy or combination therapy for 24 hours
followed by flow cytometry. Apoptotic cell populations are MMP-, PI- (bottom left quadrant);
Dead cell populations are MMP-, PI+ (Upper left quadrant); Live cells are MMP+, PI- (bottom
right quadrant). Shown are the percentages of the total population for each subpopulation.
Auranofin concentration
0nM 6nM 30nM 75nM
BS
O co
ncen
tration
0μ
M
10
μM
5
0μ
M
MMP
PI
43
Figure 2.3. Representative two parameter plots assessing the GSH content of apoptotic cells
in OCIP23. Cells were treated with either monotherapy or combination therapy for 24 hours
followed by flow cytometry. MMP is a marker for apoptosis, mBBr is a marker for GSH, and PI
is a marker for cell viability. PI positive (non-viable) cells are shown in red, and PI negative
(viable) cells are shown in grey.
Second, ROS were examined following 2 and 5 hour treatments by 2’,7’-dichlorofluorescin (DCF),
which fluoresces following oxidation by ROS. Again, treated cells were gated for PI negative
populations to restrict analysis to viable cells. At these time points, ROS were not affected by
auranofin alone. However, BSO alone and in combination with auranofin displayed a significant
dose-dependent increase (OCIP23 p<0.0001; 9A p<0.0001) in ROS levels (Fig. 2.4B). Combined,
these results indicate that the combination of auranofin and BSO leads to oxidative stress in the
Auranofin concentration
0nM 6nM 30nM 75nM
BS
O co
ncen
tration
0μ
M
10
μM
5
0μ
M
mBBr
MM
P
44
treated cells. This suggests that depletion of GSH and increase in ROS may contribute to cell death
after combination therapy.
Figure 2.4. Effect of treatment on thiol and ROS levels in 9A and OCIP23 cell lines. A) Mean
fluorescence intensity (MFI) of mBBr in PI negative cells after 24 hour treatment with BSO,
auranofin, or combination (9A n=3; OCIP23 n=3). B) MFI of DCF in PI negative cells after 2 and
5 hour treatment with BSO, auranofin, or combination (9A n=3; OCIP23 n=3). Fluorescence was
normalized to non-treated controls. Shown are the S.E.M.
Auranofin binds to TrxR1 in primary pancreatic cancer cells at concentrations associated with
oxidative stress and cell death.
We performed a cellular thermal shift assay (CETSA) to observe whether auranofin engages its
targets TrxR1 and TrxR2 in intact primary pancreatic cancer cells at different concentrations (Fig.
B) A)
45
2.5). This assay is based on thermostabilization of protein targets upon binding chemical
ligands276. It is more clinically relevant as it allows to test drug-target engagement in the complex
cellular environment, as opposed to cell-free assays. As an initial step in CETSA, we treated 9A
cells with a saturating concentration (10 µM) of auranofin and determined the optimal temperature
at which we observed maximal thermostabilization of TrxR1/2 in treated cells compared to
untreated controls. By analysis of thermal shift blots, we found 56C to be the optimal temperature
for TrxR1 and TrxR2 (Fig. 2.5A).
We then tested auranofin engagement after treating 9A and OCIP23 cells with increasing
concentrations to determine whether it binds TrxR1/2 at concentrations associated with cell death.
In both 9A and OCIP23 we observed TrxR1-auranofin binding at concentrations as low as 78nM
(Fig. 2.5B and C). Binding was observed at concentrations much below the IC50’s of auranofin
monotherapy in both cell lines. In addition, our CETSA data suggest that auranofin-mediated cell
death is attributable to targeting TrxR1, but not TrxR2, at IC50’s observed after BSO combination
(Table 2.1). Since OCIP23 and 9A cells exhibit different TrxR1 but not TrxR2 expression levels,
our CETSA data provide further explanation of greater potentiation observed with OCIP23
compared to 9A cells. Taken together, our data show that auranofin bound TrxR1 in intact primary
pancreatic cancer cells at concentrations capable of inducing cell death, and auranofin targets
TrxR1 not TrxR2 at concentrations associated with BSO-mediated potentiation.
