Aberrant Glucose Metabolism in Glioblastoma

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Aberrant Glucose Metabolism in Glioblastoma:

A New Target for Treatment

Han Shen

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy (PhD)

Cure Brain Cancer Foundation Neuro-Oncology Group

Prince of Wales Clinical School

Faculty of Medicine

University of New South Wales

March 2014

ORIGINALITY STATEMENT

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’

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Date ……………………………………………......

THE UNIVERSITY OF NEW SOUTH WALES

Thesis/Dissertation Sheet

Surname or Family name: Shen

First name: Han Other name/s:

Abbreviation for degree as given in the University calendar: PhD

School: Prince of Wales Clinical School Faculty: Faculty of Medicine

Title: Aberrant glucose metabolism in glioblastoma: a new target for treatment

Abstract

Glioblastoma (GBM) is the most common and malignant primary brain tumour in adults. Despite the significant improvements in the multimodality treatment, tumour recurrence is inevitable and the overall survival is dismal with a median survival time of just 15 months. New therapeutic strategies are urgently needed. GBM cells are dependent on both glycolysis and oxidative phosphorylation for glucose metabolism and energy production. A combination therapy targeting both forms of metabolism may be effective.

In this study, perturbation of mitochondrial function was evaluated for the first time using a novel arsenic-based mitochondrial toxin, PENAO (4-(N-(S-penicillaminylacetyl) amino) phenylarsonous acid), alone and in combination with a glycolytic inhibitor, dichloroacetate (DCA), with both in vitro and in vivo GBM models. As a single agent, PENAO demonstrated potent anti-tumour activity in GBM cells by inhibiting oxygen consumption, inducing oxidative stress and depolarising mitochondrial trans-membrane potential, which in turn activated mitochondria-mediated apoptosis in GBM cells. To suppress the compensated increase in glycolysis upon PENAO treatment, DCA was combined and synergised with PENAO to inhibit cell proliferation, cell cycle arrest and apoptosis in GBM cells. In vivo, PENAO and DCA both displayed promising anti-tumour activities individually in a heterotopic GBM xenograft by delaying tumour growth. In an orthotopic GBM model, modest survival prolongation was observed in DCA treated group, whereas no synergistic effect was observed in the combinatorial arm. Autopsy analysis revealed the concentration of PENAO in the brain was below the effective level to synergise with DCA, which would explain the absence of tumour control.

In conclusion, my thesis contributes significantly to the body of research into new therapeutic target strategies for GBM. I demonstrated the dependence of GBM cells on its abnormal glucose metabolism and the PENAO-DCA combination synergistically targeted GBM cells in vitro by perturbing both glycolytic and mitochondrial metabolism. Although the inhibitory action of this combination requires further verification in vivo using different paradigms, the findings stated in this thesis provide the first in vitro proof of concept that dual-targeting of glucose metabolism might be a novel therapeutic strategy for GBM.

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I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only).

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THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS

‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright

Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in

Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.'

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AUTHENTICITY STATEMENT

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III Abstract

Glioblastoma (GBM) is the most common and malignant primary brain tumour in adults.

Despite the significant improvements in the multimodality treatment, tumour recurrence is inevitable and the overall survival is dismal with a median survival time of just 15 months. New therapeutic strategies are urgently needed. GBM cells are dependent on both glycolysis and oxidative phosphorylation for glucose metabolism and energy production. A combination therapy targeting both forms of metabolism may be effective.

PENAO demonstrated potent anti-tumour activity in GBM cells by inhibiting oxygen consumption, inducing oxidative stress and depolarising mitochondrial trans-membrane potential, which in turn activated mitochondria-mediated apoptosis in GBM cells. To suppress the compensated increase in glycolysis upon PENAO treatment, DCA was combined and synergised with PENAO to inhibit cell proliferation, cell cycle arrest and apoptosis in GBM cells. In vivo, PENAO and DCA both displayed promising anti- tumour activities individually in a heterotopic GBM xenograft by delaying tumour growth. In an orthotopic GBM model, modest survival prolongation was observed in

DCA treated group, whereas no synergistic effect was observed in the combinatorial arm. Autopsy analysis revealed the concentration of PENAO in the brain was below the

IV effective level to synergise with DCA, which would explain the absence of tumour control.

Although the inhibitory action of this combination requires further verification in vivo using different paradigms, the findings stated in this thesis provide the first in vitro proof of concept that dual-targeting of glucose metabolism might be a novel therapeutic strategy for GBM.

Publications

1. Shen H and McDonald KL. ‘The Complexities of Resistance to Bevacizumab’

Journal of Cancer Therapy, Vol. 3 No. 5, 2012, pp. 491-503. doi:

10.4236/jct.2012.35064.

2. Wheeler H, Black J, Webb S, Shen H. ‘Dehiscence of corticosteroid-induced

abdominal striae in a 14-year-old boy treated with bevacizumab for recurrent

glioblastoma’ Journal of Child Neurology. 2012 Jul;27(7):927-9. doi:

10.1177/0883073811428007.

