Targeting energy metabolism in colorectal cancer
Transcript of Targeting energy metabolism in colorectal cancer
Targeting energy metabolism in colorectal cancer
by
Nelson Ho
A Thesis presented to
The University of Guelph
In partial fulfillment of requirements for the degree of
Doctor of Philosophy in
Biomedical Sciences
Guelph, Ontario, Canada
© Nelson Ho, August, 2015
ABSTRACT
TARGETING ENERGY METABOLISM IN COLORECTAL CANCER
Nelson Ho Advisor: University of Guelph, 2015 Dr. B. L. Coomber
A switch to aerobic glycolysis regardless of oxygen tension is a metabolic
phenotype observed in a variety of tumors, known as the Warburg effect, and is a
possible target in cancer therapy. The abnormal tumor vasculature leads to a
heterogeneous microenvironment with areas of hypoxia, acidity, and low nutrient supply.
This thesis investigated the effects of the tumor microenvironment on the expression of
glycolytic enzymes and on the efficacy of compounds targeting them. The first set of
experiments examined the molecular and metabolic changes associated with
dichloroacetate-induced cytoprotection in different human colorectal cancer (CRC) cell
lines under anoxic conditions. There was evidence of differential regulation of pyruvate
dehydrogenase phosphorylation between different cells leading to altered mitochondrial
activity following dichloroacetate exposure.
Hexokinase II (HKII) is a glycolytic enzyme often upregulated in tumors. 3-
bromopyruvate (3BP) is an alkylating agent that shows promising anti-cancer effects in
various cancer models and HKII has been suggested to be one of its principal targets. The
efficacy of 3BP against CRC cells was assessed under different media glucose
concentrations, and it was determined that 3BP was effective in reducing cell growth
through alterations in AKT signaling and that media glucose availability played a role in
cytotoxic sensitivity to 3BP. However, knockdown in HKII expression did not alter 3BP
sensitivity in different CRC cell lines.
Finally, HKII expression was assessed in colorectal cancer tissues and correlated
with clinical parameters and patient outcome. Dual immunofluorescence was used to co-
localize HKII with a marker of ischemia, carbonic anhydrase IX in 60 cases of human
CRC. HKII expression was found only in non-ischemic areas of the tumors. While HKII
expression did not correlate with any measured clinicopathlogical characteristics, patients
with low HKII-expressing tumors had a worse prognosis than those with high HKII-
expressing tumors.
Overall, this thesis presents the effects of the tumor microenvironment on both
glycolytic enzyme expression and therapeutic efficacy. This research promises to
improve our understanding of tumor metabolism in the heterogeneous microenvironment
in order to better apply this field of cancer biology for therapeutic use.
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ACKNOWLEDGEMENTS
A dissertation of this magnitude cannot be completed without the help of many. With that in mind, there are a few things I would like to say and a few people I would like to thank before half the words don’t make sense to a general audience.
To my supervisor, Dr. Brenda Coomber. I recently looked back at the first e-mail I sent you back in November of 2009, asking to see if there would be a position in your laboratory 10 months down the road. To be quite honest, reading the e-mail was quite cringe-worthy and not my best work, yet you still took a chance on me. Here we are, over 5 years later. I can’t thank you enough for all the guidance, wisdom, and support that you have provided me over the years. Your door was always open, and you were more than willing to listen to your students, whether it be a good or bad idea rolling off our tongues. The wealth of knowledge you possess regardless of topic is astonishing.
I would also like to thank the members of my advisory committee: Dr. James
Kirkland, Dr. Roger Moorehead, and Dr. Ray Lu. You all provided valuable insight and suggestions that helped shape my research and dissertation. Beyond faculty members, I would like to thank Betty-Anne McBey who played a crucial role in the early stages of my flow cytometry analysis in her training and technical support.
I would like to thank the Department of Biomedical Sciences. It was a pleasure working in an environment filled with such inspiring minds. I would like to thank the incredible office staff: Kim, Frances, Sally, and Wendy. You all kept the ship afloat and were always available for a quick social break.
I would like to thank my officemates whom I have shared many laughs with both in and out of the office. In particular, I would like to thank Amanda and Emily for putting up with my shenanigans, especially during social outings. Graham, I never would have thought back in 1st year of undergrad when I met you that we would be sharing this PhD road together, in the same department no less. Your faith in my fantasy hockey knowledge to make me your assistant GM was appreciated. I also would like to thank you for not thinking I was too terrible in volleyball to put up with me as a teammate for all these years. Lindsay, without going into too much detail, you were my partner in crime and catalyzed me interpersonally by providing me with the platforms to highlight some of my more “artistic” abilities shall we say? I would like to thank all the 4th year project students and summer research students whom I had the pleasure of supervising – Rebecca Theal, Stacey Butler, Gaelan Melanson, and Andreza Silva. I thoroughly enjoyed being a mentor to you all and I hope you enjoyed your experience. I would like to thank all the past and current lab members of the Coomber, Viloria-Petit, and Mutsaers labs for providing such a friendly and engaging working environment. To Amanda, Kaya, Nathan, Leanne, Geordon, Amy, Sonja, Divya and Richard, the “old” crew. I will never forget the memories we shared in the confines of the
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lab or the shenanigans that would ensue following a long week. The recent closing of Vinyl may be symbolic to an end of our reign. Barbs, I am not sure how better to thank you for your friendship than by saying “Ignition Remix”. Kaya, thank you for showing me the ropes when I first came into the lab and for passing down the PhD torch to me. Geo, you are the model of perseverance and will never be a duster in my book. Rich, you are an inspiration with what you have accomplished since I first met you and I am proud to have you as a friend. To my Lab Mom, Jodi Morrison. No words can describe the appreciation I have for you as a scientist and as a human being. For every bit of success or failure, you have been there for me. We were models of procaffination and I will never forget the stories we shared over those times. You were always there to answer any of my questions even though I tried my best to act like the oldest son and not bother you. I wish nothing but the best for you and your family moving forward. I would like to thank my family. To my mom, you have been an inspiration throughout my life and while you still don’t understand my research over these years, just know that your hard working attitude and perseverance has rubbed off on me. To my sister, Vanessa, thank you for taking care of mom while I have been away. Although we don’t see each other much anymore, just know that I will always be there for you. To Rico, I never thought I could love a dog as much as you, whatever mix you may truly be. Finally, I would like to thank Kate. You are my best friend, my confidant, and my biggest supporter. You never doubt my inspirations and have full faith in whatever decisions I make. I am truly blessed to have you in my life. I love you so much.
In a special tribute, I would like to thank my dad, Lawrence. You left us over 9 years ago, but I will be forever grateful for how you provided for your family even though times were never easy. You have been my inspiration over these last 5 years, and the reason for my choice to study cancer. I know you look down on me everyday and I hope that you are proud of the man that I have become. So dad, I dedicate this work to you.
Love, Son.
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DECLARATION OF WORK PERFORMED
I declare that with the exception of the items indicated below, all work reported in
this thesis was performed by me.
Under my supervision, Andreza Silva performed media-glucose reduction on
HCT116 and DLD-1 cell lines.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ............................................................................................ iv
DECLARATION OF WORK PERFORMED .............................................................. vi LIST OF TABLES ............................................................................................................ x
LIST OF FIGURES ......................................................................................................... xi LIST OF ABBREVIATIONS ........................................................................................ xii
INTRODUCTION ............................................................................................................ 1 CHAPTER I – REVIEW OF THE LITERATURE ...................................................... 3
Glucose metabolism ...................................................................................................... 3 Warburg effect .............................................................................................................. 3
Advantages of the Warburg effect to cancer cells ...................................................... 6 Regulation of glycolytic enzymes ................................................................................. 9
Hexokinase ................................................................................................................ 12 Pyruvate dehydrogenase kinase ................................................................................ 14
Metabolic regulation by oncogenes and tumor suppressors ................................... 16 Warburg effect as a therapeutic target ..................................................................... 19
Dichloroacetate ......................................................................................................... 22 3-bromopyruvate ....................................................................................................... 25
Tumor microenvironment .......................................................................................... 28 Hypoxia ..................................................................................................................... 29 HIF1 Regulation ........................................................................................................ 31 Hypoglycemia ........................................................................................................... 32
RATIONALE .................................................................................................................. 35 CHAPTER II – EFFECTS OF DCA ON ANOXIC HUMAN COLORECTAL CANCER CELLS ........................................................................................................... 37
Introduction ................................................................................................................. 37 Materials and Methods ............................................................................................... 39
Cell culture ................................................................................................................ 39 In vitro exposure to anoxia and/or DCA ................................................................... 40 Measurement of cell growth ..................................................................................... 40 qRT-PCR for PDK mRNA expression ..................................................................... 40 Protein isolation and western immunoblot analysis .................................................. 41 FACS analysis of mitochondrial function ................................................................. 42 Glucose consumption and lactate production ........................................................... 43 Statistical analysis ..................................................................................................... 43
Results .......................................................................................................................... 44 DCA reduces both normoxic and anoxic cell growth ............................................... 44 PDKs 1 and 3 are highly expressed in human CRC cells ......................................... 44 PDKs 1 and 3 are upregulated in response to treatment ........................................... 45 PDH phosphorylation status varies in response to treatment between cell lines ...... 45 Mitochondrial activity varies in response to DCA between human CRC cells ........ 46
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DCA does not significantly alter metabolic status of human CRC cells .................. 47 Discussion .................................................................................................................... 48
CHAPTER III – METABOLIC DISRUPTION VIA 3-BROMOPYRUVATE IN HUMAN COLORECTAL CANCER CELLS ............................................................. 63
Introduction ................................................................................................................. 63 Materials and Methods ............................................................................................... 65
Cell culture and media-glucose reduction ................................................................. 65 Measurement of cell growth ..................................................................................... 66 Protein isolation and western immunoblot analysis .................................................. 66 Cell death assay ......................................................................................................... 67 DNA fragmentation .................................................................................................. 68 RNA interference ...................................................................................................... 68 Statistical analysis ..................................................................................................... 69
Results .......................................................................................................................... 69 HKII expression is correlated with 3BP sensitivity in human CRC cells ................. 69 3BP-induced cell death is dose dependent ................................................................ 70 3BP induces rapid AKT phosphorylation at residue Thr308 .................................... 71 Glucose availability affects 3BP sensitivity in human CRC cells ............................ 72 HKII knockdown does not lead to changes in 3BP sensitivity ................................. 72
Discussion .................................................................................................................... 73
CHAPTER IV – HEXOKINASE II AS A POTENTIAL PROGNOSTIC BIOMARKER FOR HUMAN COLORECTAL CANCER ....................................... 87
Introduction ................................................................................................................. 87 Materials and Methods ............................................................................................... 89
Samples ..................................................................................................................... 89 Cell lines and immunoblot analysis .......................................................................... 89 Immunofluorescent staining ...................................................................................... 90 Imaging and quantification of immunofluorescence ................................................ 91 Statistical analysis ..................................................................................................... 92
Results .......................................................................................................................... 92 Patient characteristics ................................................................................................ 92 Immunoblot and immunofluorescent detection of HKII and CAIX ......................... 92 Levels of CAIX and HKII staining ........................................................................... 93 Association of CAIX and HKII expression with patient characteristics .................. 93 Association of CAIX and HKII expression with patient outcome ........................... 94 Association of stromal HKII staining with patient characteristics and outcome ...... 94
Discussion .................................................................................................................... 95 GENERAL DISCUSSION ........................................................................................... 111
SUMMARY AND CONCLUSIONS ........................................................................... 122 REFERENCES .............................................................................................................. 125
APPENDIX I – CHEMICAL LIST AND SUPPLIERS ............................................ 163 APPENDIX II – PREPARATION OF MATERIALS ............................................... 165
APPENDIX III – AV/PI STAINING OF ETOPOSIDE-TREATED SAMPLES ... 168
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APPENDIX IV – DENSITOMETRY OF AKT PHOSPHORYLATION AT DIFFERENT RESIDUES FOLLOWING 4 H 3BP EXPOSURE ............................ 169
APPENDIX V – FLOW CYTOMETRY ANALYSIS OF AV AND PI STAINING FOLLOWING 48 H 3BP EXPOSURE ....................................................................... 170
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LIST OF TABLES
Table I. Primer sequences for quantitative real-time PCR analysis of PDK isoforms ......56 Table II. Patient characteristics ........................................................................................101 Table III. Associations of CAIX and HKII staining with patient characteristics and tumor
pathology .....................................................................................................................102 Table IV. Associations of stromal HKII staining with patient characteristics and tumor
pathology .....................................................................................................................103
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LIST OF FIGURES
Figure 1. DCA reduces cell growth in a cell line-dependent manner ................................57 Figure 2. PDK1 and 3 are highly expressed in human CRC cells .....................................58 Figure 3. PDK1 and 3 levels are upregulated in response to treatment .............................59 Figure 4. Phosphorylation status of PDH at residues Ser293 and Ser300 varies in
response to treatment .....................................................................................................60 Figure 5. Mitochondrial response to DCA exposure is cell line-dependent ......................61 Figure 6. DCA does not significantly alter metabolic status of human CRC cells ............62 Figure 7. HKII expression is correlated to 3BP sensitivity ...............................................81 Figure 8. 3BP-induced cell death is cell-line dependent ....................................................82 Figure 9. Type of cell death induced by 3BP is dose-dependent .......................................83 Figure 10. 3BP treatment leads to rapid AKT phosphorylation at residue Thr308 ...........84 Figure 11. Glucose availability alter 3BP sensitivity in CRC cells ...................................85 Figure 12. HKII expression knockdown does not lead to changes in 3BP sensitivity ......86 Figure 13. HKII and CAIX immunoblotting in colorectal cancer cell lines ....................104 Figure 14. HKII and CAIX immunostaining in colorectal cancer ...................................105 Figure 15. Evaluation of CAIX immunostaining .............................................................106 Figure 16. Evaluation of HKII immunostaining ..............................................................107 Figure 17. Immunofluorescent staining of HKII and corresponding F-Scores ...............108 Figure 18. Progression-free survival and overall survival by CAIX and HKII staining .109 Figure 19. Progression-free survival and overall survival by the presence of stromal HKII
staining ........................................................................................................................110
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LIST OF ABBREVIATIONS
Abbreviation Full Name 3BP 3-bromopyruvate 5-FU 5-fluorouracil APS Ammonium persulfate AV Annexin V BSA Bovine serum albumin CAIX Carbonic anhydrase IX CRC Colorectal cancer DAPI 4’, 6’-Diamidino-2-phenylindole DCA Dichloroacetate DMEM Dulbecco’s Modified Eagle Media DMSO Dimethyl sulfoxide EDTA Ethylenediaminetetraacetic acid ETC Electron transport chain FBS Fetal bovine serum FFPE Formalin-fixed paraffin-embedded HIF Hypoxia-inducible factor HK Hexokinase HRP Horseradish peroxidase IF Immunofluorescence MA Mitochondrial activity PCR Polymerase chain reaction PBS Phosphate-buffered saline PDH Pyruvate dehydrogenase PI Propidium iodide PMSF Phenylmethylsulfonyl fluoride PVDF Polyvinylidine fluoride ROS Reactive oxygen species SDS Sodium dodecyl sulfate siRNA Small interfering RNA TAE Tris-acetate-EDTA buffer TBS Tris-buffered saline TCA Tricarboxylic acid TEMED Tetramethylethylenediamine VHL von Hippel-Lindau
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INTRODUCTION
Cancer is a group of complex diseases defined by uncontrolled cell growth. In
2012, there were an estimated 14.1 million new cancer cases and 8.2 million cancer-
related deaths worldwide (Torre et al, 2015). In 2014, it was estimated that 191,300 new
cancer cases and 76,600 cancer-related deaths occurred in Canada (Canadian Cancer
Society). This thesis will focus on colorectal cancer, which is the third most commonly
diagnosed and has the second highest mortality rate of all cancer types in Canada.
Specifically, colorectal cancer accounted for 13.9% and 11.6% of new cases among men
and women, respectively, and 12.8% and 11.5% of cancer-related deaths among men and
women, respectively, in 2014 (Canadian Cancer Society). The 5-year survival rate for
cases that are diagnosed while localized to the primary site is 90.3%. This drops
substantially to 12.5% in cases where disease has spread to distant sites by diagnosis,
accounting for 20% of all cases (Siegel et al, 2014). Thus, researchers seek to better
understand colorectal tumorigenesis and develop better screening methods and novel
treatments.
In 1926, the German scientist Otto Warburg reported that cancer cells produced
most of their energy through glycolysis, even under aerobic conditions (Warburg et al,
1927). Today, the “Warburg effect” can be described as an adaptation of cancer cells to
preferentially utilize glycolysis for energy production, even in the presence of abundant
oxygen. This adaptation is caused by alterations in signaling pathways involved in energy
metabolism, including glucose uptake, utilization and regulation of mitochondrial activity
(Koppenol et al, 2011). In 2011, its importance to tumor progression was signified by its
addition as an emerging hallmark to the revised list of hallmarks of cancer (Hanahan &
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Weinberg, 2011). Recent studies provided researchers with a further understanding of
cancer metabolism to better determine targets that may be suitable for development of
anti-cancer therapeutic strategies. Although there is a high degree of heterogeneity in
mutational status of various tumors and thus in the inability to specifically target tumors,
an underlying mechanism that most tumors possess is rewiring of metabolic pathways.
Metabolic modulators that have shown promise in their ability to target key components
of the Warburg effect and leave normal cells unharmed include dichloroacetate and 3-
bromopyruvate.
Due to the highly disorganized and chaotic nature of the tumor vasculature, cells
in solid tumors are transiently and/or chronically exposed to decreased blood flow, or
ischemia. These heterogeneous microenvironments expose cancer cells to hypoxia and
hypoglycemia (Raghunand et al, 2003). While numerous studies have documented the
effects of ischemia on metabolic signaling, the mechanisms by which it may affect the
drug efficacy of metabolic modulators in cells is not fully understood. Recent studies
have shown that hypoxic signaling may somehow play a role in protecting cancer cells
against drugs targeting metabolic signaling (Shahrzad et al, 2010).
In summary, aerobic glycolysis plays an important role in tumor progression.
While recent studies have provided further insight into the metabolic pathways essential
to the rewiring of tumor energetics, there remain many questions as to the impact of the
tumor microenvironment on targeting key metabolic pathways and targets.
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CHAPTER I – REVIEW OF THE LITERATURE
Glucose metabolism
Normal differentiated cells readily depend on mitochondrial respiration via
oxidative phosphorylation for efficient ATP production to meet energy demands,
accounting for 90% of all energy generation (Dasu et al, 2003). Glucose is initially
metabolized to pyruvate and further processed to acetyl-CoA through the TCA cycle. The
NADH-reducing equivalents generated through this process provide the hydrogen ions
required to establish an electrochemical gradient to facilitate ATP production through the
electron transport chain, in which oxygen is the final electron acceptor. Under anaerobic
conditions, glucose is converted to lactate via glycolysis. This reaction generates only 2
ATP molecules per glucose molecule, compared to the 38 ATP molecules per glucose
molecule generated through aerobic respiration (Vander Heiden et al, 2009). Thus,
aerobic respiration maximizes ATP production through mitochondrial respiration
compared to anaerobic glycolysis in the cytoplasm.
Warburg effect
The metabolic properties of cancer cells differ markedly from those of normal
cells. Dr. Otto Warburg formulated his hypothesis on cancer cell metabolism in the 1920s
when he observed that under aerobic conditions, tumor tissues metabolized glucose to
lactate approximately ten fold more than normal tissues (Warburg et al, 1927). This
ability of cancer cells to metabolize glucose to lactate regardless of oxygen tension was
coined the “Warburg effect”, or aerobic glycolysis. For example, leukemic cells are
highly glycolytic despite residing in the oxygen rich bloodstream (Elf & Chen, 2014). In
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essence, it is the reverse of the Pasteur effect, which is the inhibition of fermentation by
O2. Warburg hypothesized that this metabolic phenotype arose from mitochondrial
defects within tumor cells that inhibited their ability to effectively oxidize glucose to
carbon dioxide. While striking, his observations did not initially prompt additional
research, attributed to a poor understanding of tumor metabolism at the time. It was
understood that pyruvate metabolism through fermentation and lactate production was
energetically inefficient and would not contribute to the neoplastic phenotype. There was
also a lack of knowledge on the signaling pathways associated with the metabolic switch
from pyruvate conversion to acetyl-CoA through the mitochondria versus conversion to
lactate in the cytoplasm. It was originally thought that cancer was a disease of genetic and
epigenetic alterations in oncogenes and tumor suppressors, where altered metabolism was
considered an indirect secondary phenomenon (Levine & Puzio-Kuter, 2010).
Now in the 21st century, scientists have begun to thoroughly investigate and
accelerate our understanding of tumor metabolism. First and foremost, most tumor
mitochondria are not defective in their ability to carry out oxidative phosphorylation. It is
clear now that mitochondrial metabolism is reprogrammed to support the anabolic
products required for rapid cell growth and proliferation. This metabolic strategy
emphasizes the production of nucleotides, amino acids, and fatty acids which in turn fuel
tumor growth by maximally producing RNA/DNA, proteins, and lipids, respectively, all
necessary for rapid cell division (Vander Heiden et al, 2009). Similar alterations in
metabolic signaling are observed in normal human physiological events with the need for
rapidly proliferating cells, including embryonic development, wound healing, particularly
in liver regeneration, and in immune responses to specific antigens, where clonal
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selection provides increased cell numbers with increased immune specificity (Levine &
Puzio-Kuter, 2010). These represent appropriate responses to physiological growth
signals as opposed to constitutive cell autonomous adaptations as seen with neoplastic
growth (Newsholme et al, 1985).
In essence, increased glucose consumption is devoted to lactate conversion and
biosynthesis. While a significant portion of glucose consumed by cancer cells is directed
toward the glycolytic pathway for synthesis of anabolic precursors, oxidative
phosphorylation is utilized in most cancer cells and still a major source for ATP
generation (Guppy et al, 2002; Marin-Valencia et al, 2012). Warburg observed the rate of
O2 consumption in cancer cells remained comparable to that of normal cells (Warburg et
al, 1927). ATP concentration in tumors changes only marginally when compared to
normal tissues (Sonveaux et al, 2008). Cancer cells upregulate glucose transporters to
increase glucose uptake to support both anabolic and catabolic processes required to
sustain their rapid growth and proliferation (Macheda et al, 2005). Clinicians measure
this unique metabolic trait using 2-fluoro-2-deoxy-D-glucose positron emission
tomography (FDG-PET) as a diagnostic tool to assess various cancers including
colorectal cancer (CRC) (Artiko et al, 2015; Kruse et al, 2013; Rigo et al, 1996).
Although mitochondrial function remains active in neoplastic cells, it has been
reported that β-F1-ATPase, the catalytic subunit of the mitochondrial H+-ATP synthase
and the rate-limiting component of oxidative phosphorylation, is significantly reduced in
human tumors compared to its expression in normal tissues (Cuezva et al, 2002). This
observation was accompanied by an increase in the expression of glyceraldehyde 3-
phosphate dehydrogenase (GAPDH), a marker of glycolysis. The β-F1-ATPase/GADPH
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ratio was termed the cell’s bioenergetic signature (Cuezva et al, 2002), and its reduction
is a common trait in over 95% of the carcinomas analyzed in large cohorts of breast,
colon, and lung cancer patients (Cuezva et al, 2009). The expression of β-F1-ATPase and
the decreased bioenergetic signature of lung cancer were shown to inversely correlate
with the rate of glucose consumption as assessed by FDG-PET imaging (Lopez-Rios et
al, 2007).
Advantages of the Warburg effect to cancer cells
Hanahan and Weinberg initially proposed that six biological capabilities were
acquired during tumorigenesis and were termed the “Hallmarks of Cancer” (Hanahan &
Weinberg, 2000). In 2011, this list was expanded to include "reprogramming energy
metabolism" as an emerging hallmark of cancer, highlighting its evident importance but
also the unsolved issues surrounding its mechanisms (Hanahan & Weinberg, 2011).
However, regardless of mechanisms, the Warburg effect clearly contributes to the
malignant phenotype of cancer cells.