46
Figure 2.5. Auranofin-TrxR interaction in vitro. A) Engagement of auranofin and TrxR was
assessed by CETSA. 9A was treated with 10M auranofin for 1 hour. Following drug treatment,
cells were exposed to the indicated temperatures for 3 minutes prior to lysis and immunoblot (n=1).
B) 9A and C) OCIP23 were treated with different auranofin concentrations for 2 hours before heat
treatment at 56C and immunoblot (n=1).
GAPDH
TXRX2
TXRX1
UT T
46°C
UT T
49°C
UT T
52°C
UT T
55°C
UT T
58°C
UT T
61°C
UT T
64°C
UT T
67°C
UT T
70°C
Auranofin (µM)
TRXR1
GAPDH
TRXR2
Auranofin (µM)
TRXR1
GAPDH
TRXR2
A)
B)
C)
47
Table 2.2. Panel of markers measured by IMC using subcutaneous PDX tumours.
Antibody/Reagent Antibody Clone Metal Final Concentration
(g/mL)
Avanti Lipid N/A 115In 10M
-SMA 1A4 141Pr 1.25
Thioredoxin 2G11/TRX 146Nd 10
EF5 ELK3-51 149Sm 10
Pimonidazole N/A 155Gd 10
E-cadherin 24E10 158Gd 2.5
Pan-keratin C11 164Dy 0.6
Goat Anti-Mouse
(Erg1 detection)
N/A 165Ho 1:100
Collagen Goat polyclonal 169Tm 1.25
Intercalator N/A 191/193Ir 0.25M
Auranofin N/A 197Au 10mg/kg
Auranofin distributes within PDX tumours 24 hours after treatment, and associates with the
stroma.
Auranofin contains gold in its molecular structure, which suggested the possibility to detect it in
tissues using imaging mass cytometry (IMC), as we have recently described for platinum-based
drugs275. This novel high throughput technology detects heavy metal isotopes on a histological
section. These heavy metals can either be linked to antibodies or found within the tissue. Data
gained from this method can give information on the distribution of auranofin, its interactions with
the tumour tissue, and an approximation of its concentration within different compartments of a
tumour as long as auranofin is detectable with IMC. The antibody panel used can be seen in Table
2.2. In this pilot study we observed auranofin distributed throughout the two PDX tumours (Fig.
2.6). Auranofin appeared to be more closely associated with collagen in the stromal compartment
of the tumour (Fig. 2.6A and C), rather than E-cadherin in the epithelial compartment (Fig. 2.6B
and D). We then approximated the average number of gold ions per pixel (each pixel has a defined
volume of 5m3) in each compartment (Table 2.3). This semi-quantitative method showed a
48
preferential accumulation of auranofin in the stroma. This method can be expanded in the future
to gain a greater understanding of auranofin’s effects in vivo.
Figure 2.6. Distribution of auranofin in subcutaneous PDX tumours. Mice were treated with
10mg/kg auranofin for 24 hours, followed by pimonidazole for 5hr and EF5 for 3 hours prior to
sacrifice. Tissue section ablations were performed using IMC and markers were false-coloured.
The relation of auranofin (green) to (A and C) stromal collagen (blue), (B and D) epithelial E-
cadherin (blue), and pimonidazole (red) in OCIP83 (A and B) and 9A (C and D) tumours is shown.
Scale bars are provided for reference. Arrows highlight regions of interest.
OC
IP83
9A
A B
C
200μm
D
200μm 200μm
200μm
49
Table 2.3. Estimation of the mean ions/pixel in the stroma and epithelium of subcutaneous
tumours from auranofin treated PDX mice.
Model Ions/pixel
(Stroma)
Ions/pixel
(Epithelium)
OCIP83 4.75 2.83
9A 8.67 8.28
2.4 Discussion
New strategies are required in the treatment of pancreatic cancer. Oxidative stress is an inherent
feature in pancreatic cancer that can be potentially exploited by therapy. The described results
provide evidence that a combination of BSO and auranofin at low clinically achievable drug
concentrations278,279 yields anticancer efficacy in vitro by augmenting oxidative stress.