3. Abuhusain HJ, Matin A, Qiao Q, Shen H, Kain N, Day BW, Stringer BW,

Daniels B, Laaksonen MA, Teo C, McDonald KL, Don AS. ‘A metabolic shift

favoring sphingosine 1-phosphate at the expense of ceramide controls

glioblastoma angiogenesis’ The Journal of Biological Chemistry. 2013 Dec

27;288(52):37355-64. doi: 10.1074/jbc.M113.494740.

4. Shen H, Decollogne S, Dilda PJ, Hau E, Chung SA, Luk PP, Hogg PJ,

McDonald KL. ‘Dual-targeting of aberrant glucose metabolism in glioblastoma’

Journal of Neuro-Oncology (submitted).

5. Chung SA*, Shen H*, Luk PP*, Decollogne S, Tsoli M, Ziegler DA, Boyd AW, Day

BW, Stringer BW, Dilda PJ, McDonald KL¶, Hogg PJ¶. ‘Glioblastoma is sensitive

to perturbation of mitochondrial function’ Neuro-Oncology (submitted)

*co-first authors; ¶co-senior authors

Conference Presentations

National:

Shen H, Luk PP, Chung SA, Decollogne S, Dilda PJ, Hogg PJ, McDonald KL. PENAO, a novel arsenic-based anti-glycolytic inhibitor, has shown promising effect on glioblastoma in vitro and in vivo, Lorne, Victoria, Australia, Feb 9-11, 2012.

Poster presented at ‘The 24th Lorne Cancer Conference’

International:

Shen H, Luk PP, Chung SA, Decollogne S, Dilda PJ, Hogg PJ, McDonald KL. PENAO, a novel mitochondria-targeted agent, has shown potent antitumor effect on glioblastoma in vitro and in vivo. Washington DC, USA, April 6-10, 2013.

Poster presented at ‘AACR Annual Meeting 2013’

Shen H, Decollogne S, Dilda PJ, Chung SA, Luk PP, Hogg PJ, McDonald KL. PENAO, a novel mitochondria-targeted agent, synergizes with dichloroacetate to target aberrant glucose metabolism in glioblastoma. San Francisco, CA, USA, Nov 21-24, 2013.

Poster presented at ‘4th Quadrennial Meeting of the World Federation of Neuro-

Oncology (WFNO) held in conjunction with the 2013 Scientific Meeting and

Education Day of the Society for Neuro-Oncology (SNO)’

VII

Acknowledgements

I would like to take this opportunity to thank a number of people who have been of great support for me during my PhD. First and foremost, I would like to express my sincerely gratitude to my supervisor, Dr Kerrie L. McDonald, for all her patience, guidance, encouragement and valuable advice throughout my entire PhD process. She has selflessly dedicated her precious time and resources to ensure I had every opportunity to develop professionally and personally. She has also generously supported me in travelling all over the world for conferences, encouraged me apply for every course, award or scholarship that would further my career and offered me a unique insight into the world of science.

I would also like to extend my appreciation to all past and present members of the

Neuro-Oncology Group and Adult Cancer Program for their greatest support and friendship, particularly to Prof Philip Hogg, Dr Pierre Dilda and Dr Stephanie

Decollogne for their guidance and assistance with my animal experiments. I am grateful to Ms Sylvia Chung for her advice when I first came into the laboratory. I am also grateful to Dr Peter Luk for teaching me how to perform flow cytometry and XF24 analyser.

I also thank Rabeya Akter from the ICP-Elemental Analysis Laboratory UNSW

Australia for performing the arsenic analyses, and Fei Shang and Hayley Franklin from the Histology and Microscopy Unit UNSW Australia for preparation and staining of tumour sections.

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I would like to acknowledge the receipt of scholarships from the Prince of Wales

Clinical School, Cure Brain Cancer Foundation, TCRN, and UNSW over the years that enabled me to carry out the studies described in this thesis, attend international conferences, and complete my PhD studies within the minimum time duration.

Lastly, I wholeheartedly thank my beloved parents (Rongping and Cong), who have sacrificed enormously for me to come this far. Their contributions to my study here have been absolutely invaluable. I would also like to give a very special thanks to my loving wife Yizhou, who has always been there for me throughout this endeavour. Your endless support, encouragement, devotion, and constant love were totally indispensable for me to finish this journey. I love you!

Abbreviations

2-DG 2-deoxy-D-glucose 3-BP 3-bromopyruvate 5-ALA 5-aminolevulinic acid

A ACEC Animal Care and Ethics Committee ADP Adenosine diphosphate Akt Protein kinase B ANOVA Analysis of variance ANT Adenine nucleotide translocase ARC Animal Resources Centre ATCC American Tissue Culture Collection ATN-224 Tetrathiomolybdate ATP Adenosine triphosphate ATRA All-trans retinoic acid

B Bcl-2 B-cell lymphoma protein 2 BCNU Carmustine BMDCs Bone marrow-derived cells BMP Bone morphogenetic proteins BRC Biological Resources Centre BSA Bovine serum albumin BSO Buthionine sulphoximine

C CAO 4-(N-(S-cysteinylacetyl)amino) phenylarsonous acid CCCP Carbonylcyanide m-chlorophenylhydrazone CCND2 Cyclin D2 CCNU Lomustine CDK4/6 Cyclin-dependent kinase 4/6