Recent progress in cancer research has offered insight into the benefits and
selective advantages of the Warburg effect. As a result of rapid growth and proliferation,
cancer cells are required to increase glucose consumption and direct glucose derivatives
to anabolic pathways such as acetyl-CoA for fatty acid synthesis, glycolytic intermediates
for nonessential amino acids, and ribose for nucleotide synthesis (Vander Heiden et al,
2009). To compensate for a reduction in the levels of pyruvate available to be exhausted
through the TCA cycle and oxidative phosphorylation for ATP production, cancer cells
also rely on glutamine metabolism as an additional source of fuel (Gao et al, 2009; Wise
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et al, 2008). Glucose and glutamine account for most carbon and nitrogen metabolism in
mammalian cells (Wellen et al, 2010). Thus, cancer cells increase glutamine consumption
for use through the TCA cycle so glucose derivatives can be shunted into anabolic
pathways (DeBerardinis et al, 2007). Glutamine metabolism also contributes nitrogen for
synthesis of purines, pyrimidines, nonessential amino acids, and the reduced form of
nicotinamide adenine dinucleotide phosphate (NADPH) for synthesis of fatty acids
(Metallo et al, 2012). On top of fuel for lipid synthesis, NADPH also functions as an
antioxidant to quench ROS produced during rapid proliferation of cancer cells. Thus, it is
imperative in both anabolic processes and for the maintenance of cellular redox
homeostasis. It is suspected that NADPH production may be the rate-limiting factor for
cell proliferation (Vander Heiden et al, 2009).
Aggressive tumors acquire an ability to invade the local environment and
metastasize to distant sites. Cancer cells secrete lactate, leading to extracellular
acidification and increased motility of cells to break through the basement membrane and
metastasize (Gatenby & Gillies, 2004; Gillies et al, 2008; Martinez-Zaguilan et al, 1996).
For metastasis to occur, cells must acquire an ability to resist anoikis, a form of
programmed cell death induced by the loss of cell-matrix interaction (Frisch & Ruoslahti,
1997). Detachment from the extracellular matrix leads to upregulation and activation of
several BH3-only proteins of the BCL-2 family of proteins (Gilmore, 2005). This leads to
activation of pro-apoptotic BCL-2 family of proteins, resulting in mitochondrial outer
membrane permeabilization that facilitates the release of apoptotic factors, downstream
caspase activation, and eventual cell death via apoptosis (Llambi et al, 2011).
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Mitochondrial respiration is a major source of cellular ROS. In low levels, ROS
are able to activate various signaling pathways to stimulate cell proliferation and survival
while irreversibly damaging cellular components at higher levels that can lead to cell
death (Orrenius et al, 2007). To counteract the effects of ROS, cells express anti-oxidant
enzymes that detoxify ROS and prevent them from reaching high concentrations
(Trachootham et al, 2009). Tumors generate high ROS levels compared to normal tissue
(Haklar et al, 2001). Many cellular anti-oxidants are constitutively overexpressed in
cancer cells to counteract these effects, including the principal mitochondrial antioxidant
enzyme, manganese superoxide dismutase (Landriscina et al, 2009; Pani et al, 2004).
Matrix detachment also leads to ROS generation that can cause cell death
(Kamarajugadda et al, 2013; Li et al, 1999). Anoikis is suppressed in detached cells when
treated with antioxidants, further supporting the role of ROS generation in anoikis-
mediated cell death (Kamarajugadda et al, 2012; Schafer et al, 2009). Thus, the
metastatic potential of cancer cells is attributed to the Warburg effect and their ability to
resist cell death caused by increased ROS production as a result of matrix detachment and
anoikis-mediated cytotoxicity.
Glycolytic cancer cells are also better adapted to resist cell death through
modulation of BCL-2 family proteins (MacFarlane M 2012 Cell Cycle). Glycolysis-
mediated AKT activation stabilizes the expression of the anti-apoptotic BCL-2 protein
MCL-1 by preventing its proteasomal degradation (Coloff et al, 2011; Pelicano et al,
2006). This leads to the inhibition of pro-apoptotic proteins PUMA, BID, and Noxa and
their ability to permeabilize the mitochondrial outer membrane that would otherwise lead
to caspase-mediated apoptosis (Youle & Strasser, 2008). Furthermore, glycolysis
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maintains normal translation of MCL-1 through the AMPK/mTOR pathway (Pradelli et
al, 2010). The ability of AKT to inhibit PUMA expression is dependent upon glucose
metabolism (Coloff et al, 2011). Likewise, the BH3-only protein Noxa is regulated via
phosphorylation of a serine residue in the presence of cytoplasmic glucose (Lowman et
al, 2010).
Regulation of glycolytic enzymes
Cancer cells exhibiting the Warburg effect have been shown to upregulate several
key glycolytic enzymes to further drive aerobic glycolysis. Pyruvate kinase (PK)
catalyzes the conversion of phosphoenolpyruvate to pyruvate while concurrently
producing ATP. PKM2, an alternatively spliced variant of M1, is expressed during
embryonic development and more recently has been shown to play a key role in
promoting the Warburg effect in tumorigenesis (Christofk et al, 2008a). PKM2 is
allosterically regulated between a less active dimer and an active tetramer (Mazurek,
2011; Mazurek et al, 2005). The low-activity PKM2 dimer drives aerobic glycolysis,
while the high-activity PKM2 tetramer produces pyruvate for oxidative phosphorylation
(Christofk et al, 2008b; David et al, 2010). Regulation between the two states is dictated
by fructose-1,6-bisphosphate (FBP), which when bound to PKM2 activates
tetramerization through high affinity association (Deprez et al, 1997). Dissociation of
FBP from the PKM2 tetramer results in conversion to the PKM2 dimer (Christofk et al,
2008a). PKM2 is important for proliferation and migration of colon cancer cells (Kwon et
al, 2012; Zhou et al, 2012). PKM2 knockdown results in suppression of proliferation and
invasion in vitro and the formation of xenograft tumors in vivo (Goldberg & Sharp, 2012;
Kefas et al, 2010). Phosphoglycerate mutase 1 catalyzes the conversion of 3-
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phosphoglycerate (3PG) to 2-phosphoglycerate (2PG) during glycolysis. In many
cancers, including CRC, PGAM1 activity is increased compared with normal tissue (Liu
et al, 2008).
The important conversion of pyruvate to lactate is catalyzed by lactate
dehydrogenase (LDH). Attenuation of LDH activity impairs tumor metabolism and
subsequently proliferation (Fantin et al, 2006). Homo- and heterotetramers consisting of
gene products of LDHA and LDHB form five different isoforms (Draoui & Feron, 2011).
LDHA is upregulated by both hypoxia-inducible factor 1α (HIF1α) and MYC oncogene
(Semenza et al, 1996; Shim et al, 1997). LDH isoforms with high LDHB gene product
content catalyze the reverse conversion of lactate to pyruvate. Aberrant methylation of
LDHB promoter region, leading to transcriptional silencing, has been reported in gastric
and prostate cancers (Leiblich et al, 2006; Maekawa et al, 2003). It has been proposed
that a switch to aerobic glycolysis in colorectal cancer is due to in large part to
suppression of LDHB transcription (Thorn et al, 2009).
Unlike the glycolytic enzymes previously discussed, isocitrate dehydrogenase
(IDH) isoforms have been identified as mutated in human cancers. Sequencing studies
revealed that 12% of patients with glioblastoma multiforme have the same point mutation
in cytosolic IDH1 while up to 90% of patients with secondary gliomas have the same
mutation or the analogous mutation in mitochondrial IDH2 (Mardis et al, 2009; Parsons
et al, 2008; Yan et al, 2009). IDH normally functions to catalyze the conversion of
isocitrate to α-ketoglutarate via NADPH-dependent reduction. Mutated IDH confers
neomorphic activity in which isocitrate is covered to 2-hydroxyglutarate. This in turn
reduces α-ketoglutarate availability as a substrate for enzymes that methylate DNA and
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histones, leading to enhanced tumorigenesis through modification of the epigenome
(Dang et al, 2010; Figueroa et al, 2010; Lu et al, 2012; Ward et al, 2010).
As previously stated, Warburg’s initial hypothesis attributing aerobic glycolysis to
mitochondrial dysfunction was incorrect. It is now well established that the mitochondria
continue to play a critical role even though cancer cells heavily depend on glycolysis for
their anabolic needs. However, β-F1-ATPase is significantly reduced in human tumors
compared to its expression in normal tissues (Cuezva et al, 2002). This leads to
mitochondrial hyperpolarization and inhibits the release of cytochrome C and apoptosis
inducing factor to avoid the apoptotic cascade (Kroemer et al, 2007).
Oncogenic mutations in mitochondrial metabolic enzymes further highlight their
importance in tumorigenesis. Familial paraganglioma can be caused by mutations in any
of the 4 subunits of the succinate dehydrogenase (SDH) complex (Astuti et al, 2001;
Baysal et al, 2000; Michelakis et al, 2008; Niemann & Muller, 2000). It is the only
enzyme that participates in both the TCA cycle and electron transport chain, where it
catalyzes the oxidation of succinate to fumarate with the reduction of ubiquinone to
ubiquinol (Oyedotun & Lemire, 2004). Mutations in fumarate hydratase (FH), involved
downstream of SDH in the TCA cycle, result in uterine fibroids, leiomyoma, and
papillary renal cell cancer (Tomlinson et al, 2002).
This thesis will focus on two key glycolytic enzymes in cancer cell metabolism:
hexokinase and pyruvate dehydrogenase kinase.
12
Hexokinase
Hexokinase is responsible for the first irreversible catalytic reaction of glycolysis
in which glucose is phosphorylated to become glucose-6-phosphate in the following
reaction:
Glucose + ATP è Glucose-6-PO42- + ADP
Glucose-6-phosphate serves as a precursor for not only glycolysis, but also the pentose
phosphate pathway (NADPH and ribulose-5-phosphate), glycogenesis (glycogen), and
hexosamine biosynthetic pathway (uridine diphosphate N-acetylglucosamine) (Pedersen,
2007; Wilson, 1995). Therefore, HK plays important roles in the regulation of both
anabolic and catabolic processes. Glucose-6-phosphate subsequently is able to inhibit HK
activity thus providing a negative feedback mechanism (Ardehali et al, 1999). The
fundamental approach to FDG-PET imaging and diagnosis of metabolically active tumors
depends on HK function, as it phosphorylates FDG at the 6th carbon with ATP to become
FDG-6-G and detected by PET scanning.
To date, four HK isoforms have been identified and are all encoded at different
chromosomal loci. Isoforms I, II, III, and IV are located on chromosome arms 10q22,
2p13, 5q35, and 7p15, respectively. HK isoforms I-III have a molecular mass double that
of HKIV (~100 vs. 50 kDa) as a result of evolutionary duplication and fusion of a single
domain HK molecule (Wilson, 1995; Wilson, 1997; Wilson, 2003). As a result, HK
isoforms I-III have a 250-fold higher affinity for glucose compared to HKIV. HK
isoforms are differentially expressed in various tissues. Both HKI and III are ubiquitously
expressed, although HKI is the main isoform in the brain and HKIII is not predominant in
any tissue. HKIV is primarily restricted to the liver and pancreas. In normal tissue, HKII
13
is limited to insulin-sensitive tissues such as adipose, skeletal and cardiac muscle
(Wilson, 1995; Wilson, 2003).
HKII is upregulated in many tumors, and is considered a consequence of
metabolic re-programming in cancer. Its expression level is closely associated with tumor
grade and mortality in hepatocellular carcinoma (Kwee et al, 2012). It has also been
shown to be upregulated in glioblastoma multiforme and brain metastases of breast
cancer (Palmieri et al, 2009; Wolf et al, 2011). During tumor development in tissues
normally expressing HKIV, HKII gene expression is induced whereas HKIV is silenced
(Mathupala et al, 1997b; Mayer et al, 1997; Rempel et al, 1996). Furthermore, the
catalytic activity of both HK domains is preserved only in HKII while the carboxyl
terminal domain is the only functional domain in HKI and III. Thus, the switch to a
higher glucose affinity HK isoform may allow the neoplastic cell to meet its high-glucose
demand. HKII is able to bind to voltage dependent anion channel (VDAC), a protein
located on the outer mitochondrial membrane (Nakashima et al, 1986). This allows
neoplastic cells to preferentially utilize the rapidly producing ATP generated through
oxidative phosphorylation to drive glycolysis. In human gliomas, it was found that 69%
of HKs were mitochondrial bound, compared to 9% that were mitochondrial bound in the
normal brain (Oudard et al, 1997).
Besides its role in metabolism, HKII also functions as a protective molecule. Pro-
apoptotic BH3 only proteins BAX and BAD exert their effects through binding to free
VDAC sites on the mitochondrial membrane, leading to its oligomerization by truncated
BID. This in turn leads to the activation of the mitochondrial permeability transition pore
to facilitate the release of cytochrome c into the cytoplasm (Scorrano & Korsmeyer,
14
2003). Mitochondrial-bound HKII (mito-HKII) competitively inhibits BAX and BAD
binding to the mitochondria and prevent localization to the outer mitochondrial
membrane (Majewski et al, 2004b). Forced detachment of HKII from the mitochondria
increases the ability of BAX to induce apoptosis (Pastorino et al, 2002). Decreased HKII
level sensitizes cells to apoptotic or necrotic stimuli (Roberts & Miyamoto, 2015).
Pyruvate dehydrogenase kinase
A key branching point in the glycolytic pathway is the production of pyruvate,
which in anaerobic conditions is converted to lactate. Under normoxic conditions, the
pyruvate dehydrogenase (PDH) complex (PDC) catalyzes the oxidative decarboxylation
of pyruvate to acetyl-CoA for use in the TCA cycle for cellular respiration. PDC is
composed of three catalytic components: pyruvate dehydrogenase (E1),
dihydrolipoamide acetyl-transferase (E2), and dihydrolipoamide dehydrogenase (E3).
PDC activity is inhibited in response to site-specific phosphorylation at three residues on
the PDH (E1) subunit: Ser232, Ser293, and Ser300 (Sale & Randle, 1981).
These reactions are catalyzed by one of four pyruvate dehydrogenase kinases
(PDK) that exhibit tissue-specific expression (Kolobova et al, 2001). PDK2 is reported to
be ubiquitously expressed, while PDK1 is expressed in the heart, pancreatic islets, liver
and skeletal muscle (Bowker-Kinley et al, 1998). PDK3 expression is primarily limited to
the testis, but is also expressed at low levels in the lung, kidney, spleen, heart and brain
(Bowker-Kinley et al, 1998). PDK4 is highly expressed in skeletal muscle and heart
while weakly expressed in the kidney, liver and lung (Bowker-Kinley et al, 1998).
15
The expression of different PDK isoforms depends markedly on the cancer type.
PDK1 is upregulated in many cancer cells and has been proposed as a key regulator of
the Warburg effect (Kim et al, 2006). It has been suggested that PDK1 could be used as a
biomarker of poor prognosis in patients with gastric cancer and head and neck squamous
cell cancer (Hur et al, 2013; Wigfield et al, 2008). PDK2 is a strong, independent
prognostic factor in cervical cancers (Lyng et al, 2006). PDK3, like PDK1, is upregulated
in colon cancer and its expression level is positively associated with tumor severity and
poor disease-free survival (Lu et al, 2011). Conflicting reports on the PDK4 expression
have been published; PDK4 upregulation is observed in renal, ovarian, prostate, lung and
skin cancer cells, while its downregulation is observed in breast, ovarian, lung, and colon
tumors when compared to its corresponding normal tissues (Blouin et al, 2011; Grassian
et al, 2011; Ross et al, 2000). By extension of their function to switch cells over to
glycolysis, cells with high expression of PDK1 and 4 are more resistant to anoikis
(Kamarajugadda et al, 2012). This is presumably a result of attenuation of mitochondrial
function, thereby lowering ROS production and sensitivity.
PDK1, 3 and 4 are reported to be hypoxia-responsive proteins under the control of
transcription factor HIF-α and are often upregulated during tumorigenesis (Kim et al,
2006; Lee et al, 2012; Lu et al, 2011). Hitosugi and colleagues showed that post-
translational activation of PDK1 can occur by tyrosine phosphorylation (Y243) due to
oncogenic tyrosine kinases localized in the mitochondria, thereby increasing the
conversion of pyruvate to lactate (Hitosugi et al, 2011).
16
Metabolic regulation by oncogenes and tumor suppressors
Research over the past decade has not only further elucidated the complexity of
tumor metabolism, but also has led to an alternative view towards mutations in oncogenic
signaling pathways. While metabolism was initially thought to be an indirect secondary
effect to oncogenic mutations, there is growing evidence to suggest that the primary
functions of activated oncogenes and inactivated tumor suppressors are to reprogram
cellular metabolism. It is becoming more clear that many key oncogenic signaling
pathways converge to adapt tumor cell metabolism in order to support growth and
survival (Ward & Thompson, 2012).
Oncogenic transcription factor MYC plays several roles in cell metabolism (Dang
et al, 2009). When overexpressed, MYC can induce the glycolytic phenotype in
transgenic cardiomyocytes in vivo. The first mechanistic link reported between an
activated oncogene and altered glucose metabolism was transcriptional activation of
LDHA by MYC (Shim et al, 1997). Knockdown of LDHA expression in MYC-
dependent tumors inhibits their growth (Le et al, 2010). MYC is able to induce the
splicing factors that are involved with the processing of the PK transcript to PKM2
(David et al, 2010). Certain genes involved in glutamine metabolism are under direct
transcriptional control of MYC (Gao et al, 2009; Wise et al, 2008). It enhances
transcription of glutaminase 1 (GLS1), an enzyme that catalyzes the hydrolysis of
glutamine to glutamate (DeBerardinis et al, 2007). MYC is also able to regulate
glutaminolysis through repression of miR-23a and miR-23b, two microRNAs shown to
inhibit expression of their target protein, GLS1 (Gao et al, 2009). Furthermore, MYC
17
cooperates with HIF to activate several glucose transporters and glycolytic enzymes,
including LDHA and PDK1 (Dang et al, 2008; Kim et al, 2007).
The tumor suppressor TP53 gene is the most commonly mutated gene in human
cancer (Kandoth et al, 2013). While p53 is given the title ‘guardian of the genome’, it
also serves as a regulator of cell metabolism (Vousden & Ryan, 2009). Wild type p53
functions to stimulate mitochondrial respiration and suppress glycolysis. Cytochrome c
oxidase assembly factor SCO2 transcription is stimulated by p53, which along with
SCO1 play an integral role in biogenesis of cytochrome c oxidase subunit II of the
electron transport chain (Leary et al, 2009; Matoba et al, 2006). p53 is able to indirectly
inhibit various glycolytic pathways by upregulating expression of TP53-induced
glycolysis and apoptosis regulator (TIGAR). TIGAR causes reductions in fructose-2,6-
bisphosphate levels and blocks further glycolytic reactions (Bensaad et al, 2006). Two
functional p53 motifs exist within the HKII promoter, indicative of p53 regulation
(Mathupala et al, 1997a). While MYC is responsible for GLS1 regulation, p53 is
responsible for GLS2 regulation with interestingly opposite effects on the cell (Hu et al,
2010). GLS1 catalyzes the conversion of glutamine to glutamate while GLS2 upregulates
antioxidant glutathione levels upon oxidative stress (Lora et al, 2004; Mates et al, 2009).
p53 also suppresses glucose uptake through repression of transcription of glucose
transporters (GLUTs) 1 and 4 (Dang, 1999). Expression of phosphatase and tensin
homolog (PTEN), a negative regulator of the PI3K pathway through dephosphorylation
of phosphatidylinositol (3,4,5)-triphosphate (PIP3), is upregulated by p53, further
suppressing glycolysis (Stambolic et al, 1998). Thus, loss of p53 in cancers may be a
major driving force behind acquiring the Warburg effect.
18
The PI3K pathway is one of the most commonly altered signaling pathways in
human cancers. Its activation can be attributed to mutations to tumor suppressors, such as
PTEN, in components of the PI3K complex itself, or by aberrant signaling from receptor
tyrosine kinases (Wong et al, 2010). AKT, frequently activated downstream of PI3K,
stimulates glycolytic flux by increasing translocation of glucose transporters to the cell
surface and by phosphorylating and altering localization of glycolytic enzymes such as
HKII and phosphofrutokinase 1 and 2 (Deprez et al, 1997; Elstrom et al, 2004; Majewski
et al, 2004a; Robey & Hay, 2009). AKT overexpression has been reported as an early
event during sporadic colon carcinogenesis (Roy et al, 2002). Although mitochondrial
acetyl-CoA is committed for further processing through the TCA cycle, it can be
processed to citrate via citrate synthase and be exported back out to the cytosol, where it
can be processed back to acetyl-CoA by ATP-citrate lyase (ACL). AKT is able to
phosphorylate and activate ACL, thereby facilitating the diversion of mitochondrial
citrate to cytoplasmic acetyl-CoA production to be directed instead for anabolic pathways
(Bauer et al, 2005; Berwick et al, 2002; Hatzivassiliou et al, 2005). AKT also increases
HIF1 activity via activation of downstream kinase FKBP-rapamycin-associated protein
(FRAP), further enhancing induction of glycolysis (Laughner et al, 2001).
Approximately 30% to 50% of colorectal tumors have a mutated KRAS gene
(Wilson et al, 2010). Colon carcinoma cells under hypoglycemic conditions have been
shown to increase RAS mutation rate, which upon transcriptional activation, facilitates
glucose uptake through induction of GLUT1 (Yun et al, 2009). In a multistep, multigene
transformation study of human breast epithelial cells, it was documented that while initial
transformation by viral oncogenes and telomerase reverse transcriptase increased
19
mitochondrial function, it took KRAS activation as the final reaction step to transform
cells to exhibit a glycolytic phenotype (Ramanathan et al, 2005). It’s hypothesized that
activated RAS induces MYC activity and enhances non-hypoxic levels of HIF1, although
the mechanisms remain unclear (Kikuchi et al, 2009; Sears et al, 1999).
Taken together, alternations in regulation of oncogenes and tumor suppressors
concomitantly rewire and coordinate cellular metabolism to meet the neoplastic demands
for biosynthetic precursors for rapid growth and proliferation.
Warburg effect as a therapeutic target
The concept of exploiting cancer cell metabolism is nothing new to clinical
practice. For nearly 50 years since the introduction of chemotherapy, this strategy has
been in practice. In principle, current chemotherapeutics aim to target all rapidly dividing
cells by interfering with critical pathways that are all, on some level, driven by cellular
metabolism. Antimetabolites were among the first effective chemotherapeutic agents
further supporting the concept of targeting cancer metabolism as a treatment strategy
(Kaye, 1998).
Aminopterin, a 4-amino analog of folic acid, was the first antimetabolite used in
cancer treatment. In 1947, it was the first drug shown to induce remissions among
patients with acute lymphoblastic leukemia (Farber & Diamond, 1948). Methotrexate
soon replaced aminopterin and is still currently used in the treatment of several cancer
types (Meyer et al, 1950; Purcell & Ettinger, 2003).
5-fluoruracil (5-FU) has been in clinical use for 40 years, and is used to treat a
variety of cancer types including CRC (Longley et al, 2003; Seifert et al, 1975). It
20
inhibits thymidylate synthase and the methylation of deoxyuridine monophosphate into
deoxythymidine monophosphate, the latter of which is required for DNA replication
(Peters et al, 2002). It was the only effective drug against colorectal cancer for decades
until the late 1990s when other anti-cancer agents arrived on scene, including
capecitabine, oxaliplatin, bevacizumab, cetuximab and most recently, regorafenib
(Sridharan et al, 2014).
Over the past decade, advances in our understanding of cancer cell metabolism
have allowed scientists to better comprehend and appreciate its complexity. Development
of targeted therapies now aims to exploit metabolic vulnerabilities in cancer cells and
specifically target cancer cell-specific metabolic alterations. The Warburg effect is a
metabolic phenotype that neoplasms share with very few other cell types, as previously
discussed. Therefore, inhibition of aerobic glycolysis can reduce tumor malignancy by
decreasing proliferation and apoptotic resistance, and ultimately inducing cell death,
providing a useful therapeutic window. Furthermore, acidic extracellular environments
lead to immunosuppression (Fischer et al, 2007). Thus, switching aerobic glycolysis to
mitochondrial respiration may provide an additional benefit of improving immune
function in already immunosuppressed patients.
2-deoxyglucose (2DG) is a glucose analog initially shown to be phosphorylated
by hexokinase and cannot be further metabolized, thereby acting as a competitive
inhibitor. However, later studies revealed that the principal mechanisms of 2DG’s
anticancer effects vary (Kurtoglu et al, 2007; Zhong et al, 2008). Although studies have
not supported its anticancer potential as a single agent in vivo, 2DG synergizes with other
anti-cancer compounds such as adriamycin and cisplatin (Maschek et al, 2004; Simons et
21
al, 2007). Lonidamine is a derivative of indazole-3-carboxylic acid, and a specific
inhibitor of HKII. A phase III trial targeting prostate cancer has been completed, although
its clinical success was limited due to significant pancreatic and hepatic toxicities (Price
et al, 1996). FK11, a novel LDH inhibitor, inhibits growth of both human B-lymphoma
and pancreatic cancer xenografts (Le et al, 2010).