In this study we assessed the effect of combination therapy on cell death and the redox environment
of the cell. Through an MTS assay and flow cytometry-based assays we identified a strong
potentiation of the cytotoxic effects of auranofin by BSO. Although BSO did not contribute to cell
death on its own after 24 hour treatment, it was responsible for causing increased ROS and
decreased GSH during the combination therapy. Low auranofin concentrations did not
independently affect ROS and had a minimal effect on GSH in one cell line. However, it produced
cytotoxic effects when combined with BSO. Overall, these findings are consistent with the
predicted mechanism of action of a decreased antioxidant capacity and increased oxidative stress
following combination of two antioxidant inhibitors64,83,204. However, it was surprising to observe
that low auranofin concentrations did not independently affect ROS. Other studies have assessed
ROS following treatment with higher auranofin concentrations, and observed substantial
increases280. Our results suggest redundancy between the antioxidant pathways in that both GSH
50
and Trx moderate ROS levels, however the low auranofin concentrations are not required to affect
ROS because of the effect of BSO in this context. It is possible that only a modest increase in ROS
is required to kill the cells because of the concomitant depletion of antioxidants, although to our
knowledge literature does not exist that elucidates the amount of ROS required for cytotoxicity in
the context of antioxidant depletion. Moreover, some cytotoxicity can likely be attributed to
downstream action of TrxR. As protein disulfides accumulate under oxidative stress in the cell,
Trx is unable to remedy the damage because of impaired recycling through TrxR. Cells would lose
their ability to maintain critical functions. These data intimate that these two antioxidant systems
are critical to cancer cell survival. It is possible that other inhibitors towards these systems can be
used to achieve similar results.
When assessing the engagement of auranofin and TrxR we utilized CETSA. This assay provided
the capability to determine whether auranofin bound its target in the context of a complex
biological system276. Our observations show that auranofin engages TrxR1 at low concentrations
compatible with cell death. Engagement with TrxR2 was not observed in this assay. This data is
significant as it shows that auranofin interacts with its target in intact cells, whereas results
obtained in cell-free systems eliminate the presence of other proteins that may interfere with this
binding. This result provides greater support for auranofin’s proposed target, TrxR1, mediating the
observed cytotoxicity. Future studies to show auranofin-TrxR1 engagement in vivo can further
support this finding.
To assess the biodistribution of auranofin in the tumour we used IMC. This provided the unique
insight into auranofin’s distribution 24 hours following treatment. In this pilot study we observed
auranofin associated with collagen in the stromal compartment of the tumour. Although this has
not been previously reported, IMC has previously observed cisplatin association with collagen in
51
PDX tumours275. This finding is corroborated by the long half-life that has been observed in
patients taking oral auranofin281. Collagen may serve as a reservoir for the drug and contribute to
its slow elimination. Our semi-quantitative approach to estimate the number of ions in each
compartment trended towards an increase in the stroma compared to the epithelium, although the
data was not significant. In the future this method can be applied to gaining an in depth
understanding of the interaction of auranofin and molecular markers of oxidative stress to observe
how auranofin affects the molecular biology of the tumour.
This study is limited in a few different aspects. First, the measure of GSH by mBBr fluorescence
using flow cytometry is not specific for GSH. mBBr binds to all free thiols in cells. However, the
background we captured is consistent with previously reported results277. Second, although our
data shows that there is an interaction of auranofin and TrxR1 in the cells, there is no functional
output to measure the effect on TrxR1 activity. Therefore, it is difficult to determine how effective
the low auranofin concentrations are at inhibiting TrxR1 activity. Third, the semi-quantitative
method of determining the number of ions in the tumour can only provide an estimate, as only
small regions of stroma and epithelium in each section could be analyzed, and the number of ions
lost during acquisition cannot be accounted for.