CDKN2A/2B/2 Cyclin dependent kinase inhibitor 2A/2B/2C CI Combination index CIM Cell invasion/migration CNS Centre nervous system c-PARP Poly (ADP-ribose) polymerase cleavage CR Complete response CSCs Cancer stem cells

D DCA Dichloroacetate DDC Diethyldithiocarbamate DHE Dihydroethidium DMAPT Dimethylamino-parthenolide DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DTT Dithiothreitol

E EBRT External beam radiotherapy ECAR Extracellular acidification rate EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor EGFR Epidermal growth factor receptor EGFRvIII Epidermal growth factor receptor variant III EORTC European Organization for Research and Treatment of Cancer ERBB2 Erythroblastic leukaemia viral oncogene homolog 2 ETC Electron transport chain

F FBS Fetal Bovine Serum FDA Food and drug administration FGF Fibroblast growth factor

FGFR Fibroblast growth factor receptor

G G6P Glucose-6-phosphate GBM Glioblastoma GCAO 4-(N-(S-cysteinylglycylacetyl) amino) phenylarsonous acid GFAP Glial fibrilliary acid protein GPX Glutathione peroxidase GSAO 4-(N-(S-glutathionylacetyl)amino) phenylarsenoxide GSCs Glioma stem cells GSH Glutathione GSH-EE Glutathione ethyl ester GSSH Glutathione disulfide GST Glutathione s-transferase

H H&E Haematoxylin and eosin HA14-1 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4h-chromene-3- carboxylate HBSS Hank’s balanced salt solution HFSRT Hypofractionated stereotactic radiotherapy HGF/SF Hepatocyte growth factor/scatter factor HGG High-grade glioma HIF Hypoxia-inducible factor HK Hexokinase HK2 Hexokinase 2 HRP Horseradish peroxidase HSP90 Heat-shock protein, 90 kDa

IC50 Half maximal inhibitory concentrations ICP Inductively Coupled Plasma IDH Isocitrate dehydrogenase

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IHC Immunohistochemistry IMRT Intensity-modulated radiotherapy

J JC-1 5, 5’, 6, 6’-tetrachloro-1, 1’, 3, 3’-tetraethyl-imidacarbocyanine iodide

K Ki-67 Proliferation index

L LDH-A Lactate dehydrogenase type A LOH Loss of heterozygosity

M MCL1 Myeloid cell leukaemia sequence 1 MDM2/4 Murine double-minute 2/4 MGMT O-6-methylguanine- DNA methyltransferase MMPs Matrix metalloproteinases MPTP Mitochondrial permeability transition pore MRI Magnetic resonance imaging MRP Multidrug-resistance associated protein MTD Maximum tolerated dose mTOR Mammalian target of rapamycin mTORC1/2 Mammalian target of rapamycin complex 1/2 MTS 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2h-tetrazolium

N NAC N-acetyl cysteine NCIC The National Cancer Institute of Canada NF1 Neurofibromin 1 NOD/SCID Non-obese diabetic/severe combined immunodeficiency

XIII

NHA Normal human astrocytes

O OATP Organic ion transporter OCR Oxygen consumption rate

P PBR Peripheral benzodiazepine receptor PBS Dulbecco’s phosphate buffered saline PD Progressive disease PDGF Platelet- derived growth factors PDGFR Platelet-derived growth factor receptor PDGFRA Platelet-derived growth factor receptor a PDH Pyruvate dehydrogenase PDK Pyruvate dehydrogenase kinase PEITCs Phenyl ethyl isothiocyanates PENAO 4-(N-(S-penicillaminylacetyl) amino) phenylarsonous acid PEP Phosphoenolpyruvate PES Phenazine ethosulfate PET Positron emission tomography PFS Progression free survival PFS-6 6-month progression free survival PI Propidium iodide PI3K Phosphoinositol-3-kinase PK Pyruvate kinase PKC-β Protein kinase C-β PKM2 Pyruvate kinase isoform M2 PMSF Phenylmethanesulfonyl fluoride PPP Pentose phosphate pathway PR Partial response PS Phospholipid phosphatidylserine PTEN Phosphatase and tensin homolog PU24FCl 8-(2-chloro-3,4,5-trimethoxybenzyl)-2-ﬂuoro-9-(pent-4-ynyl)-9h- XIV

purin-6-amine PU-H58 (8-(6-bromobenzo[d][1,3]dioxol-5-ylthio)-9-(pent-4-ynyl)-9h-purin- 6-amine PU-H71 8-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-9-(3- (isopropylamino)propyl)-9h-purin-6-amine

R Rb Retinoblastoma RNA Ribonucleic acid RNS Reactive nitrogen species ROS Reactive oxygen species Real-Time Cell Analyser RTK Receptor tyrosine kinase RTV Relative tumour volume