Imatinib (Gleevec), among its many targets, inhibits constitutively active tyrosine
kinase BCR-ABL, a fusion protein found in nearly all patients with chronic myeloid
leukemia (CML) (Cambier et al, 1998; Kantarjian et al, 2011). BRC-ABL induces
increased glucose uptake and utilization through upregulation of the PI3K/AKT signaling
pathway (Elstrom et al, 2004). Imatinib reverses the Warburg effect through decreased
glucose uptake, reduced HK and G6PDH activity and increased mitochondrial
respiration. It has been approved by the US Food and Drug Administration and is used as
an anticancer drug against CML and gastrointestinal stromal tumors (Druker et al, 2006;
Druker et al, 2001; Joensuu et al, 2001).
The above examples support the significant opportunity metabolic pathways
provide for cancer control strategies. Therefore, it is imperative to continue to further our
knowledge of cancer metabolism in order to develop more effective and exclusive cancer
therapies that exploit the Warburg effect. In this thesis, exclusive focus will be placed on
examining dichloroacetate and 3-bromopyruvate as possible anti-cancer agents against
colorectal cancer.
22
Dichloroacetate
Humans have long been exposed to dichloroacetate (DCA) through water
chlorination and the metabolism of various chlorine-containing drugs or chemicals
(Stacpoole et al, 1998). It is an off-patent small molecule that has been in use for many
years to treat diseases such as lactic acidosis and inherited defects in mitochondrial
metabolism (Stacpoole et al, 2008; Stacpoole et al, 2003). In 1962, diabetic rats treated
with diisopropylammonium dichloroacetate showed a reduction in hyperglycemia and
increased respiration (Lorini & Ciman, 1962). The ability of this compound to promote
glucose oxidation exclusively in diabetic rats was thought to be due to its acid moiety,
dichloroacetate (Stacpoole & Felts, 1970). In 1973, researchers further showed that DCA
treatment led to an increase in glucose and pyruvate oxidation associated with an increase
in PDH-catalyzed acetyl-CoA levels (McAllister et al, 1973). The link between DCA and
PDK was finally elucidated in 1975 (Halestrap, 1975).
Of the 4 PDK isoforms, PDK2 is most sensitive to DCA. PDK inhibition by DCA
is a result of direct drug-protein interaction, whereby the carboxylate group of DCA
forms a salt bridge with Arg154 of PDK2 (Knoechel et al, 2006). Inhibition of PDK2 by
siRNA mimicked the physiological effect of DCA on cancer cells, and addition of DCA
in combination with PDK2 siRNA had no additional effects (Bonnet et al, 2007).
DCA is able to promote preferential oxidation of carbohydrates over fatty acids in
cardiac tissue and increase efficient energy production, thereby improving myocardial
function in vitro and in vivo (Bersin & Stacpoole, 1997). DCA is able to enhance
myocardial efficiency in patients with coronary artery disease (Wargovich et al, 1988).
This disease can lead to cardiac hypertrophy, in which cells exhibit a glycolytic shift, and
23
treatment with DCA reversed the metabolic shift and improved cardiac output (Piao et al,
2010). DCA therapy can improve post-ischemic function and recovery in rat hearts by
coupling glycolysis with glucose oxidation following cardiac hypertrophy (Wambolt et
al, 2000).
DCA had shown promise as a therapy for lactic acidosis, a potentially fatal
disease caused by buildup of lactic acid in tissues and blood (Stacpoole et al, 1988).
However, it showed little effect on the clinical outcome of patients, and further revealed
significant changes in blood pH following treatment. Thus, results have not translated
well in clinical trials (Stacpoole et al, 2006; Stacpoole et al, 1992).
In 2007, Bonnet and colleagues showed that exposing rats to DCA in drinking
water caused a significant regression of their xenografted A549 lung carcinoma cells
(Bonnet et al, 2007). Furthermore, they showed that the metabolic agent was able to
induce apoptosis through a mitochondrial-dependent pathway in cancer cells without
affecting normal cells (Bonnet et al, 2007). This metabolic shift to oxidative
phosphorylation will increase ROS production and alter mitochondrial membrane
potential, leading to the release of sequestered cytochrome c and activation of the
apoptotic-signaling cascade. Other studies have also shown DCA-induced apoptosis in
different cancer models, including ovarian (Saed et al, 2011), prostate (Cao et al, 2008),
(Madhok et al, 2010), and malignant melanoma (Populo et al, 2015).
Although several studies highlight the ability of DCA to induce apoptosis in vitro,
in vivo results show that DCA treatment slows tumor growth, rather than causes tumor
eradication. Metastatic breast cancer in vivo showed 58% decrease in lung metastases
following DCA treatment (Sun et al, 2010). The same study showed cell-line dependent
24
reduction in proliferation by 20-80% without increase in apoptosis following DCA
exposure in rat mammary adenocarcinoma cells in vitro. In multiple myeloma, certain
cell lines exhibited reduced proliferation without apoptotic induction at low doses of
DCA, while high doses of DCA led to smaller reduction in proliferation but an increase
in apoptosis (Sanchez et al, 2013). This dose effect was also observed in a separate study
on colorectal cancer cell lines (Madhok et al, 2010). However, DCA treatment of SW480
CRC xenografts led to an increase in tumor growth (Shahrzad et al, 2010). Furthermore
in HCT116 CRC cells, DCA exposure led to a reduction in proliferation with no effects
on cell survival (Delaney et al, 2015). Therefore, the effects of DCA may be cancer cell
line specific and dose-dependent.
The Warburg effect increases HIF1α levels and stimulates angiogenesis in vitro
and in vivo (Lu et al, 2002). DCA counteracts this effect in non-small cell lung cancer
and mammary carcinoma, in which a downregulation in HIF1α and decreased ability to
initiate and sustain angiogenesis in vitro and in vivo was shown (Sutendra et al, 2013).
Glioma cells stabilize HIF1α through IDH mutations that decrease cellular α-
ketoglutarate levels (Zhao et al, 2009). DCA is able to inhibit angiogenesis in
glioblastoma in vivo through increasing cellular α-ketoglutarate levels and destabilizing
HIF1α (Michelakis et al, 2010). A decrease in HIF1α levels as a result of DCA treatment
has also been reported in melanoma and T cell lymphoma (Kluza et al, 2012; Kumar et
al, 2012).
DCA treatment has been particularly successful against brain tumors such as
gliomas and glioblastomas, possibly attributed to its ability to cross the blood-brain
barrier (Duan et al, 2013; Kumar et al, 2013). The first clinical trial in glioblastoma was
25
conducted in 2010 (Michelakis et al, 2010). In freshly isolated glioblastoma tumors from
49 patients, DCA treatment depolarized mitochondria, increased mitochondrial ROS,
increased apoptosis, decreased proliferation, and decreased HIF1α expression with a
corresponding decrease in tumor vasculature. Additional Phase I trials have since been
conducted to determine drug safety and tolerability in patients (Chu et al, 2015; Dunbar
et al, 2013). In a Phase II clinical trial, 5 glioblastoma patients were treated with DCA for
15 months at a therapeutic concentration of approximately 0.5 mM (Michelakis et al,
2010). All but one of those patients, who died after three months, were clinically stable
after this regimen. However, there are significant drawbacks to all these trials. First, no
DCA-free group was included in the respective study designs. Secondly, the patients
recruited for these studies had all previously been heavily treated with chemotherapy
and/or radiation, and some were receiving standard treatment along with DCA.
Therefore, the beneficial outcomes of DCA treatment as outlined by these studies cannot
be attributed to DCA alone. Currently, clinical trials to study DCA in combination with
cisplatin for head and neck cancers, as well as DCA treatment in glioblastomas and other
neoplasms are ongoing (U.S. National Institutes of Health, 2015).
3-bromopyruvate
The pyruvate/lactate analog 3-bromopyruvate (3BP) was initially reported to
regulate enzyme activity in 1969 (Baker & Rabin, 1969). Fonda demonstrated the ability
of 3BP to alkylate cysteine residues via 3BP-mediated inactivation of glutamate
apodecarboxylase (Fonda, 1976) and its alkylating properties were outlined in a 1978
publication (Meloche et al 1978). Other subsequent studies validated the ability of 3BP to
26
inhibit other metabolic enzymes such as malate dehydrogenase, SDH, and carbonic
anhydrase (Chang & Hsu, 1973; Conroy & Maren, 1985; Sanborn et al, 1971).
In the past decade, numerous studies have highlighted the effects of 3BP on
cancer cells and its potential as an effective anti-cancer agent. 3BP-induced cell death has
been shown to be tumor-specific with limited cytotoxic effects against normal cells. 3BP
was less effective in weakly malignant pancreatic ductal adenocarcinoma cells and had
no effect on non-malignant cells (Isayev et al, 2014). A reason for its tumor-specific
actions could be due to an upregulation of monocarboxylate transporters (MCTs) in
cancer cells (Pinheiro et al, 2009; Pinheiro et al, 2010). The family of MCT proteins
helps to deliver monocarboxylates such as pyruvate and lactate across the cellular plasma
membrane. MCT1 in particular appears to be the main transporter of 3BP to glycolytic
tumors (Birsoy et al, 2013). Pretreatment of some breast cancer cell lines with butyrate
enhances MCT1 expression and sensitizes cells to 3BP treatment (Queiros et al, 2012). In
this scenario, 3BP may act as a ‘Trojan horse’ metabolite by mimicking lactate/pyruvate
but with deleterious effects once it enters the cell.
In 2001, Ko et al. initially showed the potential of 3BP to specifically kill cancer
cells in the rabbit VX2 tumor model of hepatocellular carcinoma (Ko et al, 2001). It was
later shown that the compound was able to eradicate all the tumors induced in 19 rats
tested with no obvious toxic effects or recurrence (Ko et al, 2004). Furthermore, the first
human case report was published in 2012 highlighting promising anti-tumor results in a
young adult cancer patient with fibrolamellar hepatocellular carcinoma without apparent
cytotoxicity (Ko et al, 2012). Other studies have also shown the potential of 3BP as a
therapeutic agent in different cancer models, including colorectal (Ihrlund et al, 2008;
27
Lea et al, 2013; Sanchez-Arago & Cuezva, 2011), breast (Queiros et al, 2012), ovarian
(Wintzell et al, 2012), pancreatic (Isayev et al, 2014), leukemia (Chen et al, 2009b), brain
(Davidescu et al, 2012; El Sayed et al, 2012), malignant mesothelioma (Icard et al,
2012), and malignant myeloma (Nakano et al, 2012).
The alkylating properties of 3BP allow it to react with thiol (SH) and hydroxyl
(OH) groups of various enzymes. One of its principle targets is HKII, an enzyme
upregulated in cancer cells (Ko et al, 2001). While the compound 2DG acts as a direct
competitor against glucose via HK phosphorylation, 3BP is able to inhibit mito-HKII
activity through formation of a pyruvinylated-adduct with –SH group(s) of the enzyme
(Oronsky et al, 2012). This in turn promotes the release of HKII from the mitochondria,
leading to depletion of cellular ATP levels and allowing pro-apoptotic molecules to bind
onto free VDAC sites and promote the release of apoptotic inducing factor (AIF) and
eventual cell death (Chen et al, 2009b; Kim et al, 2008). In addition, direct dissociation
of HKII from the mitochondria using a peptide was sufficient to induce cell death, but
unlike 3BP it did so without ROS induction or loss of mitochondrial membrane potential
(Chen et al, 2009b).
It is speculated that 3BP cytotoxicity is mediated through several combined
regulatory mechanisms, including ROS overproduction, ATP depletion, and modulations
to survival signaling pathways (Kim 2008, El Myiyad 2011, Calvino 2014). Researchers
believe that the rapid induction of cell death by 3BP is too fast to be simply accounted for
by the alkylation of HKII (Pereira da Silva et al, 2009). As an alkylating agent, the
effects of 3BP would not be limited to HKII. In agreement, 3BP affects various enzymes
involved in both glycolysis and mitochondrial respiration. Pereira and colleagues
28
demonstrated that exposure of HepG2 cells to 150 µM 3BP for 30 min led to significant
enzyme modifications involved in the glycolytic pathway (Pereira da Silva et al, 2009).
The activities of GADPH and phosphoglycerate kinase (PGK) were also strongly
suppressed (90 and 70% of control, respectively) while PK activity was almost twice that
of control. Furthermore, 3BP was shown to inhibit mitochondrial respiratory complex II
SDH activity. 3BP inhibits the efflux of lactate in HepG2 cells, altering the internal pH of
cells (Pereira da Silva et al, 2009). It is speculated that this is a result of MCT4
alkylation, thereby preventing the shunting of lactate out of the cell.
Several studies have shown different responses to 3BP at different doses in
various cancer types. In human myeloid leukemic cells, 3BP induced both apoptosis and
necrosis at 20-30 µM and pure cell necrosis at 60 µM (Calvino et al, 2014). El Sayed and
colleagues showed caspase-dependent cell death in C6 glioma cells with 3BP treatments
as low as 15 µM while Davidescu and colleagues demonstrated 3BP-induced autophagy
in GL15 glioblastoma cells at 80 µM (Davidescu et al, 2012; El Sayed et al, 2012). Huh-
7 hepatocellular carcinoma cells treated with 100 µM 3BP led to a significant increase in
apoptosis (Yu et al, 2012). Therefore, cellular response to 3BP appears to be dose and
cell line-dependent.
Tumor microenvironment
Tumors, like normal tissues, require a blood supply to gain access to the required
oxygen, nutrients and method for removal of waste products (Papetti & Herman, 2002).
Tumor expansion progressively distances cells from the normal vasculature, and
therefore oxygen and nutrients. During tumorigenesis, a solid tumor cannot exceed 2-3
29
mm3 without an adequate blood supply (Folkman, 1971). A fundamental hallmark of
cancer is the release of pro-angiogenic factors to stimulate the formation of new blood
vessels in a process known as angiogenesis (Holash et al, 1999). Tumor vasculature is
often characterized as disorganized, tortuous, leaky, and dilated, with uneven diameter,
and excessive branching and shunts (Carmeliet & Jain, 2000). Therefore, the variability
in blood flow within the tumor leads to a heterogeneous microenvironment with areas of
hypoxia, acidity, and low nutrient supply (Raghunand et al, 2003).
Hypoxia
Hockel and colleagues initially showed that hypoxia could be a possible
prognostic factor for cancer patients in a study of cervical cancer (Hockel et al, 1996a).
In 2005, an international multi-centre study involving 397 patients with head and neck
tumors provided further evidence of the prognostic value in tumor hypoxia (Nordsmark et
al, 2005). Hypoxia influences the effectiveness of chemotherapy by slowing cell cycle
progression and limiting drug diffusion (Brown, 1999). The expression of multidrug
resistance (MDR) genes is upregulated under hypoxic conditions, further providing
hypoxic cells with a selective advantage during chemotherapy (Wartenberg et al, 2003).
Hypoxia has also been associated with poor response to radiochemotherapy (Brizel et al,
1997; Hockel et al, 1996b; Nordsmark et al, 1996). Due to its ability to affect both
radiation and chemotherapeutic protocols, hypoxia is problematic in CRC treatment. In
CRC patients, the percentage of hypoxic cells in tissue biopsies varied from 2.2 to 37.8%
(Goethals et al, 2006). As the availability of oxygen to cancer cells is a direct function of
cell distance from a nearby functional blood supply, this leads to a gradient of diffusion-
30
limited hypoxia (Secomb et al, 1993), with both hypoxic (pO2 < normal) and anoxic (pO2
= 0 mmHg) microenvironments existing in the tumor (Vaupel et al, 1989). Differences in
vascular dependence of cancer cells are in part mediated by HIF1α expression; cells
lacking HIF1α expression were far less dependent on blood vessel proximity for their
growth and survival in vivo (Yu et al, 2001).
Hypoxia can be broadly categorized into 2 types: chronic or diffusion-limited
hypoxia, and acute, intermittent, or transient hypoxia (Brown, 1979; Thomlinson & Gray,
1955). Chronic hypoxia occurs in cells that are more than 100 µm away from blood
vessels, thus making this type of condition a function of vasculature distance/vascular
density. Acute hypoxia occurs as a result of chaotic vasculature and high interstitial
pressures within tumors, leading to vascular stasis and local fluctuations in blood
perfusion and oxygen supply. Tissue re-oxygenation/reperfusion leads to cellular damage
mediated by increased oxidative stress and ROS production. Administration of
superoxide dismutase at the time of vascular reperfusion leads to signification protection
against cell death (Parkins et al, 1995).
The ability of cells to adapt to changes in oxygen tension is in large part attributed
to HIF1 function. In 1992, Semenza and colleagues identified HIF1 and its consensus
binding sequence in the erythropoietin gene of hypoxic cells (Semenza & Wang, 1992).
Studies have shown that the activation of HIF1 has a profound effect on tumorigenesis by
activating transcription of genes involved with apoptosis resistance, invasion, metastasis
and angiogenesis (Lopez-Lazaro, 2006; Semenza, 2003; Semenza, 2006).
HIF1 is a heterodimer consisting of a constitutively expressing HIF1β subunit and
the oxygen-responsive HIF1α subunit (Jiang et al, 1996). The functionality of HIF1 is
31
dependent on the α subunit, whose synthesis is regulated through an oxygen-independent
process, whereas its degradation is oxygen-dependent (Semenza, 2003). HIF1α is able to
recognize hypoxia-responsive elements (HREs) in the promoter region of target genes
(Hu et al, 2003).
HIF1 Regulation
High HIF1α expression correlates with poor survival in numerous cancer types,
including breast (Bos et al, 2003; Gruber et al, 2004; Trastour et al, 2007; Vleugel et al,
2005), head and neck (Aebersold et al, 2001), esophagus (Matsuyama et al, 2005),
stomach (Griffiths et al, 2007), and lung (Swinson et al, 2004). HIF1α displays one of the
shortest half-lives (<5 mins) among cellular proteins in well-oxygenated cells (Berra et
al, 2001). In normoxia, HIF1α is degraded through posttranslational modification by
prolyl hydroxylation. Once hydroxylated, HIF1α will be targeted by and bound to the von
Hippel-Lindau (VHL) tumor suppressor protein – the recognition component of an E3
ubiquitin-protein ligase (Kaelin, 2003). Following its ubiquitination, HIF1α will rapidly
be degraded by proteasomes (Maxwell et al, 1999). In hypoxia, HIF1α is not modified by
oxygen-sensitive prolyl hydroxylases and thus becomes stabilized (Stiehl et al, 2006).
Other tumor suppressor effectors have been implicated in the regulation of HIF1 activity.
For instance, loss of PTEN facilitates HIF1-mediated gene expression (Zundel et al,
2000), and p53 association with HIF1α inhibits HIF1-stimulated transcription
(Blagosklonny et al, 1998).
The net result of hypoxic HIF1 activation is a shift in energy production from
mitochondria-dependent oxidative phosphorylation to mitochondria-independent
32
glycolysis. Papendreou and colleagues were the first to show that HIF1 is both necessary
and sufficient for downregulation of mitochondrial function in hypoxia (Papandreou et al,
2006). It coordinately drives glycolysis; all 12 glycolytic enzymes are in various ways
directly under HIF1 regulation (Iyer et al, 1998; Seagroves et al, 2001).
Interestingly, high levels of HIF1α have been shown in well-oxygenated tumors,
indicating potential oxygen-independent mechanisms of HIF1α stabilization (Hagg &
Wennstrom, 2005). Thus, as a result of aberrant activation of oncogenic signaling
pathways in cancers and mutations in tumor suppressor genes, HIF1 is activated even
under normoxic conditions. AKT increases HIF1 activity independent of O2 tension
(Inoki et al, 2005; Plas & Thompson, 2005). VHL is lost in renal cell carcinoma,
resulting in elevated non-hypoxic expression of HIF1α (Gordan et al, 2008). Mutations to
both SDH and FH that lead to accumulation in succinate and fumarate, respectively, have
been shown to stabilize HIF1 in normoxic cells. HIF1α hydroxylation by prolyl
hydroxylase domains involves the conversion of α-ketoglutarate to succinate. Increased
levels of succinate resulting from mutations in SDH can slow this enzyme, leading to
HIFα stabilization (Isaacs et al, 2005; Selak et al, 2005). Thus, glycolytic genes under
HIF1 regulation are transcribed even in the presence of oxygen, increasing the glycolytic
flux observed in cancer cells (Semenza, 2010).
Hypoglycemia
Like hypoxia, there is a predicted concentration gradient with glucose availability
in the extracellular space to cancer cells (Kallinowskil et al, 1985). Unlike hypoxia
however, the effects of hypoglycemia on tumor progression are not well studied. Based
33
on tumor cells’ reliance on glucose metabolism for both anabolic and catabolic processes
to sustain its rapid progression, a limitation of glucose should be expected to hinder
proliferative signaling and proliferative potential.
Cancer cells have a preference for either glucose or lactate, depending on their
microenvironment. Cells at greater distances from blood vessels rely more heavily on
glycolysis for energy production and thus show an increase in lactate production. Cells
that are located near blood vessels, and are thus aerobic, preferentially utilize lactate
secreted by glycolytic tumor cells as their main source of fuel (Sonveaux et al, 2008).
This allows the diffusion of glucose over a greater distance to reach glycolytic cells that
would otherwise be deprived of glucose. In return, these glycolytic cells produce lactate
that is effluxed out to the extracellular space to fuel aerobic cells, generating a positive
feedback loop between tumor cells across great distances of different microenvironments.
Thus, a lactate gradient is observed that mirrors the oxygen gradient in the tumor. This is
in large part controlled by MCTs 1 and 4 (Sonveaux et al, 2008). Lactate uptake by
aerobic tumor cells occurs through high-affinity lactate transporter MCT1, whereas
lactate produced by glycolysis-driven tumor cells is released through low-affinity lactate
transporter MCT4. This substitution to utilize lactate for oxidation is also observed in
ischemic brain recovery (Tanaka et al, 2004).
Previous work in our laboratory found that DCA provided a cytoprotective effect
to different human colorectal cancer cell lines when cultured under anoxic conditions
(Shahrzad et al, 2010). Furthermore, DCA-treated immune deficient mice bearing
SW480 CRC cell xenografts exhibited significantly increased tumor growth than in
untreated mice. Conversely, hypoxic conditions increased the sensitivity of colorectal and
34
lymphoma cells to 3BP (Xu et al, 2005). Strese and colleagues screened 19 commercially
available anti-cancer drugs on various cancer cell lines under different oxygen tensions.
They found that sorafenib, irinotecan, and docetaxel were in general more effective in an
oxygenated environment, while cisplatin, mitocycin c, and tirapazamine were more
effective in low oxygen environments (Strese et al, 2013). These findings suggest that the
efficacy of compounds targeting the glycolytic pathway might be dependent on the tumor
microenvironment.
In summary, the Warburg effect plays a critical role in tumor development by re-
wiring cellular metabolism to support an increased demand for anabolic processes, while
inhibiting mitochondrial driven apoptosis. Due to this 'metabolic addiction', targeting
cancer cell metabolism may provide a promising therapeutic window leading to the
development of novel anti-cancer agents. This thesis aims to explore the effects of
targeting different glycolytic enzymes shown to be upregulated in cancer progression,
with interest on the impact of the tumor microenvironment on drug efficacy.
35
RATIONALE
The ability of neoplastic cells to adapt and rewire metabolic signaling to produce
the necessary precursors to sustain continuous proliferation is a key aspect to cancer
being a disease of uncontrolled growth. Ever since 1926 when Dr. Otto Warburg made
the initial observations on the unique metabolic phenotype of cancer cells, this
relationship has gained popularity and researchers have been attempting to exploit this
avenue and find alternative therapeutics against cancer. An important aspect that has been
overlooked when examining the effects of various drugs as possible anti-cancer agents is
the tumor microenvironment. It is of particular importance when targeting metabolism as
the microenvironment has direct implications upon how neoplastic cells may regulate
their metabolic pathways.