In conclusion, we propose that targeting antioxidants can prove to be an effective new strategy in
treating pancreatic cancer. In the context of the tested combination of BSO and auranofin, we
observed that BSO potentiates auranofin by augmenting oxidative stress, allowing auranofin to
elicit its cytotoxic effects at lower concentrations. Future in vivo studies are required to assess
whether this method is feasible in the context of a living organism, as it is possible that the double
hit may cause undesired toxicities in normal tissues. However, if basal oxidative stress occurs in
the tumour then the reliance on antioxidants should make them more vulnerable to antioxidant
52
inhibition. Furthermore, normal tissues, including hepatocytes, can withstand oxidative stress
following Trx and GSH loss with dietary methionine253. This work gives further evidence to
support the growing body of evidence that suggests that cancers rely on antioxidants for their
survival. Hopefully, this can be a step towards the future development of effective treatments for
pancreatic cancer.
53
Chapter 3
Overall Discussion, Future Directions, and Conclusions
54
3.1 Overall Discussion
Oxidative stress is an interesting target for future cancer therapy development. Pancreatic cancer
relies on antioxidants to protect from oxidative stress and promote proliferation. As such, it is
sensitive to antioxidant depletion which may alter the redox environment. The reliance on
antioxidants is specific to tumour tissue, whereas normal tissues may resist antioxidant loss253.
Targeting antioxidant systems may require a double hit as they can compensate for each other’s
functions.
A common barrier to the development of novel therapies is the length of time from drug discovery
to implementation in clinics282. However, the strategy of augmenting oxidative stress avoids this
barrier with the use of auranofin, which has been used in clinics, and BSO, which has been used
in human clinical trials. Before a combination of the two can be used in human trials, preliminary
tests are required to examine their effects in vitro and in vivo. Therefore, the goal of this thesis is
to test the combination against pancreatic cancer and identify how the drugs interact.
3.1.1 BSO potentiates the acute cytotoxicity of auranofin
I first sought to examine the dose-response of primary human pancreatic cancer cells to both BSO
and auranofin. I showed that BSO potentiates auranofin’s cytotoxicity at 24 hours and that
response may depend partially on TrxR1 levels. Previous studies have examined the effect of these
drugs on cell death, in particular apoptosis. At 24 hours BSO does not induce apoptotic cell
death149. In contrast, treatment of less than 24 hours with auranofin is sufficient to induce the
mitochondrial permeability transition indicative of apoptosis258. Of the TrxR isoforms present in
the cell, TrxR1 inhibition is likely most responsible for mediating the response to auranofin, as it
has been identified as a fitness gene209.
55
Following 24 hour treatment BSO did not affect viability of either cell line, indicating that the cells
were resistant to acute GSH loss. Over the same timespan auranofin reduced viability in a dose-
dependent manner. OCIP23 was more sensitive than the 9A cell line. Since BSO was not lethal on
its own after 24 hours, synergy was excluded because by definition synergy requires an
independent effect of each drug. I then treated cells with both drugs simultaneously for 24 hours
and observed a strong potentiation of auranofin’s cytotoxic effects by BSO. Again, OCIP23 was
more sensitive. The required concentrations of both drugs was within the safe plasma
concentrations that have been achieved during human clinical trials of either drug278,279. This
supports the idea that these cytotoxic concentrations can be attained if administered to a human
patient. Although the discrepancy in sensitivity between the two cell lines can result from
numerous factors, I observed a significant decrease in TrxR1, but not TrxR2, levels in OCIP23.
As TrxR1 has been identified as a fitness gene this difference may contribute to sensitivity to this
combination therapy and may potentially be validated in the future as a biomarker to predict
response to auranofin therapy.
The MTS data showed an odd result in that low auranofin concentrations seemed to improve
viability. However, this may be an artifact of the MTS method itself, in that it measures NADH
production from mitochondrial metabolism which may accumulate in dysfunctional mitochondria
during oxidative stress283. The clonogenic assay showed that these concentrations did not improve
long-term cell growth, and when used in the combination therapy effectively inhibited
clonogenicity.