S SD Stable disease SD Standard deviation SDS Sodium dodecyl sulphate SDS-PAGE SDS-polyacrylamide gel electrophoresis shRNA Short hairpin RNA siRNA Small interfering RNA Smac/DIABLO Second mitochondria-derived activator of caspases/ direct IAP- binding protein with low pI SOD Superoxide dismutase ST1926 (e)-3-(4′-hydroxy-3′-adamantylbiphenyl-4-yl)acrylic acid STA-4783 Elesclomol

T TBST Tris-buffered saline-Tween TCA Tricarboxylic acid TCGA The Cancer Genome Atlas TGA Therapeutic goods administration

TMZ Temozolomide TNF-α Tumour necrosis factor-α

V VDAC Voltage-dependent anion channel VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor

W WHO World Health Organisation wt EGFR Wild type epidermal growth factor receptor

α-TEA Α-tocopheryloxyacetic acid α-TOS Α-tocopheryl succinate γ-GT γ-glutamyl transpeptidase

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List of Figures

Figure 1.1 Summary of molecular alterations on the progression of gliomas...... 4

Figure 1.2 Frequent genetic alterations in three critical signalling pathways...... 6

Figure 1.3 Delivery of glucose and ATP to hexokinase 2 (HK2) bound to the mitochondrial outer membrane within a malignant cell...... 33

Figure 1.4 Mechanism of action of GSAO...... 47

Figure 1.5 Mechanism of action of PENAO...... 49

Figure 2.1 A schematic design of MTS assays...... 56

Figure 2.2 Analysis of cell invasion in real-time with the cell invasion/migration (CIM)-

Plate 16...... 58

Figure 2.3 A schematic design of combination cytotoxicity assays...... 66

Figure 2.4 The procedure of tumour cell implantation...... 71

Figure 2.5 The site of injection point on the skull surface of the mouse head...... 71

Figure 2.6 Stereotactic coordinates of mouse brain for intracranial injection...... 72

Figure 3.1 Effects of glucose withdrawal on dysfunctional mitochondria in glioma cells.

...... 79

Figure 3.2 Proposed mitochondrial permeability transition pore (MPTP) complex architecture...... 81

Figure 3.3 Response of various cells to PENAO treatment...... 84

Figure 3.4 PENAO induced cell cycle arrest of GBM cells at G2/M phase...... 87

Figure 3.5 Inhibition of U251 cell invasion by PENAO...... 88

Figure 3.6 Apoptosis was induced in GBM cells dose-dependently after 24 hr PENAO treatment...... 89

Figure 3.7 Cytosolic ROS production was induced in GBM cells after 16 hr PENAO treatment...... 90

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Figure 3.8 Mitochondrial superoxide production was induced in U87 cells after 16 hr

PENAO treatment...... 91

Figure 3.9 The anti-proliferative effect of PENAO was attenuated by addition of small peptide thiols glutathione ethyl ester (GSH-EE) and N-acetyl cysteine (NAC)...... 93

Figure 3.10 Loss of mitochondrial trans-membrane potential was induced in GBM cells by PENAO treatment...... 95

Figure 3.11 PENAO-induced mitochondrial superoxide production preceded loss of mitochondrial trans-membrane potential...... 97

Figure 3.12 Blockade of mitochondrial function inhibited oxygen consumption and induced an increase in acid production in GBM cells...... 99

Figure 4.1 Response of GBM cells to DCA treatment...... 109

Figure 4.2 PENAO and DCA additively/synergistically induced proliferation arrest in

GBM cell lines...... 111

Figure 4.3 Different sensitivities to PENAO-DCA combination of GBM cells and non- cancerous cells...... 112

Figure 4.4 Cell cycle distribution of U87 cells was shifted by PENAO-DCA combination treatment...... 114

Figure 4.5 DCA enhanced PENAO-induced apoptosis in U87 cells...... 115

Figure 4.6 DCA enhanced the apoptotic activity of PENAO by elevating the production of mitochondrial superoxide and boosting the depolarisation of mitochondria...... 117

Figure 4.7 Combination treatment of PENAO and DCA suppressed both oxygen consumption and acid production in GBM cells...... 119

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List of Tables

Table 1.1 Subgroups of GBM...... 9

Table 1.2 PI3K/AKT/mTOR pathway inhibitors currently in clinical trials for GBM. .. 19

Table 1.3 Anti-angiogenic agents in trials for GBM...... 24

Table 1.4 Examples of metabolism targeting compounds...... 42

Table 2.1 Optimised seeding density for 72 hr proliferation assay...... 57

Table 2.2 List of tested drug concentrations...... 67

Table 3.1 Summary of IC50 values of PENAO on all tested GBM and non-cancerous cell lines...... 85

Table 4.1 Summary of half maximal inhibitory concentrations (IC50) of DCA under different conditions...... 110