Previous results from our laboratory showed that DCA provided a cytoprotective
effect to some human colorectal cancer cells under anoxic conditions. This led to interest
in further understanding the mechanisms that may be regulating this cytoprotection and
how the effects of the tumor microenvironment may impact the efficacy of other
metabolic-targeting compounds on human CRC cells. The first objective of this thesis is a
direct follow-up of the work on DCA-induced cytoprotection in anoxic human colorectal
cancer cells. Specifically, I assessed the expression of key glycolytic enzymes that are
directly affected by the effects of DCA: PDH and PDK. Furthermore, I also examined
changes in mitochondrial activity and metabolic output of cells following anoxic DCA
exposure. I hypothesized that the DCA-induced cytoprotection observed could be
attributed to differences in expression and activation of these molecules between various
human colorectal cancer cells.
36
The second and third objectives of this thesis looked to answer whether
hexokinase II is a viable enzyme target in human colorectal cancer. More specifically, the
second objective examined this question by treating human colorectal cancer cells with 3-
bromopyruvate and assessing its efficacy. I took into consideration the regulation of the
enzyme under varying glucose concentrations and examined the effects of the compound
under hypoglycemia conditions. I hypothesized that while 3BP would be able to induce
cell death, its efficacy would be regulated by cellular glucose availability and subsequent
regulation of HKII expression.
Based on the results of the second objective, I decided to examine hexokinase II
as a possible prognostic biomarker for human colorectal cancer. The experimental design
for the third objective utilized immunofluorescence staining of formalin-fixed paraffin-
embedded samples of human colorectal cancer tumor. I determined HKII expression
levels in these samples and correlated staining patterns to both its relation in different
microenvironments and to patient outcomes. I hypothesized that strong HKII expression
would correlate with worse outcome for patients with colorectal cancer.
In conclusion, these studies aimed to investigate how an intrinsic property of solid
tumors impacts expression of glycolytic enzymes and cellular metabolism using human
CRC as a model. Furthermore, I sought to better understand the impact of the tumor
microenvironment when targeting different aspects of tumor metabolism and the
Warburg effect.
37
CHAPTER II – EFFECTS OF DCA ON ANOXIC HUMAN COLORECTAL CANCER CELLS
A modified version of this chapter is already published:
Ho, N., Coomber, B.L. 2015. Pyruvate dehydrogenase kinase expression and metabolic changes following dichloroacetate exposure in anoxic colorectal cancer cells.
Experimental Cell Research 331: 73-81
Introduction
Cancer cells undergo a metabolic transition into a state of aerobic glycolysis
known as the “Warburg effect”, characterized by a paradoxical reliance on glycolytic
pathways for pyruvate reduction under environments of high oxygen tension (Warburg et
al, 1927). This unique metabolic profile provides cancer cells with numerous selective
advantages, including adaptation to hypoxia, resistance to mitochondria-mediated
apoptosis, and acidification of the tumor microenvironment leading to increased tumor
invasion and metastasis (Gillies & Gatenby, 2007; Gogvadze et al, 2010). Studies have
correlated the glycolytic state of tumor cells with cancer aggressiveness, further
establishing a critical role for the Warburg Effect (Simonnet et al, 2002).
Due to the highly disorganized and chaotic nature of the tumor vasculature,
regions of solid tumors are transiently and/or chronically exposed to ischemia and
reperfusion, leading to hypoxic/anoxic conditions, which are also known to be mutagenic
(Raghunand et al, 2003). The impact of the various microenvironments found in solid
tumors on the effectiveness of anti-cancer therapies is not well understood. The Warburg
effect enables cancer cells to use glycolytic pathways regardless of the
microenvironment, preventing metabolic dictation and possibly less efficient switching
between different strategies.
38
As discussed in Chapter I, an important branching point in glucose metabolism is
the production of pyruvate. Its use as a precursor for either subsequent fermentation in
the cytosol or mitochondrial uptake for processing through the TCA cycle and oxidative
phosphorylation is dictated by the function of the pyruvate dehydrogenase complex. Its
activity is regulated by site-specific phosphorylation at three serine residues of PDH:
Ser232, Ser293 and Ser300 (Sale & Randle, 1981). PDH phosphorylation is catalyzed by
the four PDK isoforms that exhibit tissue-specific expression (Kolobova et al, 2001).
PDK1, 3 and 4 are reported to be hypoxia-responsive proteins under the control of
transcription factor HIF-α and are often upregulated during tumorigenesis, playing a
crucial role in the Warburg effect (Kim et al, 2006; Lee et al, 2012; Lu et al, 2011).
Furthermore, PDK1 has been proposed as a key regulator of the Warburg Effect (Kim et
al, 2006). It has been suggested that PDK1 could be used as a biomarker of poor
prognosis in patients with gastric cancer and head and neck squamous cell cancer (Hur et
al, 2013; Wigfield et al, 2008).
Dichloroacetate (DCA) is an inexpensive small molecule that is currently being
evaluated in phase I/II clinical trials for solid tumors, including glioblastoma and glioma
(Chu et al, 2015; Dunbar et al, 2014; Yeluri et al, 2012). DCA inhibits PDK activity,
thereby indirectly activating PDH and shifting metabolism to mitochondrial glucose
oxidation (Stacpoole, 1989). Bonnet et al. initially showed that DCA exposure led to a
significant regression of tumors derived from A549 lung carcinoma cells xenografted in
rats (Bonnet et al, 2007). Other studies have also demonstrated DCA-induced apoptosis
in different cancer models, including ovarian (Saed et al, 2011), endometrial (Wong et al,
2008), neuroblastoma (Michelakis et al, 2010) and colorectal cancer (CRC) (Madhok et
39
al, 2010). However, my laboratory previously determined that DCA provided a
cytoprotective effect to some CRC cells under anoxic conditions in vitro and in vivo
(Shahrzad et al, 2010). Colorectal cancer is the third most common cancer worldwide,
and is the second leading cause of cancer-related deaths in Canada (Canadian Cancer
Society). Due to its high prevalence, a better understanding of the responses of CRC to
DCA is needed. This chapter begins to explore some of the molecular mechanisms by
which cancer cells under different microenvironmental stimuli may respond to
chemotherapeutics. In this study, I further elucidate the mechanisms by which DCA may
act on CRC cells under different oxygen tensions with particular emphasis on PDK
expression and PDH regulation.
Materials and Methods
A list of all chemicals and suppliers, and details of the materials and solutions
used for the following experiments are detailed in Appendices I and II, respectively.
Cell culture
Human colorectal cancer cell lines SW480 and LS174T and rat intestinal
epithelial cell line IEC-18 were obtained from ATCC (Manassas, VA, USA). Cells were
maintained in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, and 50
µg/ml gentamicin. Normal human colon mucosal epithelial cell line NCM460 was
obtained from INCELL and maintained in M3:D culture media supplemented with 10%
FBS. All cell lines were incubated at 37oC in a humidified atmosphere containing 5%
CO2 in room air (“normoxia”).
40
In vitro exposure to anoxia and/or DCA
Cells were seeded into plates and incubated under standard cell culture conditions
overnight. Thereafter, plates were assigned to control and anoxia treatment, with or
without 10 mM DCA (Sigma-Aldrich) for the indicated time periods. Anoxic conditions
were achieved using a Modular Incubator Chamber (Billups-Rothenberg Inc., Del Mar,
CA, USA) modified to allow continuous flushing of the chamber with a humidified
mixture of 95% N2 and 5% CO2; the oxygen content in the chamber was less than 0.1%
in all anoxic experiments.
Measurement of cell growth
Cell number was approximated through crystal violet staining. Briefly, 5 x 103
cells were seeded into a 96-well plate and cultured in the presence or absence of DCA in
normoxia or anoxia for 72 h. Post-treatment, media was aspirated and 1% crystal violet
solution was added to each well and incubated for 10 min in room temperature. Plates
were then aspirated, rinsed and left to dry overnight. 10% acetic acid was used to dissolve
the crystals and the absorbance at 570 nm was recorded. Cell numbers were extrapolated
through standard curves previously generated for each cell line. For NCM460 cells, 1 x
104 cells were seeded for the experiments described above.
qRT-PCR for PDK mRNA expression
Following 24 h treatment, RiboZol was used to homogenize cells and isolate total
RNA according to the manufacturer’s protocol. Samples were further purified using the
41
Aurum Total RNA Fatty and Fibrous Kit. iScript Reverse Transcription Supermix reagent
was used for cDNA synthesis under the following thermal cycle conditions: 25oC for 5
min, 42oC for 30 min, 85oC for 5 min. qRT-PCR was performed using the SsoFast
EvaGreen Supermix reagent and reaction conditions were optimized according to the
MIQE guidelines (Bustin et al, 2009) to achieve an assay performance efficiency of 90-
110%. The following primers for qRT-PCR analysis were obtained using Primer3
software (Rozen & Skaletsky, 1998) and outlined in Table I. qRT-PCR was conducted in
a CFX-96 Real-Time System (BioRad) under the following conditions: 95oC for 30 sec
followed by 40 cycles of 95oC for 10 sec and 59oC for 10 sec. Melt curve analysis was
performed immediately following PCR to ensure only the desired product was amplified.
Amplicons were sequenced to further validate their specificity. Gene expression was
analyzed using the CFX Manager software (Bio-Rad).
Protein isolation and western immunoblot analysis
Briefly, 5 x 105 cells were seeded in 60 mm2 plates and cultured in the presence or
absence of 10 mM DCA in normoxia or anoxia for 24 h. Following treatment, cells were
lysed on ice with lysis buffer supplemented with 2 µg/ml aprotinin, 1 mM PMSF and
phosphatase inhibitor cocktail II. Samples were centrifuged at 12,000 x g for 15 min at
4oC, and supernatant was aliquoted and stored at -80oC for future use. Protein was
quantified using the Bio-Rad DC Protein Assay Kit. 25 µg of total protein was resolved
in a 10% polyacrylamide gel using SDS-PAGE then transferred onto a polyvinylidene
difluoride (PVDF) membrane with wet transfer buffer at 100 V for 2 hr. Gels used to
subsequently detect caspase-3 expression were transferred onto PVDF membranes using
42
a Trans-Blot SD Semi-Dry Transfer Cell apparatus (Bio-Rad) with semi-dry buffer at 16
V for 30 min. Following transfer, membranes were blocked for 1 h at room temperature
in 5% non-fat dry milk diluted in 0.1% Tween 20 in TBS (TBST) followed by an
overnight incubation in blocking solution with primary antibodies. After washing,
membranes were incubated for 1 h at room temperature with the appropriate peroxidase-
conjugated secondary antibody, washed, and subjected to Luminata Forte
chemiluminescent substrate. Membranes were imaged using the ChemiDoc XRS+ system
(Bio-Rad) and densitometry was performed using Image Lab software (Bio-Rad).
Primary antibodies used included rabbit anti-caspase 3 (1:1,000), anti-PDK1(1:1,000) ,
anti-PDK2 (1:1,000), anti-PDK3 (1:1,000), anti-PDK4 (1:1,000), anti-pPDH Ser293
(1:1,000), anti-pPDH Ser300 (1:1,000), mouse anti-PDH (1:1,000), mouse anti-α-tubulin
(1:100,000), and rabbit or mouse HRP-labeled secondary antibodies (all 1:10,000).
FACS analysis of mitochondrial function
Mitochondrial activity was quantified through dual-labeling of cells with
MitoTracker dyes. Briefly, 5 x 105 cells were seeded into 60 mm2 plates and incubated
overnight. Following 24 h treatment, cells were trypsinized, and passed through a 20-
gauge needle to create a single cell suspension. Cells were centrifuged at 350 x g for 4
min, resuspended in non-supplemented DMEM with MitoTracker Green FM (50 nM) and
Orange CM-H2TMRos (300 nM) probes and incubated for 30 min at 37oC, protected
from light. Cells were then subsequently analyzed using a FACScan ™ flow cytometer
(BD Biosciences). In total, 1 x 104 events were counted per sample.
43
Glucose consumption and lactate production
Metabolic output was examined by analyzing glucose consumption and lactate
production. Briefly, 2.5x105 cells were seeded into a 24-well plate and cultured in the
presence or absence of DCA in normoxia or anoxia for 24 h. Following treatment,
conditioned media was collected and assessed for glucose consumption and lactate
production using the Amplex Red Glucose Assay and Eton Bioscience L-Lactate Assay
kits, respectively, following the manufacturer’s protocols. After collection of conditioned
media for analysis, treatment wells were stained with crystal violet as previously
described to determine cell numbers and normalize output values. Cell numbers were
extrapolated through standard curves previously generated for each cell line.
Statistical analysis
Data are presented as means of independent measurements +/- SE. GraphPad
Prism v6.0 software (GraphPad Software Inc., La Jolla, CA, USA) was used to perform
statistical analysis. Kriskal-Wallis ANOVA followed by Dunn’s post-hoc tests were
performed to determine differences between means within each cell line for cell growth,
qRT-PCR and western blot densitometric analysis. One-way ANOVA followed by
Tukey’s post-hoc tests were performed to determine differences between means within
each cell line for flow cytometry and metabolic output analysis. At least three biological
replicates were used for each analysis, and treatments were considered significantly
different if a p-value ≤ 0.05 was achieved.
44
Results
DCA reduces both normoxic and anoxic cell growth
The effects of DCA on CRC cell growth under different atmospheric conditions
were determined using crystal violet staining. Cells were treated with 0 – 20 mM DCA.
Following 72 h treatment, cells responded to DCA in a dose-dependent manner.
Significant repression on cell number was observed in all four cell lines when treated
with 20 mM DCA (Figure 1A-D). Non-tumorigenic IEC-18 cells were most sensitive to
the cell proliferation effects of DCA (Figure 1B). Anoxic exposure also led to a
significant reduction in IEC-18 cell growth while no significant changes were observed in
human normal NCM460 and CRC cells. The repressive effects of DCA on cell number
were less profound for SW480 cells compared to LS174T cells (Figure 1C,D). No
additional reduction in cell growth following DCA treatment under anoxic conditions
was observed in all cell lines examined. Anoxic exposure increased caspase-3 cleavage in
IEC-18 and LS174T cells. DCA treatment reduced caspase-3 cleavage in CRC cells
following 48 h treatment, regardless of atmospheric conditions (Figure 1E). This
suggests the observed reduction in cell growth was not a result of apoptotic induction.
Human CRC cells predominantly express PDKs 1 and 3
mRNA and protein expression of PDK isoforms in CRC cells were determined
using both western blot and qRT-PCR analysis. Both SW480 and LS174T cells highly
expressed mRNA transcripts of PDKs 1 and 3 relative to isoforms 2 and 4. (Figure
2A,B). These results correlated with protein expression detected via western blot (Figure
2C); SW480 and LS174T cells expressed PDKs 1 and 3 but not 2 and 4. LS174T cells
45
appeared to show higher expression of PDK3 than SW480 at the protein level. Normal
human NCM460 cells showed protein expression of PDK isoforms 1 – 3. Normal rat
IEC-18 cells showed protein expression of PDK isoforms 1, 3 and 4 (Figure 2).
PDKs 1 and 3 are upregulated in response to treatment
Both CRC cell lines showed an increase in PDK1 but not PDK3 mRNA levels
following anoxic exposure (Figure 3). DCA treatment led to slight increases in mRNA
levels of both PDK isoforms examined, which was further increased in combination with
anoxia. PDK3 mRNA expression was significantly upregulated when anoxic cells were
exposed to 10 mM DCA. Western blot analysis confirmed changes observed in mRNA
levels in which anoxic exposure led to increased PDK1 protein expression which further
increased in combination with DCA in LS174T cells (Figure 3B).
PDH phosphorylation status varies in response to treatment between cell lines
Following 24 h anoxic DCA exposure, PDH phosphorylation was assessed
through western blotting (Figure 4). In the non-tumorigenic cell line IEC-18, as
expected, DCA reduced and abolished phosphorylation at residues Ser293 and Ser300,
respectively, while anoxic exposure increased phosphorylation at both residues. Human
CRC cells exhibited the same phosphorylation pattern as IEC-18 cells at residue Ser300
following treatment. However, phosphorylation at residue Ser293 varied between cell
lines. In SW480 cells, neither DCA, anoxia, nor combined exposure led to significant
changes in pSer293 phosphorylation. In LS174T cells, phosphorylation only differed
46
from control cells following combination treatment (Figure 4). Native PDH levels did
not significantly change between groups over the course of treatment.
Mitochondrial activity varies in response to DCA between human CRC cells
The effects of DCA on mitochondrial activity was determined utilizing
MitoTracker probes and flow cytometry analysis. The MitoTracker Green FM probe
accumulates in the lipid environment of the mitochondria while the MitoTracker Orange
CM-H2TMRos probe does not fluoresce until it enters an actively respiring cell and is
oxidized in the mitochondria (Johnson, 2010). Representative flow plots demonstrate that
the proportion of cells with highly active mitochondria varies between cell lines (Figure
5A). Human CRC cell lines displayed a higher mitochondrial activity (MA) under
control conditions (SW480 cells: 81.1 ± 2.3%, LS174T cells: 69.4 ± 2.2%) compared to
non-tumorigenic control IEC-18 cells (57.2 ± 1.4%) (Figure 5B). In IEC-18 cells, DCA
increased MA, anoxic exposure reduced MA, and DCA restored the MA of anoxic cells
as expected. LS174T cells followed the same pattern in their response to DCA and anoxic
exposure. Surprisingly, SW480 cells differed in their mitochondrial responses to various
treatments. Unlike the other two cell lines, the MA of SW480 cells decreased following
DCA exposure in normoxia and further decreased in combination with anoxia (Figure
5B). Changes from control following anoxic DCA exposure were most profound with
SW480 compared to the other cell lines.
47
DCA does not significantly alter metabolic status of human CRC cells
Conditioned media were collected following 24h DCA exposure under different
oxic conditions to analyze glucose consumption and lactate production as outputs for
cellular metabolism. Both metabolic outputs showed similar trends between treatment
groups for each cell line (Figure 6). Cells under anoxic conditions upregulated glycolysis
and thus consumed more glucose and produced more lactate. This metabolic activity was
notably greater in SW480 and LS174T cells compared to IEC-18 cells even under
normoxia, indicative of the Warburg effect. Due to the lack of oxygen and necessity to
undergo anaerobic glycolysis, anoxic IEC-18 cells consumed approximately 4.4 fold
more glucose and produced approximately 7 fold more lactate compared to the same cells
under normoxia. The changes in metabolic outputs under low oxygen tensions were not
as profound in CRC cells: anoxic SW480 cells consumed approximately 1.6 fold more
glucose and produced approximately 1.4 fold more lactate compared to when under
normoxia, and LS174T cells consumed approximately 2.5 fold more glucose and
produced approximately 2.6 fold more lactate compared to when under normoxia (Figure
6). As expected, DCA did not significantly alter either metabolic output regardless of
atmospheric conditions in IEC-18 cells. DCA did, however, lower both glucose
consumption and lactate production in both CRC cell lines. These changes were not
statistically significant, but were more profound under normoxia than in anoxia,
presumably due to the cell's further reliance on glycolysis under these conditions.
48
Discussion
Previous studies in my laboratory demonstrated a cytoprotective effect induced by
DCA in anoxic human CRC cells (Shahrzad et al, 2010). In this chapter, I sought to
investigate the molecular mechanisms by which this cytoprotection was conferred.
Anoxic exposure led to profound changes in PDH activation, mitochondrial activity and
metabolic output. Although DCA exposure led to differences in changes in PDH
activation and mitochondrial activity between different human CRC cells, it did not lead
to differences in metabolic and growth responses.
I initially sought to determine whether DCA exposure under different atmospheric
conditions would have an effect on cell growth. Due to the hypothesized actions of DCA
on the mitochondria, I avoided commonly used cell viability assays that relied on
mitochondrial activity (i.e. MTT, WST-1) and used crystal violet staining as an indirect
method to determine cell number and growth. Normal human colon mucosal epithelial
NCM460 cells and rat intestinal epithelial IEC-18 cells were used in conjunction with
human CRC cells SW480 and LS174T to evaluate the effects of DCA treatment under
different atmospheric conditions. All cell lines responded to DCA in a dose-dependent
manner, although significant reduction in cell growth was only observed at 20 mM DCA.
While it has been hypothesized that normal cells would be unaffected by DCA treatment,
I found that IEC-18 cells were most sensitive to DCA treatment (Bonnet et al, 2007). By
altering the metabolic status of cells to enhance glycolysis for energy production as a
result of anoxic exposure, I expected that DCA treatment would further reduce cell
growth as cells are 'forced' to enter into the TCA cycle under metabolic stress. However, I
found no additional reduction in cell growth following DCA treatment under anoxic
49
conditions in all cell lines examined. The lack of caspase-3 cleavage suggests reduced
cell growth was not a result of apoptotic induction. Other studies have shown that DCA
does not need to induce apoptosis to reduce cell number in different cancer types,
including prostate (Cao et al, 2008), pancreatic (Chen et al, 2009a), breast (Sun et al,
2010), colon (Sanchez-Arago et al, 2010), and lung (Stockwin et al, 2010).
Due to the differences observed in DCA sensitivity between SW480 and LS174T
cells, I determined whether this sensitivity could be attributed to differential expression
of PDK isoforms in these two cell lines. qRT-PCR and western blot results revealed that
these two cell lines primarily expressed PDK isoforms 1 and 3. Although it has been
suggested that PDK2 is the major isoform responsible for metabolic control over PDH
activity (Bowker-Kinley et al, 1998), both mRNA and protein levels were barely
detectable in the CRC cell lines examined. These results suggest that regulation of PDH
activity in these CRC cells is through PDK isoforms 1 and 3. PDK2 is most sensitive to
inhibition by DCA (Bowker-Kinley et al, 1998), perhaps explaining the minimal change
in cell growth following DCA exposure in the cell lines examined. The addition of
exogenous pyruvate when conducting these experiments could have altered PDK
expression, although this has not been confirmed. In Baker’s study, while PDK2 activity
is reduced to <10% residual activity as a result of elevated pyruvate, its expression was
not examined (Baker et al, 2000).
PDK isoforms 1 and 3 are upregulated in response to hypoxia in CRC
Colo320DM, HT29, and HCT116 cells (Lu et al, 2011). Contrary to what has been
reported in the literature, anoxic exposure did not have a significant effect on PDK3
transcript levels in either SW480 or LS174T cells. The addition of DCA to anoxic CRC
50
cells led to significant upregulation in PDK3 mRNA expression. Furthermore, DCA
treatment led to an increase in both normoxic and anoxic PDK1 expression in LS174T
cells. The ability of cells to upregulate both PDK isoforms under anoxia coincides with
DCA-induced cytoprotection under the same conditions, leading to speculation that these
enzymes may some how play a role in this process.
Due to differences observed in PDK expression following treatment, I next sought
to determine changes in PDH expression and regulation. PDH phosphorylation by PDK
leads to enzymatic inhibition and prevents pyruvate conversion to acetyl-CoA and entry
into the mitochondria where it would enter into the TCA cycle to further produce
reducing intermediates. PDH phosphorylation occurs at 3 residues: Ser293, Ser300, and
Ser232. Each PDK isoform displays a different site-specificity for PDH phosphorylation.
While all 4 PDK isoforms are able to phosphorylate PDH at residues Ser293 and Ser300,
only PDK1 is able to phosphorylate Ser232. It has been suggested that pSer232 may have
a limited function in PDC regulation in comparison to residues Ser293 and Ser300
(Rardin et al, 2009).
Thus, I focused my efforts on examining changes to PDH phosphorylation at
residues Ser293 and Ser300. Following 24 h anoxic DCA exposure, PDH
phosphorylation was assessed through western blot analysis. As DCA is an inhibitor of
the enzyme responsible for PDH phosphorylation, it would be expected that there would
be a decrease in PDH phosphorylation. This was demonstrated with the non-tumorigenic
cell line IEC-18; DCA drastically reduced and abolished phosphorylation at residues
Ser293 and Ser300, respectively.
51
Under anoxic conditions, increases in PDK expression would subsequently lead to
increased PDH phosphorylation and inactivation, and a shift in metabolism towards
glycolytic pathways and away from mitochondrial respiration. While this pattern was
observed in IEC-18 cells, phosphorylation at residue Ser293 varied in the two human
CRC cells in response to experimental treatment. These results suggest that differences in
sensitivities to DCA between different human CRC cell lines could be attributed to PDH
phosphorylation status at residue Ser293 but not Ser300. The PDH phosphorylation
patterns at Ser293 in DCA treated anoxic CRC cells could be attributed to the differences
in PDK1 regulation at the protein level. While LS174T cells are able to upregulate PDK1
expression following anoxic DCA exposure, this upregulation was not observed in
SW480 cells. Despite these differences, responses in cell growth following various
treatments appeared the same between SW480 and LS174T cells, suggesting PDH
phosphorylation at residue Ser300 still plays a role in maintaining PDH activity. Residue
Ser293 is the predominant PDH inactivation site while residues Ser300 and Ser232 may
play a limited role (Sale & Randle, 1981). Pharmacological manipulation of the
phosphorylation status of PDH with various known inhibitors of oxidative
phosphorylation or signaling pathways showed no changes at residue Ser232 (Rardin et
al, 2009).