3.1.2 Apoptosis contributes to cell death
I also identified that apoptosis contributes to the mechanism of cell death of the combination
therapy. Previous work has shown apoptosis caused by BSO following treatment of 48 hours and
56
longer149, and apoptosis following short-term auranofin treatment258. The combination also
displayed apoptosis as shown by the mitochondrial permeability transition. However, this cannot
exclude other mechanisms of cell death as contributors, including senescence and ferroptosis.
Ferroptosis in particular may likely contribute as a result of augmented oxidative stress but was
not assessed using the current assays284.
3.1.3 The drugs engage their expected targets
An important observation when assessing a therapy is to determine whether the drugs enact their
expected primary effect. In the case of BSO, GCL inhibition is expected to decrease GSH levels
as previously observed83. I identified that in the primary pancreatic cancer cells there was a
decrease in GSH following both BSO monotherapy and in combination with auranofin. Thus, BSO
enacted its primary role of preventing GSH synthesis. Auranofin is expected to decrease TrxR
activity through direct interactions with TrxR. Although this thesis does not include an assay for
TrxR activity, it is well-established that auranofin can reduce or eliminate TrxR activity83,254.
Instead I used CETSA to examine drug-target interaction in the context of the complex cellular
environment. I observed that auranofin engaged TrxR1 at low concentrations compatible with cell
death during combination therapy, indicating that auranofin does in fact bind its proposed target
in the cell when used at low concentrations.
3.1.4 Oxidative stress follows combination therapy
The downstream effects following target inhibition are important to understand when assessing a
potential new therapy. I observed that simultaneous inhibition of GSH and Trx augments oxidative
stress by increasing ROS. Previous studies have observed this phenomena following both BSO
and auranofin monotherapy83,149,254. As such, the studied combination therapy caused the expected
downstream effect. Of note, the increase in ROS was modest and of lower magnitude than
57
previously reported with higher auranofin concentrations83,149. However, the observed increase in
ROS was still capable of inducing cell death. Unfortunately, it is difficult to make comparisons to
other work. This is because other studies mostly focus on the amount of exogenous ROS required
to kill cells, rather than ROS from endogenous sources285. Furthermore, the amount of ROS
required to kill in the context of a cell with compromised antioxidant defences has not been
previously determined. It is likely that antioxidant depletion allows for this modest acute spike in
ROS to elicit cytotoxic effects. Lastly, it should be noted that higher auranofin concentrations (ie.
monotherapy IC50 concentrations) have been shown to induce ROS83, which would resolve their
cytotoxic effects as a monotherapy in light of my results showing that low auranofin concentrations
do not independently augment ROS.
3.1.5 IMC is an effective tool to examine auranofin in vivo
Powerful analytical tools are required to understand the full effect of a therapy in vivo. Auranofin’s
unique chemistry provides the potential of IMC identification in tissues, as has been reported with
cisplatin275. My pilot study determined that gold from auranofin can be identified by IMC 24 hours
after drug administration. As with cisplatin, it displays a preferential association with collagen in
the PDX tissue stroma versus E-cadherin in the epithelium275. However, this data does not provide
insight to the biodistribution at shorter time points and may be suggestive of the auranofin reservoir
following elimination of most auranofin molecules. This would be consistent with auranofin’s
exceptionally long total body elimination (55-80 days) observed during clinical trials286. A larger
number of samples are required to prove these results, as this was only a pilot study. Lastly, it
should be noted that IMC cannot distinguish between auranofin and atomic gold. Therefore, the
observations of auranofin distribution in the stroma may be indicative of gold following auranofin
58
breakdown. However, this is consistent with previous methods that restrict detection to atomic
gold when evaluating auranofin287.
The significance of this finding is that IMC can be a tool for the assessment of a larger cohort to
develop an in-depth study of auranofin’s effects in mouse and/or human clinical trials. An antibody
panel can be developed to examine auranofin’s interactions with different tissue structural features,
cell proliferation or growth arrest, hypoxia, cell signaling, and many other potential features of
interest.