XIX

Table of Contents

ORIGINALITY STATEMENT ...... I

Dissertation Sheet ...... II

Abstract ...... IV

Publications ...... VI

Conference Presentations ...... VII

Acknowledgements ...... VIII

Abbreviations ...... X

List of Figures ...... XVII

List of Tables ...... XIX

Table of Contents ...... XX

1 CHAPTER 1: Literature Review ...... 1

1.1 Introduction ...... 1

1.1.1 Background ...... 1

1.1.2 Classification and grading of gliomas ...... 1

1.1.3 Genetic characteristics of GBM ...... 5

1.2 Treatment update on GBM ...... 10

1.2.1 Standard of care...... 10

1.2.1.1 Surgical treatment ...... 10

1.2.1.2 Radiotherapy ...... 11

1.2.1.3 Chemotherapy ...... 11

1.2.2 Novel therapeutic approaches ...... 14

1.2.2.1 EGFR-targeted therapy ...... 15

1.2.2.2 Inhibition of PI3K/Akt/mTOR signalling pathway ...... 16

1.2.2.2.1 PI3K inhibitors ...... 17

1.2.2.2.2 Akt inhibitors ...... 18

1.2.2.2.3 mTOR inhibitors ...... 18

1.2.2.3 Targeting glioma stem cells ...... 20

1.2.2.4 Anti-angiogenic therapy ...... 22

1.3 The Warburg effect and metabolic remodelling of GBM ...... 27

1.4 Inhibition of aerobic glycolysis, a potential therapeutic strategy to target aberrant glucose metabolism in GBM ...... 29

1.4.1 Tumour microenvironment and HIF-1 ...... 29

1.4.2 PI3K/Akt pathway ...... 31

1.4.3 Hexokinase 2 (HK2) ...... 32

1.4.4 Lactate dehydrogenase type A (LDH-A) ...... 34

1.4.5 Pyruvate kinase isoform M2 (PKM2) ...... 35

1.4.6 Pyruvate dehydrogenase kinase (PDK) ...... 36

1.4.6.1 Inhibition of PDK by dichloroacetate ...... 37

1.4.6.2 DCA in cancer: pre-clinical work and clinical trials ...... 37

1.5 Targeting mitochondria as a therapeutic strategy in cancer treatment ...... 39

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1.6 PENAO: a novel synthetic mitochondrial toxin with both anti-angiogenic and