Recent studies have revealed emerging roles of alternate post-translational
modifications to PDH that would further our understanding of its molecular regulation
beyond its activation via dephosphorylation. Individual global acetyl-proteomic and
lysine desuccinylation analyses revealed several lysine acetylation and succinylation sites
on PDH (Fan et al, 2014; Park et al, 2013). Therefore, although some differences were
52
observed in PDH phosphorylation, the response of CRC cells to DCA exposure via PDH
activation is unclear unless more experiments are conducted to examine these novel post-
translational modifications.
The effects of DCA treatment under different atmospheric conditions on
mitochondrial activity and the cell metabolic status were also examined. I opted to
determine changes in mitochondrial activity through mitochondrial staining and
subsequent flow cytometry analysis. Results showed that control CRC cells displayed a
higher MA compared to non-tumorigenic control IEC-18. This could be attributed to the
presence of hyperpolarized mitochondria in the cancer cells as a result of the Warburg
effect (Bonnet et al, 2007). SW480 cells differed in their mitochondrial responses to
various treatments. This coincides with the unique PDH phosphorylation pattern
observed at residue Ser293 following DCA exposure in SW480 cells. These results
suggest that the differences in regulation of PDH phosphorylation at residue Ser293
between different cell lines could be affecting mitochondrial activity.
I then investigated whether the changes observed in mitochondrial activity
correlated with the metabolic output of treated cells. As expected, CRC cells consumed
more glucose and produced more lactate as a result of the Warburg effect. Under anoxic
conditions, IEC-18 cells had a greater increase in glucose consumption and lactate
production compared to CRC SW480 and LS174T cells. These numbers suggest that as a
result of the Warburg effect, the CRC cells have no need to heavily re-route their
metabolic pathways to compensate for lack of oxygen in order to undergo further
glycolysis. DCA was shown to reduce glucose consumption and lactate production in
normoxic CRC cells with no apparent effect on IEC-18 cells, indicative of a re-wiring of
53
the Warburg effect (Stacpoole et al, 1998; Warburg et al, 1927). However, this change
was diminished in anoxic CRC cells, presumably due to an increased reliance on
glycolysis under these conditions.
An issue with interpreting results from DCA studies conducted in vitro is the
super-physiological doses that are used to induce cellular responses. This is required due
to a lack of effective cellular uptake and localization inside mitochondria (Stockwin et al,
2010). SLC5A8 is the plasma membrane transporter responsible for DCA uptake. This
transporter is expressed in normal cells but limited in tumor cells, leading to an increased
resistance to the antitumor effects of DCA and the detrimental side effects involving the
nervous system associated with high doses in clinical studies (Babu et al, 2011; U.S.
National Institutes of Health, 2015). Derivatives of DCA have been shown to be more
potent in cancer cells. Mito-DCA (Pathak et al, 2014) and mitaplatin (Dhar & Lippard,
2009) are two new DCA-derived molecules that are now under investigation. Mito-DCA
is more potent and selective towards cancer cells than DCA. The incorporation of a
lipophilic triphenylphosphonium cation through a biodegradable linker allows
mitochondrial targeting in Mito-DCA. Pathak and colleagues showed that this compound
was able to re-route metabolism from lactate production to oxidative phosphorylation,
which subsequently led to induction of apoptosis in affected cells (Pathak et al, 2014).
These effects were found to be cancer specific and had no significant effect on normal
cells. Mitaplatin in essence is a complex formed by combining cisplatin and DCA in one
molecule (Dhar & Lippard, 2009). Its potency lies in its ability to attack both nuclear
DNA via cisplatin and aerobic glycolysis via DCA. The authors showed that mitaplatin
was able to significantly increase apoptosis vs. either cisplatin or DCA alone (55, 172 and
54
1.2%), while also significantly reducing the cytotoxic effects observed in normal lung
fibroblasts in response to cisplatin when treated with mitaplatin. (2.8 down to 73.6%)
(Dhar & Lippard, 2009).
In summary, human CRC cells SW480 and LS174T express predominantly PDK
isoforms 1 and 3, which are upregulated in response to DCA and anoxia. I found
evidence for differential phosphorylation of PDH at residue Ser293 that was not reflected
in changes in cell proliferation. Activation of PDK on Ser293 could possibly lead to
differences in mitochondrial activity, however, the metabolic changes following anoxic
DCA exposure did not differ between cell lines. This study confirms previous findings
that the effects of DCA on CRC cells are heterogeneous, which may limit the usefulness
of this putative anti-cancer agent.
The results presented here suggest there is still potential for DCA to be used as a
chemosensitizing agent. DCA was able to alter mitochondrial activity and mitochondrial
membrane potential. Since reducing the polarization of the mitochondria of cancer cells
could lead to facilitated release of pro-apoptotic signaling molecules, this could potentiate
current chemotherapeutics. For instance, Sanchez et al. (Sanchez et al, 2013) showed an
increase in sensitivity to bortezomib in combination with DCA in myeloma-bearing mice,
and Kumar et al. (Kumar et al, 2013) reported that DCA was able to amplify the effects
of bevacizumab in a glioblastoma-derived xenograft mouse model. Activation of
mitochondrial respiration by DCA can sensitize can cells to sorafenib in hepatocellular
carcinoma cells (Shen et al, 2013). Reduction in lactic acid secretion and
immunosuppression in thymoma and melanoma tumors following DCA exposure in vivo
highlights another pathway towards chemosensitization (Ohashi et al, 2013). The results
55
presented suggest different colorectal cancer cell lines have diverse responses to the
presence of DCA under different atmospheric conditions. Since tumors are under
constant changes in oxygen levels due to the tumor microenvironment, it is important to
take into account the atmospheric conditions which cells are being treated with possible
anti-cancer agents.
56
Table I. Primer sequences for quantitative real-time PCR analysis of PDK isoforms.
Gene Primer Sequence
PDK1 Forward 5'-CTATGAAAATGCTAGGCGTCT-3'
Reverse 5'-AACCACTTGTATTGGCTGTCC-3'
PDK2 Forward 5'-AGGACACCTACGGCGATGA-3'
Reverse 5'-TGCCGATGTGTTTGGGATGG-3'
PDK3 Forward 5'-GCCAAAGCGCCAGACAAAC-3'
Reverse 5'-CAACTGTCGCTCTCATTGAGT-3'
PDK4 Forward 5'-TTATCATACTCCACTGCACCA-3'
Reverse 5'-ATAGACTCAGAAGACAAAGCCT-3'
β-actin Forward 5'-AAGATCAAGATCATTGCTCCTC-3'
Reverse 5'-CAACTAAGTCATAGTCCGCC-3'
57
Figure 1. DCA reduces cell growth in a cell line-dependent manner. Crystal violet staining assay was used to examine cell growth following 72 h 0 – 20 mM DCA and/or anoxic (< 0.1% O2) treatment in non-tumorigenic NCM460 (A), IEC-18 (B) and human CRC SW480 (C) and LS174T (D) cell lines. Cell numbers were determined and normalized to control groups. Caspase-3 cleavage was determined through western blot analysis in IEC-18, SW480 and LS174T cells following 48 h 10 mM DCA and/or anoxic (<0.1% O2) treatment (E). (a p ≤ 0.05 vs. control, b p ≤ 0.05 vs. anoxia-treated)
58
Figure 2. PDK1 and 3 are highly expressed in human CRC cells. qRT-PCR analysis was performed to determine mRNA expression of PDK isoforms 1-4 in human CRC cell lines SW480 (A) and LS174T (B). Expression levels were normalized to β-Actin reference gene to determine relative mRNA expression, as shown. Protein levels of PDK isoforms in non-tumorigenic NCM460, IEC-18 and human CRC SW480 and LS174T cell lines were assessed via western blotting (C); α-tubulin was used as a loading control. Expected molecular weights are noted. (* p ≤ 0.05 vs. PDK2 and 4)
59
Figure 3. PDK1 and 3 levels are upregulated in response to treatment. qRT-PCR was used to assess PDK mRNA expression in human CRC cells SW480 (A) and LS174T (B) following 24 h 10 mM DCA exposure in normoxia and anoxia (< 0.1% O2). PDK mRNA expression levels were normalized to β-Actin reference gene, and then normalized to control samples to determine relative fold expression. Samples from 24 h treatments were subjected to western blot analysis to determine PDK1 and PDK3 protein expression. (a p ≤ 0.05 vs. control, b p ≤ 0.05 vs. DCA-treated, c p ≤ 0.05 vs. anoxia-treated)
60
Figure 4. Phosphorylation status of PDH at residues Ser293 and Ser300 varies in response to treatment. Western blot analysis was used to examine PDH phosphorylation following 24 h 10 mM DCA and/or anoxic (< 0.1% O2) treatment. Representative blots are shown (A) above graphical outputs for densitometric analysis of pPDH normalized to native PDH at residues Ser300 and Ser293 (B). (a p ≤ 0.05 vs. control, b p ≤ 0.05 vs. DCA-treated, c p ≤ 0.05 vs. anoxia-treated)
61
Figure 5. Mitochondrial response to DCA exposure is cell line-dependent. Mitochondrial activity (MA) of cells exposed to 10 mM DCA in normoxia and anoxia (< 0.1% O2) following 24 h was determined through mitochondrial staining using MitoTracker probes and subsequent flow cytometry analysis. Representative plots are shown in (A). Values displayed in each plot represent the % of cells with active mitochondria (i.e. in the upper right quadrant). Quantification of values from three biological replicates ± SEM is shown in (B). (a p ≤ 0.05 vs. control, b p ≤ 0.05 vs. DCA-treated)
62
Figure 6. DCA does not significantly alter metabolic status of human CRC cells. Conditioned media was collected from cells treated with 10 mM DCA under normoxia and anoxia (< 0.1% O2) following 24 h. Glucose consumption (A) and lactate production (B) was determined using commercially available kits. Graphs represent the average amount of glucose consumed and lactate produced per 104 cells from each cell line in various treatments from three biological replicates ± SEM. (a p ≤ 0.05 vs. control, b p ≤ 0.05 vs. DCA-treated)
63
CHAPTER III – METABOLIC DISRUPTION VIA 3-BROMOPYRUVATE IN HUMAN COLORECTAL CANCER CELLS
Introduction
Tumors upregulate key glycolytic enzymes to utilize and rely on the glycolytic
pathway for energy production regardless of oxygen tension in a phenomenon called the
Warburg effect (Koppenol et al, 2011; Warburg et al, 1927). This metabolic strategy
promotes tumorigenesis by re-routing glycolytic intermediates to fuel anabolic processes
to sustain rapid proliferation (Vander Heiden et al, 2009). Furthermore, highly glycolytic
tumors are associated with tumor aggressiveness (Simonnet et al, 2002).
The family of hexokinase enzymes function to catalyze the first irreversible step
of glycolysis in which glucose is phosphorylated to glucose-6-phosphate. While HK
isoforms are differentially expressed in various tissues, HKII is upregulated in many
tumors (Kwee et al, 2012; Palmieri et al, 2009; Wolf et al, 2011). Mito-HKII promotes
metabolic coupling of glycolysis and oxidative phosphorylation while also playing a
cytoprotective role by maintaining mitochondrial integrity (Majewski et al, 2004b;
Nakashima et al, 1986; Pastorino et al, 2002).
HKII expression is regulated by the AKT signaling pathway. AKT, or protein
kinase B, is a serine/threonine kinase that is often hyperactivated in human cancers. AKT
phosphorylates and stimulates the activity of target molecules involved in glucose
metabolism, cell proliferation, migration, and survival (Miyamoto et al, 2009a;
Miyamoto et al, 2009b; Scheid & Woodgett, 2001; Sussman et al, 2011; Thompson &
Thompson, 2004). AKT is able to phosphorylate HKII at residue Thr473 and increase
subsequent mitochondrial localization (Majewski et al, 2004a; Roberts et al, 2013).
64
Furthermore, the anti-apoptotic effects of AKT require mitochondrial-bound HKII
(Gottlob et al, 2001).
In Chapter II, I demonstrated that while DCA exposure in CRC cells led to
changes in metabolic signaling and variations in mitochondrial activity, 1) it did not
ultimately lead to significant changes in cellular metabolism and growth and 2) the doses
used were above physiological tolerance. While the effects of DCA at physiologically
doses have not shown promising results in pre-clinical studies, other compounds that
target metabolism in cancer cells in vitro have led to dramatic outcomes in preclinical and
human trials. 3-bromopyruvate (3BP) is a pyruvate analog with alkylating properties that
depletes cellular ATP levels and induces rapid cell death in tumor cells with limited
cytotoxic effects against normal cells. 3BP treatment led to eradication of tumors in all 19
rats tested without apparent cytotoxic effects, and the first human case report suggested
that 3BP was able to prolong survival in a cancer patient diagnosed with hepatocellular
carcinoma in 2012 (Ko et al, 2004; Ko et al, 2012). As previously mentioned, 3BP has
been reported to effectively kill other cancer cell types in vitro. 3BP is able to dissociate
and inhibit mitochondrial HKII function, thereby reducing ATP production and free
binding sites for pro-apoptotic molecules previously occupied by HKII to promote the
release of cytochrome c into the cytosol and induce eventual cell death (Chen et al,
2009b; Kim et al, 2008). However, the effects of 3BP on AKT signaling have not been
well studied.
In most solid tumors, imbalances between cancer cell proliferation and the
angiogenic response lead to some cancer cells being at a great distance from blood
vessels. This produces patchy regions of ischemic tumor microenvironment, due to a
65
reduction in both oxygen and nutrient availability, including glucose. This ischemia
ultimately leads to changes in metabolic pathways within cancer cells in response to a
reduction in glucose consumption that would otherwise be upregulated in well-perfused
regions of the solid tumor. Thus, it is important to study not only the impact of oxygen
tension on chemotherapeutic resistance, but the effects of hypoglycemia as well. Xu and
colleagues have shown that both HCT116 CRC cells and Raji lymphoma cells grown
under hypoxic conditions were more sensitive to 3BP than in normoxia (Xu et al, 2005),
but the effects of glucose exposure on 3BP responses are not well examined.
In this chapter, I examined the effects of 3BP on different human colorectal
cancer cell lines, with particular emphasis on examining the HKII/AKT signaling axis. I
also investigated whether glucose availability may play a role in targeting CRC cells with
3BP. I hypothesized that HKII expression will correlate with sensitivity to 3BP exposure
in human colorectal cancer cells and that a knockdown in its expression will decrease this
sensitivity. Furthermore, decreasing glucose availability in culturing conditions will lead
to an increase in 3BP resistance due to alterations in metabolic signaling pathways.
Materials and Methods
A list of all chemicals and suppliers, and details of the materials and solutions
used for the following experiments are detailed in Appendices I and II, respectively.
Cell culture and media-glucose reduction
Human colorectal cancer cell lines HCT116, CaCo2, SW480 and DLD-1 were
obtained from ATCC. Cells were maintained in high glucose DMEM supplemented with
66
10% FBS and incubated at 37oC in a humidified atmosphere containing 5% CO2 in room
air. Stock 1 M 3BP was prepared in H2O, filter-sterilized and aliquots were stored at -
80oC for future use.
Cells were routinely maintained under logarithmic growth by passaging them
every 3-4 days at a 1/8 dilution. At each passage, media-glucose concentrations were
reduced by 2.5 mM, by supplementing no glucose DMEM with 10% FBS and the desired
glucose concentration using a sterile stock of glucose. Cells were maintained at each
reduced media glucose concentration for at least 3 additional passages before further
experimentation.
Measurement of cell growth
Cell growth was approximated through crystal violet staining. Briefly, 5 x 103
cells were seeded into a 96-well plate and incubated overnight. Cells were then treated
with 1 – 100 µM 3BP for up to 72 h. Post-treatment, media was aspirated and 1% crystal
violet solution was added to each well and incubated for 10 min in room temperature.
Plates were then aspirated, rinsed and left to dry overnight. 10% acetic acid was used to
dissolve the crystals and the absorbance at 590 nm was recorded.
Protein isolation and western immunoblot analysis
Sodium orthovanadate (1 mM) was added 15 min prior to protein extraction.
Following treatment with 3BP or 50 µM etoposide, cells were lysed on ice with lysis
buffer supplemented with 2 µg/ml aprotinin, 1 mM PMSF and phosphatase inhibitor
cocktail II. Samples were centrifuged at 12,000 x g for 15 min at 4oC, and supernatant
67
was aliquoted and stored at -80oC for future use. Protein was quantified using the Bio-
Rad DC Protein Assay Kit. 30 µg of total protein was resolved in a 10% polyacrylamide
gel using SDS-PAGE then transferred to a PVDF membrane with wet transfer buffer at
100 V for 2 hr. Gels used to subsequently detect caspase-3 expression were transferred
onto PVDF membranes using a Trans-Blot SD Semi-Dry Transfer Cell apparatus (Bio-
Rad) with semi-dry transfer buffer at 16 V for 30 min. Following transfer, membranes
were blocked for 1 h at room temperature in 5% non-fat dry milk diluted in TBST
(blocking solution) followed by an overnight incubation in blocking solution with
primary antibodies. After washing, membranes were incubated for 1 h at room
temperature with the appropriate peroxidase-conjugated secondary antibody, washed, and
subjected to Luminata Forte chemiluminescent substrate. Membranes were imaged using
the ChemiDoc XRS+ system and densitometry was performed using Image Lab software.
Primary antibodies used included rabbit anti-pAKT Ser473 (1:1,000), anti-pAKT Thr308
(1:1,000), anti-pan-AKT (1:1,000), anti-caspase 3 (1:1,000), anti-HKII (1:5,000), anti-
pPDK1 (1:1,000), anti-PDK1 (1:1,000), and anti-PTEN (1:1,000), mouse anti-α-tubulin
(1:100,000), and rabbit or mouse HRP-labeled secondary antibodies (all 1:10,000).
Cell death assay
Cell death was determined through annexin V/PI staining using the Alexa Fluor®
488 annexin V/Dead Cell Apoptosis Kit following the manufacturer’s protocol. Briefly, 5
x 105 cells were seeded onto 6-well plates and incubated overnight. Following 48 h
treatment with 3BP, cells were trypsinized, washed, resuspended in annexin-binding
buffer containing AV and PI dyes and left to incubate for 15 min at room temperature,
68
protected from light. Cells were subsequently analyzed using a FACScanTM flow
cytometer. In total, 1 x 104 events were counted per sample. Fluorescence scatter plots
were gated and analyzed using the Cyflogic software (Terho & Korkeamaki, 2008) to
determine the percentage of viable and dead cells.
DNA fragmentation
Following 48 h treatment with 3BP or etoposide, cells were trypsinized, pelleted
and stored at -80oC. DNA extractions were carried out using the Omega Bio-Tek Tissue
DNA Kit according to the manufacturer’s protocol with the following changes: 1)
samples were left to precipitate in ethanol overnight, 2) samples were subsequently
centrifuged at 6,000 x g for 1 min, 3) DNA Wash Buffer step was carried out once and
centrifuged for 3 min at 20,000 x g for 3 min, 4) samples were eluted in 50 µl elution
buffer. DNA samples were run on either a 0.8% or 2% agarose gel at 150 V for 2 hr or
100 V for 1.5 h, respectively, and visualized using the ChemiDoc XRS+ system.
RNA interference
Knockdown experiments were performed by transient transfection of HKII
siRNA using INTERFERin according to the manufacturer’s protocol. Briefly, 5 x 104
cells were seeded into 12-well plates and incubated overnight. Cells were transfected
with 10 nM siRNA for 24 h in Opti-MEM and subsequently allowed to recover in high
glucose DMEM for the times indicated. The sense sequences of double-strand siRNA
used were previously published (Yuan et al, 2008):
siHKIIa: 5’-GGAUAAGCUACAAAUCAAA[dT][dT]-3’ siHKIIb: 5’-CGGGAAAGCAACUGUUUGA[dT][dT]-3’
69
3BP dose response following HKII knockdown was conducted as follows. 2 x 105
cells were seeded into 60 mm2 plates and incubated overnight. Cells were transfected
with 10 nM siRNA for 24 h in Opti-MEM and subsequently allowed to recover in high
glucose DMEM for 24 h. A mixture of both HKII siRNA sense sequences was used.
Following its recovery, a 4 h 3BP dose response was conducted.
Statistical analysis
Data were presented as means of independent measurements +/- SE. GraphPad
Prism v6.0 software was used to perform statistical analysis. One-way ANOVA followed
by Tukey’s post-hoc tests was performed to determine differences between means within
each cell line for flow cytometry and cell growth. Non-parametric Kriskal-Wallis
ANOVA followed by Dunn’s post-hoc tests was performed to determine differences
between means within each cell line for western blot densitometric analysis. At least
three biological replicates were used for each analysis unless otherwise specified, and
treatments were considered significantly different if a p-value ≤ 0.05 was achieved.
Results
HKII expression is correlated with 3BP sensitivity in human CRC cells
The protein expression of HKII by various human CRC cell lines was screened
via western blot analysis. All four cell lines showed HKII expression to varying degrees
(Figure 7A). HCT116 and CaCo2 cells showed moderate and low HKII expression,
respectively, while SW480 and DLD-1 cells showed high HKII expression.
Corresponding IC50 values were determined through 72 h crystal violet cell growth assays
70
(Figure 7B) and were as followed: HCT116 = 22.5 ± 0.7 µM, CaCo2 = 36.6 ± 2.1 µM,
SW480 = 16.9 ± 1.0 µM, DLD-1 = 16.9 ± 1.3 µM. 3BP sensitivity of HCT116, SW480,
and DLD-1 cells were statistically different from CaCo2, but not from each other (p <
0.05). Thus, high HKII-expressing SW480 and DLD-1 cells were significantly more
sensitive to the effects of 3BP than low HKII-expressing CaCo2 cells.
3BP-induced cell death is dose dependent
To determine the cytotoxic effects of 3BP on CRC cells, cell death was measured
using AV/PI staining and subsequent flow cytometry analysis. Following 48 h 3BP
treatment, cell death was observed in all cell lines examined (Figure 8). Cells staining
positive for AV was indicative of an apoptotic event while PI positivity correlated to a
necrotic event. Cells treated with 50 µM etoposide served as a positive control for
apoptosis and are shown in APPENDIX III. In all cell lines examined, both forms of cell
death were induced upon 3BP exposure. Limited cell death was observed with 5 µM 3BP
treatment. At higher doses (30 and 50 µM), the percentage cell death observed in each
cell line was associated with levels of HKII expression previously examined. In moderate
and low HKII expressing HCT116 and CaCo2 cell lines, significant cell death was only
observed following 50 µM 3BP treatment (56.8 and 26.9%, respectively). In high HKII
expressing SW480 and DLD-1 cell lines, significant cell death was observed at both 30
µM (31.1 and 78.1%, respectively) and 50 µM 3BP treatment (90.1 and 92.9%,
respectively) (Figure 8).
SW480 cells showed considerable caspase-3 cleavage even under control
conditions. HCT116 cells were most resistant to etoposide-induced caspase-3 cleavage
71
compared to CaCo2, SW480, and DLD-1 cells. Following 3BP exposure, no cleaved
caspase-3 was observed in HCT116 or CaCo2 cells while limited caspase-3 cleavage was
observed in SW480 and DLD-1 cells (Figure 9A). However, cells exposed to 50 µM 3BP
showed overall reduced cellular protein as evidenced by reduced full length caspase-3 but
no detectable cleaved product, and reduced α-tubulin expression. This observation,
suggestive of cellular necrosis, was verified using DNA fragmentation analysis, where 50
µM 3BP treatment on SW480 cells led to DNA smearing on the agarose gel, and no
obvious nucleosome laddering expected with apoptotic cell death (Figure 9B, C).