3.2 Future Directions
A number of different avenues can be explored following the work described in this thesis. The
first possibility would be to study the mechanism of action greater detail. As previously mentioned,
apoptosis contributes to cell death following the combination therapy. However, the presented data
cannot exclude other mechanisms of death such as ferroptosis, which can result from excessive
lipid peroxidation that occurs during oxidative stress284. The fluorescent probe C11-BODIPY can
be used to assess the extent of lipid peroxidation in a convenient flow cytometry assay that includes
probes for ROS, GSH, viability, and MMP288. Another avenue could be to perform experiments to
further support the current data. The current method for GSH measurement uses mBBr
fluorescence, which also detects protein thiols. A modified version of the Tietze enzymatic
recycling assay can provide a direct biochemical measurement of GSH following BSO
treatments289. The current method to assess auranofin efficacy detects whether auranofin binds its
target. However, this does not specify the extent of TrxR activity loss. An insulin reduction assay
can be performed to assess TrxR activity at the same concentrations290,291.
59
To optimize therapeutic results alterations to the dosing regimen can be tested. For example, BSO
concentrations can be decreased while increasing auranofin. This can be changed to ensure that
doses of each drug that are easiest to achieve in humans are used. Furthermore, alternative
treatment lengths can be tested. This may maximize cytotoxic effects of BSO that have been
observed when used for 48 hours and longer149. Lastly, the strategy for therapy can be altered. The
current study uses simultaneous administration of BSO and auranofin. However, since GSH
depletion by BSO requires time to commence it is possible that an alternative strategy may take
advantage of this. BSO pre-treatment for 24 hours may possibly maximize the effect of auranofin
as the second hit would be administered in a GSH knockdown system. Other alterations to the
strategy may include additions to the current combination. For example, lower doses of BSO and
auranofin may sensitize tumours to chemo or radiotherapy83,202. Chemotherapy drugs like
gemcitabine or ionizing radiation can be used in combination with reduced BSO and auranofin
doses to optimize responses.
Lastly, the most significant option for future studies would be to examine the efficacy of this
combination therapy in orthotopic PDX mice. This approach would allow for testing in a model
that is the most representative of tumours seen in clinics. First, a few mice can be used to determine
the safety of the combination, as adverse events can be unpredictable. If the therapy is deemed
safe a larger experiment can be performed. The design of such an experiment would place mice in
4 treatment arms: control, auranofin, BSO, and combination therapy. Numerous endpoints can be
assessed including survival, changes in tumour volume, and number of metastases. In addition, a
comparison can be made to the current standard of care if another group is used. It is possible that
sensitivity to therapy may depend on TrxR1 levels. As such, tumours bearing different amounts of
TrxR1 can be tested.
60
This experiment also provides the opportunity to assess the effects of auranofin using IMC.
Various questions can be postulated and answered if the correct antibody panel is used on the
tumour tissue. This can also compare differences between monotherapy and combination therapy.
In conjunction, a shorter time-course experiment can examine the timing of different effects
following therapy. These experiments would provide unique insights into how auranofin affects
tumour tissue in vivo, and whether this combination therapy is safe and effective enough to receive
attention in human clinical trials. An additional benefit of running a trial like this would be that
CETSA can be performed on tumours to assess auranofin engagement with TrxR in vivo.
3.3 Conclusions
Oxidative stress is a hallmark of pancreatic cancer that deserves consideration in the development
of new therapies against pancreatic cancer. I conclude in chapter 2 that the current study supports
the efficacy of a combination therapy using BSO and auranofin to treat pancreatic cancer. In this
combination BSO potentiates auranofin’s cytotoxic effects. This therapy results in both antioxidant
depletion and augmentation of oxidative stress. Furthermore, auranofin is detectable by IMC
analysis. Future work can further characterize the effects of this combination and identify whether
it is feasible for clinical use in human pancreatic cancer patients.
61
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