anti-tumour effects ...... 45

1.7 Aims of this thesis ...... 50

2 CHAPTER 2: Materials and Methods ...... 52

2.1 Introduction ...... 52

2.2 In vitro methods ...... 52

2.2.1 Cell culture ...... 52

2.2.1.1 Maintenance of cell lines ...... 52

2.2.1.2 Cryopreservation of Cell Lines ...... 53

2.2.2 Preparation of PENAO and dichloroacetate (DCA) ...... 54

2.2.3 Trypan blue cell counting...... 54

2.2.4 Cell proliferation assay ...... 55

2.2.5 Cell cycle analysis ...... 57

2.2.6 Invasion assay ...... 57

2.2.7 Oxidative stress detection assays ...... 59

2.2.7.1 Cytosolic production of reactive oxygen species (ROS) ...... 59

2.2.7.2 Mitochondrial superoxide production ...... 60

2.2.8 Mitochondrial trans-membrane potential detection assay...... 61

2.2.9 Apoptotic assays ...... 62

2.2.9.1 Annexin V and PI staining ...... 62

2.2.9.2 Poly ADP-ribose polymerase (PARP) cleavage detection ...... 63

2.2.10 Preparation of cell lysates and western blotting ...... 63 XXII

2.2.10.1 Preparation of cell lysates ...... 63

2.2.10.2 Protein concentration quantification ...... 64

2.2.10.3 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western

blotting ...... 64

2.2.11 PENAO-DCA combination treatment ...... 65

2.2.12 Oxygen consumption and acid production assays ...... 68

2.3 In vivo methods ...... 69

2.3.1 Mice...... 69

2.3.2 Heterotopic tumour implantation (subcutaneous xenograft) ...... 69

2.3.3 Orthotopic tumour implantation (intracranial xenograft) ...... 70

2.3.3.1 Preoperative preparation ...... 70

2.3.3.2 Anaesthesia induction ...... 70

2.3.3.3 Intracranial injection ...... 70

2.3.3.4 Post-operative recovery ...... 73

2.3.4 Drug administration ...... 73

2.3.4.1 Administration of PENAO...... 73

2.3.4.2 Administration of DCA ...... 74

2.3.5 Tumour volume measurements ...... 74

2.3.6 Assessment of neurological dysfunction ...... 74

2.3.7 Measurement of arsenic levels in the plasma and brain ...... 74

2.3.8 Immunohistochemistry (IHC) ...... 75

2.4 Statistical analysis ...... 76 XXIII

3 CHAPTER 3: PENAO, a Mitochondrial Toxin Targeting Glucose Metabolism of

GBM Cells In Vitro ...... 77

3.1 Introduction ...... 77

3.1.1 The role of mitochondria in gliomas ...... 77

3.1.2 Targeting mitochondria with arsenic-based compounds...... 79

3.2 Hypothesis and aims of this chapter ...... 82

3.3 Results ...... 83

3.3.1 PENAO triggered proliferation arrest of GBM cells ...... 83

3.3.2 PENAO induced cell cycle arrest of GBM cells ...... 86

3.3.3 PENAO inhibited invasion of GBM cells ...... 87

3.3.4 PENAO induced apoptosis in GBM cells ...... 88

3.3.5 Mechanism of action of PENAO on GBM cells ...... 89

3.3.5.1 Inhibition of mitochondrial function in GBM cells by PENAO

triggered production of ROS ...... 89

3.3.5.2 The ROS production triggered by PENAO in GBM cells was

quenched by addition of small peptide thiols ...... 92

3.3.5.3 PENAO disrupted the mitochondrial trans-membrane potential in

GBM cells ...... 94

3.3.5.4 PENAO-induced mitochondrial superoxide production preceded loss

of mitochondrial trans-membrane potential ...... 96

3.3.6 PENAO inhibited the mitochondrial glucose oxidation in GBM cells ..... 98

3.4 Discussion ...... 100

XXIV

4 CHAPTER 4: Dual-Targeting of Glucose Metabolism in GBM cells In Vitro..... 104

4.1 Introduction ...... 104

4.1.1 Dual-targeting of glycolysis and mitochondrial oxidative phosphorylation

in cancer cells as a promising therapeutic strategy ...... 105

4.2 Hypothesis and aims of this chapter ...... 106

4.3 Results ...... 108

4.3.1 The effect of DCA as a single agent on GBM cells in vitro ...... 108

4.3.2 Synergistic anti-proliferative effect of PENAO and DCA on GBM cells ....

...... 110

4.3.3 PENAO and DCA synergised to induce cell cycle arrest of GBM cells at

the G2/M phase ...... 113

4.3.4 Combination of PENAO and DCA led to increased apoptosis ...... 114

4.3.5 PENAO in combination with DCA increased mitochondrial superoxide

production and loss of mitochondrial trans-membrane potential ...... 116

4.3.6 PENAO in combination with DCA inhibited oxygen consumption and

acid production in GBM cells simultaneously ...... 118

4.4 Discussion ...... 120

5 CHAPTER 5: Targeting Glucose Metabolism in GBM In Vivo ...... 125

5.1 Introduction ...... 125

5.1.1 GBM rodent models ...... 125

5.1.1.1 U251 GBM xenograft model ...... 126

5.1.1.2 U87 GBM xenograft model ...... 127

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5.1.1.3 Patient-derived orthotopic GBM xenograft models...... 128

5.2 Hypothesis and aims of this chapter ...... 129

5.3 Results ...... 130

5.3.1 Discrepant characteristics between GBM cell line (U87) and patient-

derived primary GBM cells (RN1) in orthotopic mouse models...... 130

5.3.2 PENAO and DCA inhibited heterotopic U87 tumour growth as single

agents ...... 133

5.3.3 PENAO-DCA combination was well-tolerated by BALB/c nude mice at

the maximum effective doses of each individual drugs ...... 136

5.3.4 Effect of the combination therapy on orthotopic U87 model ...... 138

5.4 Discussion ...... 141

6 CHAPTER 6: Summary and Future Directions ...... 146

XXVI

1 CHAPTER 1: Literature Review

1.1 Introduction

1.1.1 Background

Tumours of the central nervous system (CNS) are difficult to treat and the overall survival time for patients with high grade tumours is short. Glioblastoma (GBM) is by far the most common and deadliest primary brain tumour in adults. More than 50,000 primary brain tumours are diagnosed in the United States each year, 31% of which are gliomas [1]. Of these, more than half are GBM [1]. Based on its histological characteristics, the World Health Organisation (WHO) classifies GBM as a grade IV astrocytoma [2].

1.1.2 Classification and grading of gliomas

Primary brain tumours are classified according to the cell type of origin and are graded using the WHO classification [2] (Figure 1.1). Gliomas are the most common neuroepithelial tumours in adults and are classified histologically, immunohistochemically, and/or ultrastructurally as astrocytomas, oligodendrogliomas, or tumours with morphological features of both astrocytes and oligodendrocytes, termed oligoastrocytomas. Gliomas are graded on a WHO scale of I to IV according to their degree of malignancy as judged by various histological features accompanied by genetic alterations [2]. Grade I gliomas (pilocytic astrocytomas) are generally considered to be biologically benign and are curable by surgical resection. Grade II gliomas (diffuse 1 astrocytomas) are low-grade malignancies that may follow long-term clinical courses, but their property of diffuse infiltration into the surrounding parenchyma renders them incurable by surgery. Compared to grade II gliomas, Grade III gliomas (anaplastic astrocytomas) display higher cellularity, increased mitoses and nuclear atypia. Their proliferations are faster than grade II gliomas and thus are more rapidly fatal. Grade IV gliomas (GBMs) are the most malignant gliomas that exhibit more advanced features of malignancy, including highly mitotic activity, endothelial proliferation and necrosis.

Due to their resistance to chemoradiotherapy, patients with GBM generally die within

15 months after initial diagnosis. For grading purpose, pathological diagnosis is based on the most malignant part of the tumour.

On the basis of clinical presentation, GBMs have been further subdivided into the primary or secondary GBM subtypes. Primary GBMs account for the great majority of

GBM cases in elderly patients (45-70 years old), while secondary GBMs are quite rare and tend to occur in younger patients [3]. Primary GBMs develop rapidly de novo with no clinical or histological evidence of a less malignant precursor lesion. In contrast, secondary GBMs progress consistently from the progressive transformation of lower grade astrocytomas (low-grade astrocytoma or anaplastic astrocytoma). They have a lesser degree of necrosis, are preferentially located in the frontal lobe, and carry a significantly better prognosis [4].