3BP induces rapid AKT phosphorylation at residue Thr308
Through a 24 h time course study of 3BP on AKT activation, maximum
phosphorylation of AKT at residue Thr308 was observed after 4 h 3BP treatment (Figure
10A). Dose-dependent changes in AKT phosphorylation following 3BP treatment were
observed at residue Thr308 with no changes at residue Ser473 (Figure 10B,C;
APPENDIX IV). Native AKT expression was not affected. The trends in AKT
phosphorylation at residue Thr308 matched the sensitivity to 3BP in different cell lines;
phosphorylation occurred at lower doses in cell lines highly expressing HKII (SW480
and DLD-1) (Figure 10C). In CaCo2 cells, AKT phosphorylation appeared to remain
constitutively active regardless of 3BP treatment (Figure 10B). Phosphorylation of AKT
at residue Thr308 occurs through PIP3-mediated recruitment of AKT and
phosphoinositide-dependent kinase-1 (PDK1) to the plasma membrane (Alessi et al,
1997). Similar to AKT phosphorylation, changes in the expression of these proteins
occurred in a dose-dependent manner. Changes in PTEN expression were limited to
72
SW480 and DLD-1 cells, where a decrease in its expression was observed at higher 3BP
doses (Figure 10C). At 3BP doses that induced cytotoxic effects, the molecular weight of
PDK1 shifted by approximately 3 kDa. In SW480 and DLD-1 cells, an increase in PDK1
expression at higher 3BP doses was observed.
Glucose availability affects 3BP sensitivity in human CRC cells
HCT116 and DLD-1 cells were adapted and maintained in DMEM with varying
glucose concentrations. Cells were maintained in DMEM containing 25, 5.5 and 1 mM
glucose for subsequent experimentation, denoted as 'high', 'normal' and 'low' glucose
conditions, respectively. Western blot analysis showed that HKII levels decreased with
lower media glucose levels in both cell lines examined (Figure 11A,B). Differences in
HKII expression between cells grown under high vs. normal glucose were more profound
in DLD-1 cells. Both CRC cell lines proliferated exponentially under high and normal
glucose concentrations, but growth was limited in cells cultured in low glucose over 72 h
(Figure 11C). Cells grown in normal glucose showed an increase in 3BP resistance vs.
cells grown in high glucose. Significant differences in cell growth were observed
between high and normal glucose for HCT116 cells treated with 30 and 40 µM 3BP and
DLD-1 cells treated with 20 and 30 µM 3BP (Figure 11D). This pattern is consistent
with the finding that DLD-1 cells are more sensitive to 3BP than HCT116 cells.
HKII knockdown does not lead to changes in 3BP sensitivity
To determine whether HKII was the reason for the differences observed in 3BP
sensitivity between different CRC cells, HKII expression was knocked down in HCT116
73
and DLD-1 cells using transient RNAi transfection. Effective knockdown of HKII
expression was observed in both cell lines (Figure 12A). After 24 h siRNA transfection,
treatment media was replaced with DMEM to allow subsequent assessment of 3BP
effects in standard treatment media. Protein levels remained knocked down for 48 h post-
transfection with a slight increase in HKII expression after 72 h of post-transfection
recovery (Figure 12B). Given this stability in HKII knockdown for at least 48 h post-
transfection, a 4 h 3BP dose-response was conducted on both HCT116 and DLD-1 cells
after the 24 h recovery period (i.e. samples were collected after 28 h knockdown). There
were no differences in 3BP sensitivity as revealed by AKT phosphorylation when
comparing scrambled controls to HKII knockdown cells in either HCT116 (Figure 12C)
or DLD-1 cells (Figure 12D). The dose-response patterns observed mimicked those
previously seen in parental HCT116 and DLD-1 cells (Figure 10B,C), suggesting that,
contrary to expectation, a reduction in HKII expression did not lower 3BP sensitivity in
the cell lines examined.
Discussion
While the promising anti-cancer effects of pyruvate/lactate analog 3-
bromopyruvate on cancer cells in vitro have been well documented, the molecular
mechanisms surrounding its potent effects remain understudied. In this chapter, I sought
to investigate the potential link between HKII expression and the sensitivity of human
colorectal cancer cells towards 3BP, and whether glucose availability may factor into
these cytotoxic effects. I found that although limiting glucose availability in the culturing
conditions led to a reduction in HKII expression and a decrease in 3BP sensitivity, the
74
expression of HKII alone cannot account for the differences in 3BP sensitivity observed
amongst the different CRC cells examined.
Due to its high affinity for glucose compared to other isoforms, HKII is
upregulated during tumorigenesis. This increases glucose consumption and alleviates the
metabolic requirements for glycolytic intermediates, thus supporting key anabolic
processes that sustain rapid cellular proliferation (Kwee et al, 2012; Palmieri et al, 2009;
Wolf et al, 2011). I examined the expression of HKII in 4 different CRC cell lines and
observed that the expression of HKII correlated with their reported aggressiveness in vivo
(Flatmark et al, 2004). Western blot results reflected the data generated from 3BP IC50
determination via crystal violet staining, where high HKII-expressing SW480 and DLD-1
cells were significantly more sensitivity to the cytotoxic effects of 3BP versus low HKII-
expressing CaCo2 cells. Like in Chapter II, crystal violet was used to monitor the effects
on cell growth as 3BP has the potential to interfere with the tetrazolium reduction assays
(i.e. MTT, WST-1) generally used to evaluate cytotoxicity (Ganapathy-Kanniappan et al,
2010).
I next sought to determine the mechanisms of 3BP-induced cytotoxicity. AV/PI
labeling was used to determine whether 3BP triggered cell death in the cells examined. I
observed that, much like the results from crystal violet staining, the amount of cell death
induced in CRC cells was correlative to HKII levels. In all cell lines treated, a significant
portion of cells following > 50 µM 3BP exposure appeared in the AV+/PI+ quadrant,
signifying either late apoptosis or necrosis (Kroemer et al, 1998). Analyzing AV and PI
staining individually did not provide further insight into the mechanisms of cell death
induced by 3BP (See APPENDIX V).
75
As AV/PI labeling was insufficient to fully elucidate the route of 3BP-induced
cell death, I performed additional assays including detection of caspase-3 cleavage and
DNA nucleosome fragmentation, both characteristics of apoptotic events (Zhivotovsky et
al, 2001). Activation of initiator caspases (caspase 8 and 9) by either ‘extrinsic’ or
‘intrinsic’ apoptotic signaling pathways, respectively, leads to the cleavage and activation
of executioner pro-caspase 3, an common event for both 'intrinsic' and 'extrinsic'
apoptotic pathways (McIlwain et al, 2013). In high HKII expression CRC cells, I found a
slight induction of caspase-3 cleavage following 30 µM 3BP treatment. This effect
however was lost with 50 µM 3BP treatment, in which total protein was also reduced,
presumably due to microtubule degradation during necrotic cell death. Calvino and
colleagues observed the same trend in myeloid leukemia cells: increase in cleaved
caspase-3 following 30 µM 3BP exposure which was lost at 60 µM (Calvino et al, 2014).
Programmed cell death is not solely dependent on the cleavage and activation of
caspases. BH3 domain-only proteins, such as tBID, BIM and BAD can kill cells
independently of caspase activation (Cheng et al, 2001). A common feature in all forms
of programmed cell death is DNA fragmentation (Zhivotovsky et al, 2001). However, I
was unable to visualize the presence of small DNA fragments (representing nucleosomes)
in 3BP-treated samples, suggesting minimal apoptotic induction following 30 µM 3BP
treatment. However, there was visible DNA smearing in 50 µM 3BP treated SW480 cells,
suggesting a necrotic event (Zhivotovsky et al, 2001). Combining the AV/PI labeling,
caspase-3 cleavage, and DNA fragmentation results is thus consistent with apoptosis
induction under lower doses of 3BP and necrosis at higher doses.
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The specific dose at which 3BP was able to induce these effects was dependent on
the cell line. Opinions on the mechanisms of 3BP-induced cell death vary in the
literature. Necrosis was induced in both hepatocyte and cancer cell lines following 50 µM
3BP treatment (Ihrlund et al, 2008; Kim et al, 2008). Moreover, a 60% reduction in
intracellular ATP levels was observed in hepatoma cells, further suggesting that 3BP
induces ATP depletion-dependent necrosis at higher concentrations (Kim et al, 2008). In
myeloid leukemic cells, both apoptosis and necrosis were induced at 20-30 µM and a
pure necrotic response was seen at 60 µM (Calvino et al, 2014). Other studies report the
ability of 3BP to induce apoptosis at 100 µM (Gwak et al, 2005; Yu et al, 2012). The
variation observed in 3BP sensitivity and mechanisms of cell death induction may thus be
attributed to both the dose and the metabolic phenotype of the cell models analyzed.
AKT plays a role in the localization of HKII to the mitochondria (Majewski et al,
2004a). In addition to HKII phosphorylation, AKT is able to phosphorylate and inhibit
glycogen synthase kinase-3β (GSK3β), a constitutively active serine-threonine kinase
able to phosphorylate VDAC and prevent HKII binding. Thus, inhibition of GSK3β leads
to increased mitochondrial localization of HKII (Gall et al, 2011; Miura & Tanno, 2012;
Pastorino et al, 2005; Rasola et al, 2010). I examined the effects of 3BP on
phosphorylation of AKT and its upstream effectors. Initial studies using an approximate
IC50 dose for DLD-1 showed that the serine residue 473 remained constitutively
phosphorylated. However, maximum AKT phosphorylation was induced after 4 h of 3BP
exposure at residue Thr308. Furthermore, the effects of 3BP on AKT phosphorylation
occurred at residue Thr308 not residue Ser473 in all 4 CRC cell lines examined. Rapid
induction of AKT phosphorylation following 3BP treatment was previously shown at
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residue Ser473 in myeloid leukemic cells (Calvino et al, 2014). These changes in AKT
phosphorylation correspond with expression of the upstream signaling molecule PTEN in
my CRC cells. Phosphorylation of PIP2 to activated PIP3 recruits AKT and PDK1 to the
plasma membrane. This in turn causes a major conformational change in AKT to allow
PDK1 to phosphorylate residue Thr308 in the activation loop of AKT (Alessi et al, 1996;
Alessi et al, 1997; Calleja et al, 2007). Maximum AKT activity is dependent on
phosphorylation of both Thr308 and Ser473 residues, the latter of which is
phosphorylated by mTORC2 (Alessi et al, 1996; Sarbassov et al, 2005). PTEN
dephosphorylates PIP3 and prevents AKT membrane recruitment and phosphorylation
(Stambolic et al, 1998).
Interestingly, I observed a shift in the molecular weight of PDK1 by
approximately 3 kDa at cytotoxic 3BP doses in cell lines. In SW480 and DLD-1 cells,
PDK1 expression increased in a dose-dependent manner. Scheid and colleagues have
previously shown that a shift in the molecular weight of PDK1 is a result of
hyperphosphorylation in response to survival signaling. Treatment of HEK 293 cells with
IGF-1 caused a decrease in PDK1 mobility, and co-treatment with calf alkaline
phosphatase reversed this effect, supporting a phosphorylation event (Scheid et al, 2005).
PDK1 has shown to be constitutively phosphorylated on at least five serine residues.
Residue Ser241 is located in the activation loops of the PDK1 kinase domain, and its
phosphorylation is required for PDK1 activity (Casamayor et al, 1999). Native PDK1
levels reflected the changes observed in PDK1 phosphorylation at residue Ser241. This
would suggest that there was an increase in PDK1 levels as well as an increase in
phosphorylation (or possibly other posttranslational modification) at various residues. In
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agreement with this finding, Schied and colleagues also observed changes in PDK1
phosphorylation patterning changes in AKT phosphorylation at residue Thr308 (Scheid et
al, 2005).
As previously discussed, the in vitro conditions may have a profound effect on the
outcome when testing the efficacy of anti-cancer agents. The concentration of glucose
present in my culturing media (4.5 g/L) is nearly 5 fold greater than what is considered
physiological blood sugar levels. As HKII expression is a function of the rate of glucose
consumption, I wanted to determine whether glucose availability would alter HKII
expression and subsequently change 3BP sensitivity. HCT116 and DLD-1 cells
maintained in high, normal and low media glucose concentrations were subjected to 3BP
treatment. The normal glucose concentration (5.5 mM) used for these studies is
equivalent to that of normal human physiological steady-state conditions (Freckmann et
al, 2007). I observed a decrease in HKII expression with decreasing glucose
concentrations. This change also led to a change in 3BP sensitivity; cells treated in
normal glucose concentrations were most resistant to the effects of 3BP. It has previously
been shown that HCT116 cells induced to exhibit an increased glycolytic phenotype via
oligomycin treatment were also more sensitive to the effects of 3BP (Sanchez-Arago &
Cuezva, 2011).
To definitively confirm whether levels of HKII expression alone can explain the
differences observed in 3BP sensitivity, I elected to knock down HKII expression in
HCT116 and DLD-1 cells. I hypothesized that reduction in HKII expression would lead
to increased resistance against 3BP. Surprisingly, effective and sustained HKII
knockdown in both cell lines did not lead to any changes in AKT phosphorylation
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induced by 3BP. Thus, although high HKII-expressing CRC cells were more sensitive to
the effects of 3BP versus low HKII-expressing CRC cells, this observation cannot
explain the differences in 3BP-sensitivity. 3BP affects various enzymes involved in both
glycolysis and mitochondrial respiration, including SDH, GADPH and PGK (Pereira da
Silva et al, 2009). It has previously been suggested that an ester derivative of 3BP, 3-
bromopyruvate propyl ester, exhibits a more potent inhibitory effect on GADPH than on
HKII in CRC cells HCT116 and HT29 (Tang et al, 2012). The fact that 3BP can affect
multiple glycolytic enzymes may explain the lack of change in 3BP sensitivity in HKII
knockdown cells. The differences in 3BP sensitivity when cells were treated under
different glucose concentrations may thus be explained by changes in the activity of these
other glycolytic enzymes.
Along with its ability to inhibit tumor growth as a single agent, 3BP has also been
shown to exert potentiating effects in combination with platinum drugs cisplatin and
oxaliplatin (Ihrlund et al, 2008) and with doxorubicin (Nakano et al, 2011). Wintzell and
colleagues showed that cisplatin-resistant ovarian carcinoma SKOV3 cells were more
sensitive to 3BP than parental cells, and in both cell lines, 3BP was able to potentiate the
effects of cisplatin in vitro (Wintzell et al, 2012). Similarly, Isayev and colleagues
showed that 3BP exposure reverted gemcitabine resistance in various pancreatic ductal
adenocarcinoma cell models and provided an additive effect in combination with
gemcitabine in vivo (Isayev et al, 2014). The mechanisms of 3BP potentiation are not
known, but can be speculated upon. The loss of intracellular ATP upon 3BP exposure
may affect a number of processes that are critical to chemoresistance, including efficient
DNA repair or the function of multi-drug resistance ATP-binding cassette transporters
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(Nakano et al, 2011). It is also possible that 3BP-induced dissociation of HKII from the
mitochondria may lower mitochondrial integrity and sensitize cells to pro-apoptotic
factors binding more freely onto open sites on VDAC upon exposure to other anti-cancer
drugs.
In summary, 3BP is a promising anti-cancer agent capable of inducing rapid ATP
loss and cell death in numerous cancer types. Since HKII has been proposed to be one of
its principal targets, I hypothesized that HKII expression would correlate with 3BP
sensitivity in CRC cell lines, and reduction in HKII expression would reduce cell
sensitivity. I showed that high HKII-expressing CRC cells displayed increased 3BP
sensitivity, and that alteration of culture conditions via glucose availability affected HKII
expression. However, RNAi studies showed that HKII expression could not explain for
the variability in 3BP sensitivity between different cell lines. The mechanism by which
lowered glucose availability increases 3BP resistance is thus not known. Elucidating the
alterations in metabolic pathways that may lead to the observed resistance could provide
better understanding of the impact of the tumor microenvironment on colorectal cancer
cells, and clinical insight to the mechanisms of chemoresistance.
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Figure 7. HKII expression is correlated to 3BP sensitivity. HKII protein expression in CRC HCT116, CaCo2, SW480, and DLD-1 cells was assessed via western blot (A). Crystal violet staining assay was used to determine 3BP IC50 values in HCT116, CaCo2, SW480, and DLD-1 cells (B). IC50 values shown represent the means of three biological replicates ± SEM.
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Figure 8. 3BP-induced cell death is cell-line dependent. Following 48 h 3BP treatment, cell death was assessed through AV/PI labeling and subsequent flow cytometry analysis. Representative plots are shown in (A) and quantification of live vs. dead cells from three biological replicates ± SEM are shown in (B). Viable cells appear in the lower left quadrant. 3BP induced significant cell death in all four cell lines. (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001)
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Figure 9. Type of cell death induced by 3BP is dose-dependent. Caspase-3 cleavage and DNA fragmentation was assessed to further elucidate the mechanisms of 3BP-induced cell death. Caspase-3 cleavage was examined through western blot analysis (N=3) (A). An image of a longer exposed membrane to better detect cleaved caspase-3 is shown below each full caspase-3 blot. DNA fragmentation was examined through gel electrophoresis in SW480 cells following 48 h 3BP treatment using 0.8% (B) and 2% (C) gels (N=2). Etoposide (E; 50 µM) was used as a positive control for caspase-3 cleavage and DNA fragmentation; note nucleosome laddering in etoposide treated cells in (C) indicative of apoptosis.
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Figure 10. 3BP treatment leads to rapid AKT phosphorylation at residue Thr308. Time course assessment of AKT phosphorylation in response to 3BP. Example from DLD-1 cells shown in (A). Western blot analysis of proteins involved in AKT phosphorylation following 4 h 3BP dose-response in HCT116, CaCo2, SW480, and DLD-1 cells (B,C).
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Figure 11. Glucose availability alter 3BP sensitivity in CRC cells. HCT116 and DLD-1 cells were treated with 3BP under different media glucose concentrations. HKII expression was assessed via western blot analysis. Representative blots from three biological replicates (A) and graphical outputs for densitometric analysis of HKII expression normalized to α-tubulin from three biological replicates ± SEM are shown (B). Growth curves for CRC cells grown under different media glucose concentrations in the presence or absence of 3BP over 72 h (C). Dose-effects of 3BP on cell growth following 72 h treatment in CRC cells under different media glucose concentrations (D). (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 vs. control (0 µM 3BP))
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Figure 12. HKII expression knockdown did not lead to changes in AKT phosphorylation following 3BP exposure. HKII expression in HCT116 and DLD-1 cells were knocked down using RNA interference. Both siRNA sequences used showed efficient knockdown of HKII expression following 24 h transfection (A). Representative blots from DLD-1 cells are shown. Recovery time of CRC cells following siRNA transfection (B). Expression of AKT and related proteins following 4 h 3BP dose response on HCT116 (C) and DLD-1 cells (D). siRNA experiments were performed twice with equivalent results.
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CHAPTER IV – HEXOKINASE II AS A POTENTIAL PROGNOSTIC BIOMARKER FOR HUMAN COLORECTAL CANCER
Introduction
CRC is the second leading cause of cancer-related deaths and the third most
common cancer in Canada (Canadian Cancer Society). In 2015, it is estimated that CRC
will account for 17.6% of all new cancer cases and 25.3% of all cancer-related deaths
(Siegel et al, 2015). Decreasing mortality rates have been attributed to early screening,
reduced prevalence of risk factors, and/or improved treatments (Bosetti et al, 2011;
Edwards et al, 2010). However, outcome for patients with advanced and metastatic
disease remains poor.
In 2011, deregulating cellular energetics was identified as an emerging hallmark
of cancer (Hanahan & Weinberg, 2011). Many tumors upregulate key glycolytic enzymes
to utilize and rely on the glycolytic pathway for energy production regardless of oxygen
tension in a phenomenon called the Warburg effect (Warburg et al, 1927). Studies have
correlated the glycolytic state of cancer cells with tumor aggressiveness (Simonnet et al,
2002). Measurement of this unique metabolic change specific to cancer cells using FDG-
PET is widely used as a diagnostic tool for assessing various cancers including CRC
(Artiko et al, 2015; Kruse et al, 2013). Therefore, specific glycolytic enzymes may serve
as possible biomarkers of CRC aggressiveness, providing useful prognostic tools.
As discussed in Chapter III, upregulation of HKII is considered a consequence of
metabolic re-programming in cancer, and has been shown in breast carcinoma (Sato-
Tadano et al, 2013), laryngeal squamous cell carcinoma (Chen et al, 2014), glioblastoma
multiforme (Wolf et al, 2011), and gastric adenocarcinoma (Qiu et al, 2011). Its
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assessment in CRC has been very limited to date; immunohistochemical analyses on 104
surgically resected CRC samples showed a positive correlation between HKII expression
with extensive tumor diameter, advanced tumor depth, presence of lymph node
metastasis, and shorter recurrence-free survival (Hamabe et al, 2014).
The rapid and disorganized proliferation associated with tumorigenesis leads to
ischemic regions within solid tumors (Raghunand et al, 2003). Cells within these regions
are exposed to poor blood flow leading to transient or chronic hypoxic and hypoglycemic
conditions. The impact of these microenvironments on the effectiveness of anti-cancer
therapies is not well understood. HKII expression is upregulated under hypoxic
conditions as a result of stabilized HIF1α transcription factor binding to the promoter
region of HKII, activating gene expression (Srinivas et al, 2001). Results from Chapter
III showed that limiting glucose availability in vitro led to a decrease in HKII expression.
The family of carbonic anhydrase (CA) enzymes catalyzes the hydration of carbon
dioxide to bicarbonate and proton (Potter & Harris, 2004). CAIX is a HIF-inducible gene
that has been proposed as an ischemic marker and is overexpressed colorectal cancer
(Loncaster et al, 2001; Niemela et al, 2007).
Although I demonstrated that HKII expression does not affect cellular sensitivity
to 3BP, there is still therapeutic potential in targeting HKII due to its importance in
aerobic glycolysis. Therefore, it is still worth examining the HKII expression in CRC
samples and determining its value as a prognostic marker and also as a chemotherapeutic
target. In this study, I evaluated the expression of HKII and CAIX in FFPE specimens of
colorectal cancer tumors and assessed its relationship to clinicopathologic or prognostic
relevance. Based on findings from other studies, I hypothesized that high HKII
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expression in colorectal cancer tumor samples would be associated with shorter
progression-free status and worse outcome.
Materials and Methods
A list of all chemicals and suppliers, and details of the materials and solutions
used for the following experiments are detailed in Appendices I and II, respectively.
Samples
Sixty FFPE colorectal cancer samples used for this study were gathered by the
Ontario Tumor Bank, Ontario Institute for Cancer Research. Samples were collected
between 2005 and 2011. The patients’ medical records were reviewed and general
information, including sex, age range at diagnosis, histological grade, and TNM staging
(Edge & Compton, 2010), are provided in Table 3. Follow-up data were retrieved for
survival analysis. The last follow-up was May, 2013. Cases were anonymized and I was
blinded to tumor and patient details until completion of image capture and analysis.
Cell lines and immunoblot analysis
Human colorectal cancer cell lines HCT116 and DLD-1 were obtained from
ATCC and were maintained in high glucose DMEM supplemented with 10% FBS and
incubated at 37oC in a humidified atmosphere containing 5% CO2 in room air. Anoxic
conditions were achieved using a Modular Incubator Chamber modified to allow
continuous flushing of the chamber with a humidified mixture of 95% N2 and 5% CO2.
Cells were lysed on ice with lysis buffer supplemented with 2 µg/ml aprotinin, 1 mM
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PMSF and phosphatase inhibitor cocktail II, following the manufacturer’s protocol.
Protein was quantified using the Bio-Rad DC Protein Assay Kit. 30 µg of total protein
was resolved in a 10% polyacrylamide gel using SDS-PAGE then transferred to a PVDF
membrane with wet transfer buffer at 100 V for 2 hr. Membranes were blocked for 1 h at
room temperature in 5% non-fat dry milk diluted in TBST followed by an overnight
incubation in blocking solution with primary rabbit anti-HKII (1:5,000), mouse anti-
CAIX (1:1,000) and mouse anti-α-tubulin (1:100,000). HRP-conjugated goat anti-rabbit
or mouse antibodies (1:10,000) were incubated for 1 h at room temperature. Luminata
Forte chemiluminescent substrate was used for visualization.