Histologically, primary and secondary GBMs are largely indistinguishable, but their genetic and epigenetic profiles are disparate. Generally, primary GBMs are genetically characterised by loss of heterozygosity (LOH) on chromosome 10q, amplification of 2 epidermal growth factor receptor (EGFR), loss of cyclin-dependent kinase inhibitor 2A

(CDKN2A) and phosphatase and tensin homolog (PTEN) mutations, whereas mutations in TP53, loss of functional Rb1 and genetic alterations in platelet-derived growth factor receptor (PDGFR) and isocitrate dehydrogenase 1 (IDH1) are identities of secondary

GBMs [4-6] (Figure 1.1).

Figure 1.1 Summary of molecular alterations on the progression of gliomas.

WHO grade I gliomas (pilocytic astrocytomas) are characterised by duplication/fusion or point mutation of the BRAF gene on 7q34. WHO grade II/III gliomas (astrocytic, oligodendroglial and oligoastrocytic gliomas) and WHO grade IV secondary GBMs frequently carry mutations in IDH1 or IDH2, indicating that they may share a common cell of origin. WHO grade II diffuse astrocytomas carry mutations of TP53, whereas loss of 1p/19q is associated with oligodendrogliomas. Most oligoastrocytomas are characterised by either of these alterations. Molecular alterations associated with progression to WHO grade III anaplastic gliomas primarily include 9p loss and inactivation of the CDKN2A, p14ARF and CDKN2B genes on 9p21, whereas progression to secondary GBM is normally linked with frequent loss of 10q and loss of DCC expression. WHO grade IV primary GBMs display complex chromosomal, genetic, and epigenetic alterations, including the RTK/MAPK/PI3K pathway (e.g. EGFR, MET, PDGFRA, ERBB2, NF1, PTEN, PIK3R1, PI3KCA, CTMP), the p53 pathway (e.g. TP53, p14ARF, MDM2, MDM4), and the pRb1 pathway (e.g. CDKN2A, CDKN2B, CDK4, CDK6, RB1). Additionally, primary GBMs frequently show monosomy 10, trisomy 7 and gains of 19q and 20q. BRAF: v-raf murine sarcoma viral oncogene homolog B1, CDK4: 4 cyclin-dependent kinase 4, CDKN2A/2B: cyclin-dependent kinase inhibitor 2A/2B, CTMP: carboxyl-terminal modulator protein, DCC: deleted in colorectal carcinoma, EGFR: epidermal growth factor receptor, ERBB2: erythroblastic leukaemia viral oncogene homolog 2, HDM2: human double-minute 2, IDH1: isocytrate dehydrogenase 1, p14ARF: p14 alternate reading frame, PDGFR: platelet-derived growth factor receptor, PI3KCA: phosphatidylinositol 3-kinase catalytic alpha polypeptide, PI3KR1: phosphatidylinositol 3-kinase regulatory alpha subunit, PTEN: phosphatase and tensin homologue, RB1: retinoblastoma susceptibility protein, WHO: World Health Organisation. This figure was redrawn from ‘Molecular diagnostics of gliomas: state of the art’ [7].

1.1.3 Genetic characteristics of GBM

The classification of primary GBM into clinical subtypes has been made possible through improved understanding of glioma molecular pathways. In particular, The

Cancer Genome Atlas (TCGA) Research Network has accelerated our understanding of gene mutations and expression profiles of GBM [6]. Multiple alterations in the expression levels of genes and/or proteins have been identified in GBMs, including activation of oncogenes and/or silencing of tumour-suppressor genes. Based on copy numbers, expression analyses and DNA sequencing, TCGA has confirmed that three signalling pathways are commonly disrupted in GBMs, including (1) alterations in pathways related to receptor tyrosine kinase (RTK)/Ras/PI3K, (2) TP53 and (3)

Retinoblastoma (Rb) tumour suppressor pathways [6] (Figure 1.2). Alterations in these three pathways are essential in the development of GBM, but it remains possible that other canonical pathways will be uncovered through different types of molecular analyses.

Figure 1.2 Frequent genetic alterations in three critical signalling pathways.

EGFR, epidermal growth factor receptor; ERBB2, erythroblastic leukaemia viral oncogene homolog 2; PDGFRA, platelet-derived growth factor receptor A; NF1, neurofibromin 1; PI3K, phosphoinositol-3-kinase; PTEN, phosphate and tensin homolog; CDKN2A/2B/2C, cyclin-dependent kinase inhibitor 2A/2B/2C; MDM2/4, murine double-minute 2/4; CDK4/6, cyclin-dependent kinase 4/6; CCND2, cyclin D2; RB1, retinoblastoma 1. This figure was extracted from The Cancer Genome Atlas Research Network.