Immunofluorescent staining
FFPE histological sections were deparaffinized in xylene, rehydrated in
isopropanol, and washed with H2O. Antigen retrieval was performed by incubating
samples in 10 mM sodium citrate buffer (pH 6.0) at 95oC for 15 min and subsequently
left to cool at room temperature for 30 min. Sections were blocked with 1:1 solution of
5% goat serum in PBS + 0.1% Tween (PBST) and protein block solution for 1 hr at room
temperature. Sections were subsequently exposed to primary antibodies, rabbit mAb
HKII (1:400) and mouse mAb CAIX (1:400), diluted in PBST and incubated at 4oC
overnight in a humidified chamber. Sections were washed with PBST and then incubated
for 1 hr at room temperature with goat anti-rabbit Alexa 488-conjugated (1:300) and goat
anti-mouse Cy3-conjugated (1:200) antibodies diluted in PBST. Slides were
counterstained using DAPI and washed with PBS twice for 5 min, and coverslips were
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mounted with Fluorescence Mounting Media. Slides were stored flat in the dark at 4oC
and imaged the following day.
Imaging and quantification of immunofluorescence
Slides stained with HKII and CAIX antibodies were photographed with a QICAM
camera (QImaging, BC, Canada) with a 40X objective at 1.4 megapixel resolution, using
excitation and barrier filter sets for fluorescein (green emission), rhodamine (red
emission) and DAPI (blue emission). Images were saved as TIFF files. In total, 5 images
were captured per slide, covering different regions of the sample in an unbiased,
systematic fashion. Brightness and contrast adjustments were applied using Photoshop
CS6 (Adobe) on all images; images from the same slides were adjusted using the same
parameters. Fiji (Schindelin et al, 2012) was used to process and analyze the images to
determine HKII and CAIX staining. For CAIX staining in all images, a minimum
threshold value of 20 was used in the software and the percent area stained was
subsequently calculated. The Mean and SD from the 5 images were calculated for each
sample and used for subsequent analysis. A variation of the semi-quantitative H-Score
immunohistochemistry evaluation method (Hatanaka et al, 2003) was used to assess
HKII staining, and given the term “F-Score”. The percent area of HKII staining was
determined at 2 minimum threshold values (25 and 85), and the areas were added
together to form the F-Score as denoted in the following equation:
F-Score = (% area stained at threshold=25) + (% area stained at threshold=85)
The Mean and SD from the 5 images per sample were calculated and used for subsequent
analysis.
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Statistical analysis
GraphPad Prism v6.0 software was used for statistical analysis. Correlations
between staining parameters and patients’ characteristics were analyzed with the Fisher’s
exact test. Progression-free survival and overall survival were calculated per the Kaplan-
Maier method and were compared between two groups by using the logrank test. Patients
lost to follow-up, and those still progression-free and/or still alive at last follow-up were
right-censored for survival analysis. Results were considered significantly different when
a two-sided p value ≤ 0.05 was achieved.
Results
Patient characteristics
Clinicopathological characteristics of the study cohort are shown in Table II.
Briefly, there were slightly more men (N=34, 57%) than women in this study. The cohort
contained both low grade (I & II; N=30, 50%) and high grade (III & IV; N=28, 47%)
tumors; grading for two cases was not available. Sixty three percent of patients presented
with low TNM tumors (Stage 1, 2 & 3; N=38) while 22% presented with high TNM
tumors (Stage 4; N=13). TNM stage was not available for 15% (N=9) of the cases.
Immunoblot and immunofluorescent detection of HKII and CAIX
Human colorectal cancer cells showed an upregulation in both HKII and CAIX
expression following 24 h anoxic exposure in vitro (Figure 13). IF revealed
heterogeneous staining for both HKII and CAIX in FFPE sections of human CRC;
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representative staining patterns are shown in Figure 14. Both markers were localized to
the cytoplasm in neoplastic cells. HKII staining was predominantly located in areas of
low CAIX staining, suggesting HKII expression is primarily present in non-ischemic
regions of the tumors.
Levels of CAIX and HKII staining
Computational analysis was used to examine both CAIX and HKII staining.
Representative analyses for areas staining low or high for CAIX are shown on the left
and right panels of Figure 15, respectively. The median area stained for CAIX was
15.5% with a minimum of 1.3% and maximum of 65.5%, and this median value was used
to dichotomize the cases into ‘low’ and ‘high’. For HKII staining the F-Score was
obtained by the summation of percent area HKII staining at two minimum thresholds, of
25 and 85, as represented in Figure 16. Examples of HKII staining and corresponding F-
Scores are shown in Figure 17. The median F-Score of the five images per sample was
24.7 with a minimum of 3.4 and maximum of 71.5. This median F-Score was used to
dichotomize the cases into ‘low’ and ‘high’.
Association of CAIX and HKII expression with patient characteristics
Age was significantly associated with 'high' CAIX expression (p=0.038). The
odds of patients 70 years of age or older staining high for CAIX expression was
approximately 3.5 times greater than patients younger than 70. HKII expression was not
statistically associated with sex, age at diagnosis, histological grade, TNM stage, nor
primary tumor size (Table III).
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Association of CAIX and HKII expression with patient outcome
No significant differences in PFS were observed between patients with low CAIX
expression (median PFS, 2132 days) and those with high expression (median PFS, 548
days); p = 0.16) (Figure 18A). Likewise, no significant differences in OS were observed
between patients with low CAIX expression (median OS, undefined) and those with high
expression (median OS, 1011 days); p = 0.085) (Figure 18B), although both PFS and OS
trended towards better outcome with low CAIX. Kaplan-Meyer survival analysis showed
a significant association between HKII expression and median survival duration of CRC
patients (Figure 18C,D). Specifically, patients who scored high for HKII had
significantly longer progression-free survival (Undefined vs. 497 days; p=0.016) and
overall survival (Undefined vs. 1060 days; p=0.025) than patients who scored low for
HKII.
Association of stromal HKII staining with patient characteristics and outcome
Intense stromal HKII staining was present in at least 1 of 5 images in 30 of the 60
samples analyzed (shown by arrows in Figure 17E). This stratification was used to
dichotomize the cases into “stromal positive” and “stromal negative”. Stromal HKII
staining was not statistically associated with sex, age at diagnosis, histological grade,
TNM stage, nor primary tumor size (Table IV). Samples positive for stromal HKII
staining were associated with larger primary tumor sizes, although not significantly (p =
0.119). Kaplan-Meyer survival analysis showed that stromal HKII staining was
associated with median survival duration of CRC patients (Figure 19). Specifically,
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patients whose tumors presented with stromal HKII staining had significantly shorter
progression-free survival (Figure 19A; 879 vs. 2132 days; p=0.049) and overall survival
(Figure 19B; 1011 vs. Undefined days; p=0.024) than patients whose tumors did not
present with stromal HKII staining.
Discussion
The results of this study revealed that low HKII scoring was associated with
shorter progression-free and overall survival in human CRC patients. During
hepatocellular carcinogenesis, the normal expression of HKIV in the liver is silenced
whereas HKII gene expression is induced, thereby promoting the glycolytic phenotype
(Mayer et al, 1997). Results from Chapter III suggested HKII is upregulated in more
aggressive CRC cell lines, but the impact of ischemia on cell signaling in the context of
solid tumors is poorly understood. In this study, I sought to quantify HKII expression in
FFPE samples of human colorectal cancer using immunofluorescence. This approach was
utilized to allow for co-localization between staining of HKII and of carbonic anhydrase
IX, a marker of tissue ischemia (Potter & Harris, 2004). My findings were subsequently
correlated to clinicopathological characteristics of the patients, and to patient outcome.
I initially wanted to verify the correlation between low oxygen conditions and the
induction of CAIX expression by exposing human CRC cells to anoxia in vitro. I found
that in HCT116 and DLD-1 cells, both HKII and CAIX expression was upregulated
under anoxic conditions (Figure 13). Upregulation of CAIX expression under low
oxygen conditions has been reported in breast and prostate cancer (Li et al, 2009; Wykoff
et al, 2001). Significant correlation between HKII and HIF1α, a marker of hypoxia, has
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also been reported in hepatocellular and gastric carcinoma tissues (Qiu et al, 2011;
Yasuda et al, 2004). However, those studies did not examine HKII expression within
different tumor microenvironments via co-localization.
The abnormal vasculature that develops as a result of tumor progression leads to
ischemic areas that consist of both hypoxic and hypoglycemic conditions (Raghunand et
al, 2003). While the effects of hypoxia on tumor progression have been well documented,
the effects of hypoglycemia have been less studied. My results reveal that HKII staining
was present primarily in regions negative for CAIX staining, indicating that HKII is
predominantly expressed in non-ischemic regions of these CRC tumors. This suggests
that while hypoxia and hypoglycemia may have opposite effects on HKII expression, the
availability of glucose to cancer cells may ultimately determine the expression of this
enzyme. Previous work in our laboratory using preclinical models has established the
importance of hypoglycemia in CRC progression, including its ability to influence
mutation rate, DNA repair, and DNA methylation (Plumb et al, 2009; Shahrzad et al,
2005; Skowronski et al, 2014; Skowronski et al, 2010).
Since I found that HKII and CAIX expression were not co-localized, the
assessment of their expression patterns was performed separately. A number of studies
have shown that hypoxia-related overexpression of CAIX in the tumor is associated with
poor survival rates, including esophageal cancer (Birner et al, 2011), endometrioid
ovarian cancer (Choschzick et al, 2011), and oral cavity squamous cell carcinoma
(Brockton et al, 2012). CAIX is generally upregulated in the tumor compared to normal
intestinal epithelium (Kivela et al, 2001; Saarnio et al, 1998a; Saarnio et al, 1998b). In
the present study, CAIX expression was found to only be significantly associated with
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age and none of the other clinicopathological characteristics. Increased CAIX expression
was associated with shorter progression free and shorter overall survival in our patient
set. However, these results were not statistically significant, perhaps reflective of the
small sample size.
Increased HKII expression has been observed via immunohistochemistry when
comparing hepatocellular carcinoma to non-dysplatic cirrhosis (Guzman et al, 2015).
HKII is a potential prognostic marker in breast carcinoma (Sato-Tadano et al, 2013),
laryngeal squamous cell carcinoma (Chen et al, 2014), glioblastoma multiforme (Wolf et
al, 2011), and gastric adenocarcinoma (Qiu et al, 2011). In the present study, I
hypothesized that high HKII expression would correlate with worse patient outcome (as
has been previously reported for CRC; Hamabe et al, 2014), due to an increased capacity
to increase glucose consumption and produce the necessary glycolytic intermediates to
sustain rapid proliferation. HKII expression was not associated with any of the
clinicopatholigical characteristics assessed. Of most interest, lower HKII expression was
found to be significantly associated with shorter progression-free status and worse overall
survival. While this is the first such observation in colorectal cancer, Lyshchik et al had
also observed this correlation in pancreatic cancer, where patients with high HKII
expression survived longer than those with low HKII expression (Lyshchik et al, 2007).
I was interested in optimizing a protocol that would allow for automated analysis
of immunofluorescence staining of any target through computational algorithms that can
be used as a rapid prognostic tool in the future. I therefore developed a variation of the H-
Score used for HKII staining, denoted as the F-Score, which took into account the 2
distinct staining intensities that were observed. This approach took into consideration
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different expression levels of HKII throughout the tumor sections analyzed. As
mentioned, there are conflicting reports on the impact of HKII expression on cancer
progression. This, combined with the observation of intense HKII staining in the stroma
of 50% of the cases in my study, suggested that HKII may prove a more useful
prognostic marker using stromal vs. neoplastic cell staining as a focal point of analysis.
It has become increasingly apparent that the tumor microenvironment plays a
critical role in tumorigenesis. Stratification of colon cancer patients based on tumor
stroma revealed a correlation between patients with high stromal content and shorter
progression free and overall survival (Mesker et al, 2007). Stromal fibroblasts that
undergo myofibroblast differentiation lose caveolin 1 (Cav-1) expression which induces a
cancer-associated fibroblast phenotype, characterized by an increase in glycolysis
(Pavlides et al, 2009). This observation has been termed the ‘reverse Warburg effect’ in
which there is metabolic coupling between supporting glycolytic stromal cells and
oxidative tumor cells. Loss of stromal Cav-1 expression was associated with both shorter
disease-free and overall survival in CRC (Zhao et al, 2015). Cav-1 null stromal cells also
upregulate key glycolytic genes, including HKII (Pavlides et al, 2009).
My study showed intense HKII stromal staining in 30 of 60 cases. Based on the
trend between positive stromal HKII staining and increased tumor sizes, my results are
consistent with the possible role of the tumor stroma in feeding neoplastic cells and
supporting tumor growth. Further analysis revealed that patients with tumors presenting
stromal HKII staining had significantly shorter progression-free and overall survival than
patients with tumors lacking stromal HKII staining. This association of survival and
tumor aggressiveness (as revealed by outcome) with stromal HKII is novel for CRC and
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indicates the possibility of the reverse Warburg effect occurring in some cases of
colorectal cancer. Stromal HKII may be a more informative prognostic marker than
overall HKII expression within the tumor. Further study with a larger number of patients
is needed to confirm these findings. Additional studies are needed to identify which
stromal cell type(s) are expressing HKII, and whether that expression is associated with
changes in Cav-1 as shown previously (Pavlides et al, 2009).
My study had several limitations. The retrospective nature of this study is a major
limitation, due to possible confounding factors and bias associated with case selection.
The samples for this study were gathered from different tumor bank sites around Ontario.
Thus, the fixation protocols may have been slightly altered leading to possible variability
in the staining outcome and the sample size is relatively small. Further studies using
tumor microarrays can be used to control staining protocols and increase sample size.
In conclusion, I have demonstrated that HKII is expressed in non-ischemic
regions of tumors and that lower expression by neoplastic cells is associated with worse
outcome in patients with colorectal cancer. HKII was also highly expressed in stromal
cells of some cases, where it was associated with worse outcome, suggesting an
important role for HKII in glycolytic stromal cells. Until recently, the Warburg effect
phenomenon was thought to occur in most cancer cells (Gatenby & Gillies, 2004). There
is now a growing appreciation for the complexity of tumor metabolism and the interplay
between the tumor and adjacent stromal tissue (Pavlides et al, 2009). Identifying
glycolytic enzymes that are upregulated in the stromal compartment, such as HKII, may
help to improve the efficacy of targeting tumor metabolism as therapeutic strategy for
human CRC. These findings may be widely applicable, as many different cancer subtypes
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exhibit an increased glycolytic phenotype and in many cases a large proportion of the
tumor mass is stromal in nature.
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Table II. Patient characteristics.
Variable N %
Sex
Male 34 57
Female 26 43
Age at diagnosis (years)
40-49 6 10
50-59 12 20
60-69 13 22
70-79 17 28
80-89 9 15
90-94 3 5
Histological Grade
I 5 8
II 25 42
III 19 32
IV 9 15
Not available 2 3
TNM Stage
1 2 3
2 16 27
3 20 33
4 13 22
Not available 9 15
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Table III. Associations of CAIX and HKII staining with patient characteristics and tumor pathology.
Variable High CAIX (N=30) High HKII F-Score (N=30)
N n % P value n % P value
Sex
Male 34 16 47.1 0.795 17 50.0 1.000
Female 26 14 53.8 13 50.0
Age at diagnosis (years)
< 70 31 11 35.5 0.038* 14 45.2 0.797
≥ 70 29 19 65.5 16 55.2
Histological Grade
I/II 30 13 43.3 0.600 15 50.0 0.799
III/IV 28 15 53.6 13 46.4
TNM Stage
1-3 38 19 50.0 0.336 24 63.2 0.057
4 13 9 69.2 4 30.8
Primary tumor size (cm)
≤ 5 27 14 51.9 1.000 13 48.1 1.000
> 5 33 16 48.5 17 51.5
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Table IV. Associations of stromal HKII staining with patient characteristics and tumor pathology.
Variable Presence of Stromal HKII (N=30)
N n % P value
Sex
Male 34 16 47.1 0.795
Female 26 14 53.8
Age at diagnosis (years)
< 70 31 13 41.9 0.302
≥ 70 29 17 58.6
Histological Grade
I/II 30 15 50.0 0.799
III/IV 28 15 53.6
TNM Stage
1-3 38 17 44.7 1.000
4 13 6 46.2
Primary tumor size (cm)
≤ 5 27 10 37.0 0.119
> 5 33 20 60.6
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Figure 13. HKII and CAIX immunoblotting in colorectal cancer cell lines. HKII and CAIX expression was detected by western blot analysis. Two human colorectal cancer cells were exposed to either normoxia (C; 20% O2) or anoxia (A; < 0.1% O2) for 24 hours. In response to anoxia, both cell lines showed upregulation in HKII and CAIX expression.
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Figure 14. HKII and CAIX immunostaining in colorectal cancer. Each row in descending order represents immunostaining of HKII (green), CAIX (red), DAPI nuclear counterstain (blue), and merged images. CAIX was used as a marker of ischemia. The left panel shows images from a non-ischemic region of a tumor sample, noted by the lack of CAIX staining. The right panel shows images from an ischemic region of a different tumor sample, noted by the presence of CAIX staining. Images were taken at 40X objective.
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Figure 15. Evaluation of CAIX immunostaining. Images from CAIX staining, as shown on the top panel, were subjected to computational analysis. Images were taken at 40X objective. Bottom panel shows analysis of CAIX staining when the detection threshold was set at 20. Percent area stained (outlined in yellow) is calculated by the amount of staining set by the threshold value and numerically shown on the bottom right corner. Examples of low and high CAIX staining from two different tumors are shown on the left and right panels, respectively.
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Figure 16. Evaluation of HKII immunostaining. Images from HKII staining from two different CRC specimens, as shown on the top panel, were subjected to computational analysis. Images were taken at 40X objective. Middle and bottom panels show analysis of HKII staining when the detection threshold was set at 25 and 85, respectively. Percent area stained (outlined in yellow) is calculated by the amount of staining set by the threshold values, and numerically displayed on the bottom right corner.
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Figure 17. Immunofluorescent staining of HKII and corresponding F-Scores. Images were taken at 40X objective. The F-Score is calculated by summation of the two values generated by the percent area stained at the two thresholds. Images in the top panel with a F-Score of < 25 were considered to have a low F-Score. Images in the bottom panel with a F-Score of > 25 were considered to have a high F-Score. Arrows in E indicate stromal cell staining.
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Figure 18. Progression-free survival and overall survival by CAIX and HKII staining. Kaplan-Meier curves of PFS (A, C) and OS (B, D) in patients with CRC based on CAIX (A, B) and HKII (C, D) staining. Ticks indicate censored subjects.
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Figure 19. Progression-free survival and overall survival by the presence of stromal HKII staining. Kaplan-Meier curves of PFS (A) and OS (B) in patients with CRC based on the presence of stromal HKII staining. Ticks indicate censored subjects.
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GENERAL DISCUSSION
Metabolic reprogramming is an important aspect of tumorigenesis that remains to
be fully understood. Initial observations on lactate buildup in tumors under normal
oxygen tension have been further characterized through alterations in metabolic signaling
pathways in cancer cells, a phenomenon known as the Warburg effect (Warburg et al,
1927). These alterations are a result of mutations in key glycolytic enzymes and are
aberrantly regulated by oncogenic signaling (Koppenol et al, 2011). The tumor
microenvironment is an important modulator of tumor behavior (Raghunand et al, 2003;
Subarsky & Hill, 2003). What remains poorly understood are the effects of the tumor
microenvironment on drug sensitivity, especially pertaining to treatments that affect
metabolic pathways. Previous data generated in our laboratory suggesting that DCA, a
potential anti-cancer agent, provided a protective effect to ischemic CRC cells. I therefore
sought to further understand this phenomenon and to determine the effects of compounds
targeting metabolic pathways in CRC.
Over the course of the 21st century, much research interest has focused on better
comprehending the metabolic pathways that permit rapid tumor growth. A major issue
with studies elucidating the mechanisms of cancer metabolism lies in the experimental
models used. It is well known and widely accepted that results collected through in vitro
experimentation are not necessarily reproducible in vivo, and that therapeutic success in
preclinical models and early phase studies rarely translates to advanced trials. One
possible reason for this disconnect is the influence of the natural microenvironment seen
in solid tumors. The effect of the tumor microenvironment on drug sensitivity is
dependent on both the therapeutic agent and the cells being studied (Strese et al, 2013).
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This is one reason why targeting various aspects of cancer metabolism may show anti-
cancer potential in vitro but not show any benefit in vivo.
Our laboratory previously showed that DCA provided a cytoprotective effect to
human CRC cells under anoxic conditions. In contrast, Xu and colleagues found that
targeting lymphoma and CRC cells with another compound, 3BP, was more effective
under hypoxic conditions (Xu et al, 2005). I wanted to follow up our previous study and
determine the molecular mechanisms by which DCA-induced cytoprotection was
conferred. In Chapter II, I found differential regulation of PDH phosphorylation between
different human CRC cells led to differences in mitochondrial activity following DCA
exposure. However, these effects did not result in significant changes in cellular
metabolism nor growth. This suggests that DCA may only be beneficial in treating a
subset of tumor types based on their molecular profiles of different PDK isoform
expression.
As with hypoxia, glucose concentration varies in a gradient fashion resembling
what is observed with oxygen levels in the extravascular space of solid tumors
(Kallinowskil et al, 1985). Tumor hypoxia is a common occurrence, and since it is
primarily due to insufficient blood flow, reduced nutrient supply such as low glucose
availability is likely also associated with those conditions. As previously discussed,
aerobic cancer cells are able to utilize lactate as fuel and allow glucose to support
glycolytic cells at greater distances from supporting blood vessels (Sonveaux et al, 2008).
Therefore, a tumor with inadequate vascularization would experience both hypoxic and
hypoglycemic conditions, although glucose may be able to diffuse over greater distances
due to the ability of aerobic cells to preferentially metabolize lactate.
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The bulk of research examining tumor ischemia has focused on hypoxia and the
effects of hypoglycemia on tumor progression are not well studied. This issue would be
particularly important when studying the effects of compounds that may alter cellular
metabolism, which is heavily reliant on glucose consumption. In particular, 3BP is a
pyruvate analog that inhibits HKII activity and effectively kills cancer cells (Ko et al,
2001). Therefore, the effects of this drug on cytotoxicity may be altered by glucose
availability. I found that human CRC cells were far more sensitive to the cytotoxic effects
of 3BP when cultured under high glucose conditions versus normal glucose conditions.
The IC50 value against 3BP in SKOV3 cells increases by over 3 fold when treated in
galactose medium vs. glucose medium, suggesting the effects of simply treatment
medium alone affects drug response in vitro (Wintzell et al, 2012).
Glucose-dependent alteration of 3BP-sensitivity led me to suspect that my
observations could be explained by the increase in HKII expression I saw under high
glucose conditions. However, HKII knockdown using RNA interference resulted in no
differences in 3BP sensitivity. Thus, I believe that the differences observed in 3BP
sensitivity in CRC cells under altered glucose availability conditions cannot be explained
by HKII expression alone. Since 3BP acts as an alkylating agent and affects other
glycolytic enzymes, it is possible that the main effects of 3BP in CRC are through other
metabolic pathways. Although HKII expression did not ultimately correlate with 3BP
sensitivity, it is still worthwhile to continue examining the therapeutic potential of 3BP
against colorectal cancer. The potent effects of 3BP on colorectal cancer have been
shown in a xenograft mouse model, where 3BP treatment over 6 days led to over 50%
tumor regression (Sanchez-Arago & Cuezva, 2011).
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Under glucose limiting conditions, cancer cells including glioma (Beckner et al,
2005; Bouzier et al, 1998), cervical, breast carcinoma, hepatoma, and pancreatic (Plecita-
Hlavata et al, 2008; Rossignol et al, 2004; Smolkova et al, 2010) switch from aerobic
glycolysis to oxidative phosphorylation. Furthermore, removal of glucose, or inhibition
of glycolysis by iodoacetate in HeLa cells led to a rapid switch toward glutamine
utilization (Brand et al, 1994). These findings suggest the flexibility of cancer cells to
utilize different metabolic pathways and survive in various microenvironments.
The majority of metabolic enzymes implicated in tumorigenesis are not mutated
and furthermore are expressed in both transformed and normal cells (Koppenol et al,
2011). This leads to a considerable set of challenges regarding target specificity when
using metabolic modulating approaches. However, regardless of enzyme expression, the
differences observed between transformed and normal metabolism provides a window for
therapeutic intervention. Furthermore, the additional issue of the tumor
microenvironment and its impact on the outcome from therapeutic intervention further
adds to the complexity of cancer metabolism (Pavlides et al, 2009). Future work studying
the effects of DCA and 3BP in CRC cells would include evaluating the combination of
both hypoxic and hypoglycemic conditions to better recapitulate what may occur in the
tumor microenvironment. Several other environmental factors present in solid tumors
may be studied beyond oxygen and nutrient supply, including interaction with stromal
cells (such as vascular endothelial cells, infiltrating immune cells, and tumor fibroblasts)
and acidity (in part secondary to hypoxia, and metabolism). These factors can be studied
using models that better resemble the in vivo conditions by the use of spheroid or
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organoid cultures (Gao et al, 2014; Santini & Rainaldi, 1999). Treating cells for
prolonged incubation times beyond confluency could mimic possible contact inhibition
effects and study confluence dependent resistance in vitro (Pelletier et al, 1990).
While the ability of cancer cells to increase their glucose consumption has been
confirmed through FDG-PET scanning in vivo, the shuttling of glucose for further
processing through either glycolysis or oxidative phosphorylation cannot be evaluated
using this technique. Several glioma cell lines are highly dependent on oxidative
phosphorylation for ATP production (Griguer et al, 2005). 50-80% of ATP production in
various cancer cell lines was generated through oxidative phosphorylation (Guppy et al,
2002; Kallinowski et al, 1989; Kallinowski et al, 1988; Zu & Guppy, 2004). With
increasing reports outlining the importance of oxidative phosphorylation in cancer
metabolism, the Warburg effect is currently being challenged by other theories.
Growing evidence suggests the tumor microenvironment plays a pivotal role in
tumor progression and metastasis. A hypothesis gaining traction is termed the ‘reverse
Warburg effect’ formulated by Dr. Michael Lisanti; it suggests that aerobic glycolysis
occurs in the fibroblastic tumor stromal compartment rather than in tumor cells (Pavlides
et al, 2009). These cancer cells induce the Warburg effect in neighboring stromal
fibroblasts, resulting in differentiation of cancer-associated fibroblasts (CAFs) to
myofibroblasts. These tumor-associated myofibroblasts express muscle-specific actin,
and show an increased ability to secrete and remodel the extracellular matrix (Ronnov-
Jessen et al, 1996; Shekhar et al, 2003). The glycolytic end-products, pyruvate and
lactate, are then secreted by CAFs and taken up by adjacent cancer cells to sustain their
proliferative capacity and continued use of oxidative metabolism for energy production.
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In human breast cancer tissues lacking Cav-1 expression, 2 key glycolytic enzymes,
PKM2 and LDH, are prominently expressed in CAFs and not within adjacent cancer
cells, consistent with the reverse Warburg effect hypothesis (Pavlides et al, 2009).
The reverse Warburg effect has also been independently confirmed by Pol and
colleagues, where it was also observed that Cav-1 deficient murine fibroblasts had
dysfunctional mitochondria and that restoring its expression recovered mitochondrial
function (Bosch et al, 2011). CAFs have been shown to express MCT4 for lactate
secretion while adjacent breast cancer cells express MCT1 for lactate uptake (Whitaker-
Menezes et al, 2011).
Under monolayer culturing conditions, MCF7 breast cancer cells exhibit very low
mitochondrial mass, a conventional aerobic glycolysis phenotype. However, when these
cells were co-cultured with fibroblasts, it led to a significant increase in the mitochondrial
mass of the MCF7 cells. Lactate administration to MCF7 cells also led to an increase in
mitochondrial mass, suggesting lactate produced by the fibroblasts may promote
mitochondrial biogenesis and increase mitochondrial respiration (Martinez-Outschoorn et
al, 2010). In a breast cancer mouse model, oncogenically driven human organoids
develop into tumors only if co-injected with immortalized fibroblasts and not with normal
primary fibroblasts, indicative of stromal activation in breast cancer formation (Wu et al,
2009). These results suggest that cross talk between transformed tumor cells and the
adjacent stroma exists.
Metabolic cooperation between adjacent cell compartments is not unique to
tumors. During ovarian folliculogenesis, the oocyte controls cumulus granulosa cell
metabolism to compensate for its lack of critical metabolic functions, including
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cholesterol synthesis and amino acid uptake (Su et al, 2009; Sugiura et al, 2007).
Glycolytic astrocytes generate lactate to support oxidative phosphorylation in adjacent
neurons (Magistretti & Pellerin, 1996; Tsacopoulos & Magistretti, 1996). Likewise,
glycolytic fast-twitch muscle fibers produce lactate that is secreted for slow-twitch fibers
to utilize as oxidative phosphorylation substrates (Connett et al, 1984; McCullagh et al,
1997).
In Chapter IV, I observed the presence of strong HKII staining in the stromal
compartment and weaker staining in the adjacent tumor epithelial cells. This would
suggest the potential presence of the reverse Warburg effect in some CRC tumors. Other
studies found that colon cancer patients with high tumor stromal content show
dramatically increased tumor progression and poor survival (Benita et al, 2009; Mesker
et al, 2007). The observed HKII staining pattern provides further evidence to suggest that
cell culture experiments in vitro might not reflect what occurs in an in vivo experimental
system, in which the tumor microenvironment would affect metabolic strategies and
cellular responses. Another interesting finding from Chapter IV was the negative
correlation between HKII expression and survival measures, a finding that has also been
reported in pancreatic cancer (Lyshchik et al, 2007). This could suggest that aggressive
tumors have adapted an ability to better utilize other carbon fuels for its metabolic
processes, thereby reducing expression of glucose-related processing molecules such as
HKII. Glucose and glutamine account for most carbon and nitrogen metabolism in
mammalian cells (Wellen et al, 2010). Using ovarian cancer cell lines, Yang and
colleagues discovered a correlation between glutamine dependence and invasiveness;
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high-invasive ovarian cancer cells were markedly glutamine dependent versus low-
invasive ovarian cancer cells (Yang et al, 2014).
A chronic disease characterized by hyperglycemia is diabetes mellitus. Globally,
approximately 387 million people aged between 20 to 79 years have diabetes. Type 2
diabetes, characterized by insensitivity to insulin, accounts for 85-95% of prevalent cases
(International Diabetes Foundation, 2014). Metformin is the first-line therapy for type 2
diabetes (Alexander et al, 2008). This metabolic modulator decreases plasma glucose
concentrations by decreasing hepatic gluconeogenesis and increasing cellular glucose
uptake (Iozzo et al, 2003; Wollen & Bailey, 1988). This is accomplished through
inhibition of complex I of the electron transport chain, thereby increasing AMP:ATP
ratio and activating AMPK signaling (El-Mir et al, 2000).
In 2005, Evans and colleague found a 23% reduction in total cancer incidence in
diabetic patients treated with metformin (Evans et al, 2005). Several meta-analyses also
show a correlation between metformin use and lowered cancer incidence (Gandini et al,
2014). Metformin inhibits proliferation and induces apoptosis in cancer cells, presumably
due to the re-wiring of cancer metabolism to better utilize mitochondrial respiration and
increase ATP production via AMPK activation (Luo et al, 2012; Owen et al, 2000; Zhou
et al, 2001). Similarly, numerous epidemiological studies suggest an association between
type 2 diabetes and cancer, including increased risk and mortality rates (Joung et al,
2015).
However, increased focus on metformin use and cancer risk has led to conflicting
data arising from different epidemiological, human, and animal studies. The variation in
estimated risks may differ due to the characteristics of the study population, including
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genetic susceptibility, life-style behavior, and environmental exposure (Habib & Rojna,
2013). In a systematic review and meta-analysis on studies controlling for body mass
index, prospective studies, and studies without time-related biases, Gandini and
colleagues found that the association between metformin use and decreased overall
cancer incidence was considerably smaller than observed without such adjustments (10-
18% vs. 31%) (Gandini et al, 2014). High glucose media concentrations reduced the
effectiveness of metformin on breast cancer cell proliferation (Wahdan-Alaswad et al,
2013). The relationship between diabetes and cancer remain unclear; the hyperglycemic
conditions associated with diabetes may facilitate neoplastic proliferation, which is
reduced upon metformin exposure. This cancer-diabetics association may also be indirect
and due to common risk factors such as obesity (Gallagher & LeRoith, 2015). Clinical
trials are needed to determine whether metformin can in fact be applicable to non-
diabetic populations. Results from pre-clinical and retrospective studies support testing
the use of metformin as an adjuvant in chemotherapy (Zhang et al, 2013).
An alternative to directly targeting glycolytic enzymes with metabolic modulators
is to exploit the regulation of these enzymes. A field of molecular biology with rapidly
growing interest is the role of small interfering RNAs. In particular, microRNAs are non-
coding 18-24 nucleotide-long RNAs that match and specifically bind onto 3’ untranslated
regions of target mRNA, resulting in altered gene expression of a suite of target genes
(Flynt & Lai, 2008; Vasudevan et al, 2007). Over the past few years, a large number of
miRNAs have been implicated in tumor metabolic regulation and the Warburg effect
(Bienertova-Vasku et al, 2013; Chen et al, 2012; Hatziapostolou et al, 2013). miRNAs
have been shown to regulate several glycolytic enzymes, including GLUTs, GAPDH,
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LDHA and PKM2 (Chan et al, 2015). HKII expression in particular is regulated by
numerous miRNAs. miR-143 expression is downregulated in human glioblastoma, and its
overexpression leads to glycolytic inhibition via HKII downregulation (Zhao et al, 2013).
Downregulation of HKII via miR-143 and inhibition of glucose metabolism was also
shown in head and neck squamous cell carcinoma (Peschiaroli et al, 2013), breast cancer
(Jiang et al, 2012), lung cancer (Fang et al, 2012), and colon cancer (Gregersen et al,
2012). Jiang and colleagues showed that miR-55 promoted HKII transcription through
activation of signal transducer and activator of transcription 3 (STAT3) in hepatocellular
carcinoma cells (Jiang et al, 2014). miR-125b was also found to regulate HKII
expression, and this finding was also observed in lung cancer (Fang et al, 2012) and in
chronic lymphocytic leukemia (Tili et al, 2012). Kim and colleagues demonstrated that
miR-21 and -34 regulates HKII expression in bladder cancer and lung cancer,
respectively, while both miRNAs were capable of repressing HKII expression in colon
cancer (Kim et al, 2013). Through bioinformatics analysis, it was revealed that miR103/7
might have the ability to target PDK4 (Wilfred et al, 2007).
As some miRNAs have been implicated in tumorigenesis and thus preferentially
expressed in cancer and not normal cells, therapeutic strategies can be developed to target
these small RNA molecules. Studies show that miRNA deregulation leads to reduction in
cancer cell growth with no effects on normal cells, suggesting its therapeutic potential
(Drakaki et al, 2013; Hatziapostolou et al, 2013). Thus, either downregulating miRNAs
important to cancer malignancy or upregulating those that would otherwise limit cancer
progression may be a potential anti-cancer strategy. A limitation to this approach is our
current lack of depth of knowledge in this field. Since miRNAs have numerous targets
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and thus functions in cell biology, it is important to first comprehend the ability of the
various miRNAs that play a role in cancer progression before contemplating its
therapeutic potential. Furthermore, the effectiveness of in vivo delivery of miRNAs/anti-
miRs is still a major hurdle in clinical studies (Park et al, 2011).
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SUMMARY AND CONCLUSIONS
The research described in this thesis highlighted the effects of dichloroacetate and
3-bromopyruvate on human colorectal cancer cells under varying culture conditions in
attempts to mimic aspects of the tumor microenvironment. The goals were to determine
how different in vitro conditions might alter metabolic signaling and ultimately affect
drug sensitivity in human CRC cells. In addition, I assessed the expression of a key
glycolytic enzyme, hexokinase II, in colorectal cancer tumor samples to better understand
its regulation within the microenvironment in vivo.
I initially studied the molecular mechanisms surrounding DCA-induced
cytoprotection in CRC cells. PDKs 1 and 3 were determined to be the predominant
isoforms expressed by different CRC cell lines. This in part could account for the
resistance to DCA-induced apoptosis observed, as PDK2 is reported to be most sensitive
to the effects of DCA. I found changes in expression of PDK isoforms under anoxia that
led to slight differences in PDH phosphorylation at residue Ser293 and mitochondrial
activity. However, these molecular changes did not lead to significant changes in cell
metabolism or proliferation. I propose that DCA may only be beneficial in treating a
subset of tumor types based on their molecular profiles of different PDK isoforms.
The next compound I focused on was 3-bromopyruvate and its proposed target,
hexokinase II. I found that the variation in HKII expression between different CRC cell
lines correlated to their sensitivity towards 3BP. Exposure to 3BP at lower doses led to
induction of apoptosis while necrotic cell death was observed at higher doses. Studies
involving the manipulation of glucose availability in culture conditions showed that cells
were less sensitive to the effects of 3BP when cultured under normal glucose vs. high
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glucose concentrations. However, HKII knockdown experiments revealed no differences
in 3B sensitivity, even with robust HKII knockdown. This suggests that 3BP sensitivity
could be correlated to the bioenergetic signature of cancer cells and that HKII expression
is a surrogate marker for this signature. Such a situation explains the correlation found
between HKII expression and 3BP sensitivity while manipulations to HKII expression
led to no changes in 3BP sensitivity.
Although other enzymes are targeted by 3BP in CRC cells, HKII still plays an
important role in aerobic glycolysis. Thus, I examined its expression in FFPE samples of
CRC tumors with known clinical outcomes to determine whether HKII expression is a
prognostic marker for patient outcome. HKII expression was detectable only in non-
ischemic regions of tumors. Surprisingly, I found that low HKII expression correlated
with both shorter disease free progression and with worse overall survival in patients.
Stromal cells sometimes exhibited high HKII expression, agreeing with a novel theory on
cancer metabolism termed the ‘reverse Warburg effect’, in which the glycolytic
phenotype is exhibited by the stromal cells within the tumor and not the malignant cells
themselves.
In conclusion, these studies have provided a better understanding of how the
tumor microenvironment plays a role in altering the sensitivity of cancer cells to various
metabolic modulators. Both oxygen and glucose deprivation can alter the effects of
compounds such as DCA and 3BP. An issue with in vitro experimentation involves the
lack of external cues otherwise provided by the microenvironment in in vivo settings. A
better understanding of how the tumor microenvironment may affect the susceptibility of
cancer cells to metabolic re-wiring will allow researchers to develop and identify more
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effective anti-cancer compounds and will provide better insight into the possible
translational effects in vivo.
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APPENDIX I – CHEMICAL LIST AND SUPPLIERS
Chemical Supplier 3BP Sigma-Aldrich, St. Louis, MO Acetic acid, glacial Fisher Scientific, Nepean, ON Acrylamide/Bis solution, 40% Bio-Rad Laboratories, Hercules, CA Agarose Life Technologies, Carlsbad, CA α-Tubulin mouse mAb; T5168 Sigma-Aldrich, St. Louis, MO Amido Black Bio-Rad Laboratories, Hercules, CA Ammonium persulfate Sigma-Aldrich, St. Louis, MO Amplex Red Glucose Assay Life Technologies, Carlsbad, CA Alexa Fluor® 488 Annexin V/Dead Cell Life Technologies, Carlsbad, CA Apoptosis Kit Aprotinin Sigma-Aldrich, St. Louis, MO pan-AKT rabbit mAb; 4691 Cell Signaling Technology, Danvers, MA pAKT (S473) rabbit pAb; 9271 Cell Signaling Technology, Danvers, MA pAKT (T308) rabbit mAb; 4056 Cell Signaling Technology, Danvers, MA Aurum Total RNA Fatty and Fibrous Kit Bio-Rad Laboratories, Hercules, CA BSA AMRESCO, Solon, OH CAIX mouse mAb; ab107257 Abcam plc, Cambridge, UK Caspase-3 rabbit pAb; 9662S Cell Signaling Technology, Danvers, MA Cell lysis buffer, 10X Cell Signaling Technology, Danvers, MA Crystal violet Fisher Scientific, Nepean, ON DC protein assay Bio-Rad Laboratories, Hercules, CA DCA Sigma-Aldrich, St. Louis, MO DMEM Sigma-Aldrich, St. Louis, MO DNA Ladder, 50 Kb SibEnzyme, Russia Etoposide Sigma-Aldrich, St. Louis, MO FBS Life Technologies, Carlsbad, CA Fluorescence Mounting Media Dako, Denmark Gentamicin Sigma-Aldrich, St. Louis, MO Glycine Fisher Scientific, Nepean, ON Goat anti-mouse Cy3 Ab Life Technologies, Carlsbad, CA Goat anti-mouse HRP Ab; A9917 Sigma-Aldrich, St. Louis, MO Goat anti-rabbit Alexa 488 Ab Life Technologies, Carlsbad, CA Goat anti-rabbit HRP Ab; A0545 Sigma-Aldrich, St. Louis, MO Goat serum Sigma-Aldrich, St. Louis, MO HKII rabbit mAb; 2867 Cell Signaling Technology, Danvers, MA Hydrogen peroxide, 30% Fisher Scientific, Nepean, ON INTERFERin Polyplus-transfections, France iScript Reverse Transcription Supermix Bio-Rad Laboratories, Hercules, CA Kplus DNA Ladder RTU FroggaBio, Toronto, ON L-Lactate Assay Kit I Eton Bioscience, San Diego, CA Luminata Forte Millipore, Billerica, MA M3:D culture media INCELL, San Antonia, TX Methanol, 100% Fisher Scientific, Nepean, ON
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MitoTracker Green FM Life Technologies, Carlsbad, CA MitoTracker Orange CM-H2TMRos Life Technologies, Carlsbad, CA Opti-MEM Life Technologies, Carlsbad, CA PBS Sigma-Aldrich, St. Louis, MO PDH mouse mAb; ab110334 Abcam plc, Cambridge, UK PDK1 rabbit pAb; 3062 Cell Signaling Technology, Danvers, MA PDK1 rabbit pAb; NB100-2384 Novus Biologicals, Littleton, CO PDK2 rabbit mAb; NBP1-44428 Novus Biologicals, Littleton, CO PDK3 rabbit pAb; NBP1-32581 Novus Biologicals, Littleton, CO PDK4 rabbit pAb; NBP1-07047 Novus Biologicals, Littleton, CO pPDH (S293) rabbit pAb; NB110-93479 Novus Biologicals, Littleton, CO pPDH (S300) rabbit pAb; AP1064 Millipore, Billerica, MA pPDK1 (S241) rabbit mAb; 3438 Cell Signaling Technology, Danvers, MA Phosphatase Inhibitor Cocktail 2 Sigma-Aldrich, St. Louis, MO PMSF Sigma-Aldrich, St. Louis, MO Protein block solution Dako, Denmark PTEN rabbit mAb; 9188 Cell Signaling Technology, Danvers, MA PVDF membrane Roche, Laval, QC ReBlot Plus (Mild) Millipore, Billerica, CA RedSafeTM Nucleic Acid Staining Solution FroggaBio, Toronto, ON RiboZol AMRESCO, Solon, OH Scrambled siRNA Sigma-Aldrich, St. Louis, MO SDS, 20% Fisher Scientific, Nepean, ON Sodium chloride Fisher Scientific, Nepean, ON Sodium orthovanadate Fisher Scientific, Nepean, ON Sodium pyruvate Sigma-Aldrich, St. Louis, MO SsoFast EvaGreen Supermix Bio-Rad Laboratories, Hercules, CA TAE Buffer, 50X Stock Eppendorf, Hamburg, Germany TEMED Sigma-Aldrich, St. Louis, MO Tissue DNA Kit Omega Bio-Tek, Norcross, GA Tris base, powder Fisher Scientific, Nepean, ON Tris-HCl buffer, 0.5 M, pH 6.8 Bio-Rad Laboratories, Hercules, CA Tris-HCl buffer, 1.5 M, pH 8.8 Bio-Rad Laboratories, Hercules, CA Trypan blue solution, 4% Sigma-Aldrich, St. Louis, MO Tween-20 Fisher Scientific, Nepean, ON Xylene Fisher Scientific, Nepean, ON
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APPENDIX II – PREPARATION OF MATERIALS
Acrylamide SDS-PAGE Gels (2 x 1.5 mm) Resolving gels of various % acrylamide were prepared as follows:
Acrylamide 7.5% 10% 12% 15% Distilled water 11 ml 9.7 ml 8.7 ml 7.2 ml 40% acrylamide/bis solution 3.72 ml 5 ml 6 ml 7.5 ml 1.5 M Tris-HCl buffer, pH 8.8 5 ml 5 ml 5 ml 5 ml 10% SDS 200 µl 200 µl 200 µl 200 µl 10% APS* 240 µl 240 µl 240 µl 240 µl TEMED 10 µl 10 µl 10 µl 10 µl
*1 g APS dissolved in 10 ml distilled water Once resolving gel was polymerized, stacking gel was prepared as follows:
Distilled water 3.2 ml 40% acrylamide/bis solution 500 µl 0.5 M Tris-HCl buffer, pH 6.8 1.26 ml 10% SDS 50 µl 10% APS 50 µl TEMED 5 µl
Plastic combs (1.5 mm) were added to the gels following addition of stacking gel. Agarose Gels (1.5%) 1.5 g of agarose/100 ml of 1X TAE buffer was added to a flask. Solution was heated in a microwave until completely dissolved and allowed to slightly cool. Once cooled, 5 µl of SafeRed was added, and te prepared solution was poured into a casting apparatus. Amido Black Stain Amido black 10B 0.5 g Glacial acetic acid 50 ml Methanol 200 ml H2O 250 ml Mix all components and store at RT. Citrate Buffer Stock Solution A (0.1 M Citric acid): 1.92 g in 100 ml H2O Stock Solution B (0.1 M Sodium citrate dihydrate): 14.7 g in 500 ml H2O Mix 9 ml of Stock Solution A with 41 ml of Stock Solution B. Top up with 450 ml of H2O and adjust pH to 6.0 with HCl. Store at 4oC
166
Crystal Violet Stain Crystal violet 5 g Methanol 100 ml H2O 400 ml Mix all components and store at RT. Electrophoresis Buffer 10X Tris base 30.25 g Glycine 144.1 g 20% SDS 50 ml H2O 600 mL Mix all components and adjust pH to 8.3 with HCl. Adjust final volume to 1 L. Store at RT. PBS-T 10X PBS 100 ml H2O 900 ml Tween-20 1 ml Mix all components and store at RT. Protein Loading Buffer 8X 400 mM Tris-HCl (pH 6.8) 16% SDS 0.8% Bromophenol blue 40% Glycerol 0.4 M DTT Store at -20oC. Once thawed, store in the dark at RT. TBS 10X Tris base 24.2 g NaCl 80 g H2O 600 ml Mix all components and adjust pH to 7.6 with HCl. Adjust final volume to 1 L. Store at RT. TBS-T 10X TBS 100 ml H2O 900 ml Tween-20 1 ml Mix all components and store at RT. Towbin Solution Tris base 30.25 g Glycine 141.1 g Dissolve in H2O to a final volume of 1 L. Store at RT.
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Transfer Buffer (Semi-dry) Tris base 5.82 g Glycine 2.93 g 10% SDS 3.75 ml Methanol 200 ml Dissolve in H2O to a final volume of 1 L. Store at RT. Transfer Buffer (Wet) H2O 770 ml Towbin solution 80 ml 20% SDS 200 µl Methanol 150 ml Mix all components and store at 4oC.
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APPENDIX III – AV/PI STAINING OF ETOPOSIDE-TREATED SAMPLES
Ctrl 50 µM Etoposide
Annexin V
Propidium Iodide
HC
T116
C
aCo2
SW
480
DLD
-1
169
APPENDIX IV – DENSITOMETRY OF AKT PHOSPHORYLATION AT DIFFERENT RESIDUES FOLLOWING 4 H 3BP EXPOSURE
Western blot analysis was used to examine AKT phosphorylation following 4 h 3BP treatment. Graphical outputs for densitometric analysis from three biological replicates of pAKT normalized to native AKT at residues Thr308 and Ser473 are shown above. Representative blots are shown in Figure 10B and C. (* p ≤ 0.05 vs. control (0 µM 3BP))
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APPENDIX V – FLOW CYTOMETRY ANALYSIS OF AV AND PI STAINING FOLLOWING 48 H 3BP EXPOSURE
Quantification of AV and PI staining following 48 h 3BP treatment from three biological replicates ± SEM. As dead cells stained mostly in the top right quadrant (AV+/PI+), the trends observed when assessing AV and PI staining separately are similar to its combined analysis (e.g. see Figure 8B). These results therefore do not clarify the mechanism of 3BP-induced cell death in CRC cells. (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001)
HCT116 CaCo2 SW480 DLD-10
20
40
60
80
100
Cell Line
% A
V +
0 µM5 µM30 µM50 µM
****
*
**
****
[3BP]
HCT116 CaCo2 SW480 DLD-10
20
40
60
80
100
Cell Line
% P
I +
0 µM5 µM30 µM50 µM
****
*
*
**
**
[3BP]