To identify clinically relevant subtypes of GBM, a robust gene expression-based molecular classification of GBM into 4 subgroups (Classical, Mesenchymal, Proneural, and Neural) has been established using integrated genomic analysis [8] (Table 1.1). The

Classical subtype generally displays consistent amplification of chromosome 7 paired with loss of chromosome 10 and frequent focal loss on chromosome 9p21.3. These chromosomal events lead to amplification of the EGFR gene and loss of the PTEN and

CDKN2A gene loci. Alterations in the TP53, NF1, PDGFRA or IDH1 genes are nearly all absent. Classical GBM demonstrates sensitive response to the classical chemoradiotherapies, likely because the p53 DNA damage response is intact in this subtype [9]. The second subgroup, Mesenchymal, is characterised by an expression profile of genes associated with the mesenchyme and angiogenesis. The Mesenchymal group overexpresses the CHI3L1/YKL40 and MET genes, as well as astrocyte markers

CD44 and MERTK along with genes in the TNF super family and NF-κB pathways. In addition, Mesenchymal subtype has frequent inactivation of the NF1 (37%), TP53

(32%), and PTEN (32%) genes [8]. These tumours exhibit response to aggressive chemoradiotherapies and may be sensitive to Ras, PI3K, and anti-angiogenic therapies

[9]. Proneural tumours frequently show an overexpression of PDGFRA and IDH1 gene mutation [8]. The presence of IDH1 gene mutations suggests that this subtype may be largely subscribed by secondary GBM [10]. Frequent mutations in TP53 (54%) and

PIK3CA/PIK3R1 (19%) genes are also observed, whereas amplification of chromosome

7 and loss of chromosome 10 were significant (>50%) but not as frequently occurring as found in the Classical subtype [8]. Patients belonging to the Proneural group are younger in age and it has been suggested that they may be most responsive to inhibitors of the hypoxia-inducible factor (HIF), PI3K, and PDGFRA pathways. The overall survival of the Proneural subtype is slightly better than that of the other 3 subtypes.

However, these tumours are the most resistant to classical chemotherapies such as temozolomide (TMZ) [9]. There is a fourth Neural subtype described by Verhaak’s group but not confirmed by other studies [11]. The Neural subtype is less defined and has gene expression signatures that appear to be more similar to those found in normal brain tissue, with expression of neuronal markers such as NEFL, GABRA1, SYT1, and

SLC12A5. These tumours exhibit a low degree of infiltration into normal surrounding

7 parenchyma and their expression signature is suggestive of cells with a differentiated phenotype [8, 9].

Table 1.1 Subgroups of GBM.

GBM subtype Molecular signatures

ERFG amplification; Ras activation; Loss of PTEN and Classical CDKN2A; Absence of TP53 mutation

NF1 mutation; Loss of TP53 and PTEN; Activation of TNF

Mesenchymal and NF-κB; CHI3L1/YKL40, MET, CD44 and MERTK

amplification

IDH-1 mutation; PDGFRA amplification; Loss of TP53, Proneural PTEN, and CDKN2A

Neural Gene expression profile resembles normal brain

EGFR, epidermal growth factor receptor; PDGFRA, platelet-derived growth factor receptor A; NF1, neurofibromin 1; PTEN, phosphate and tensin homolog; CDKN2A, cyclin-dependent kinase inhibitor 2A; MDM2/4, murine double-minute 2/4; CDK4/6, cyclin-dependent kinase 4/6; CCND2, cyclin D2; TNF, tumour necrosis factor.

Although classification into subtypes may prove useful in identifying different classes of tumour, it is of importance to realise that all subtypes ultimately demonstrate disturbance of three main pathways as discussed above. The RTK/Ras/PI3K signal cascade controls proliferation and invasion of glioma cells while the TP53 and Rb tumour suppressor pathways control progression through the cell cycle [6].

1.2 Treatment update on GBM

1.2.1 Standard of care

1.2.1.1 Surgical treatment

Surgical treatment is a critical component of the standard of care, allowing maximum resection of tumour to reduce mass effect and tumour burden. Surgery also reduces intracranial pressure, which, depending on the location of the tumour, may result in recovery of the CNS function or decrease in usage of corticosteroid. The extent of resection of glioma directly and significantly affects patients’ median survival time independent of age, degree of disability, WHO grade, or subsequent treatment modalities [12]. Maximal survival benefit is reported when resection volume is greater than 98% [13] and a complete resection of all gadolinium enhancing tumour significantly improves the survival effect when treated with adjuvant chemoradiotherapy [14]. However, achieving complete resection is a challenge when using conventional white light microsurgical techniques. Resections are limited by the difficulty in accurately eliminating glioma infiltration from normal brain intraoperatively, and the concerns about resecting and damaging normal brain. Although many attempts have been made to increase the rate of complete resection, conclusive evidence supporting the efficacy of these approaches is limited. The use of intraoperative magnetic resonance imaging (MRI) has gained popularity with some neurosurgical centres with reports of increased complete resection rates [15, 16] and improved survival [16, 17]. In addition, the use of 5-aminolevulinic acid (5-ALA), a fluorescent label that accumulates in high grade gliomas after being ingested, which allows the surgeon to more easily distinguish and accurately resect the tumour, also

10 assists the surgeon in achieving a radiographic gross total resection of the contrast- enhancing portion of GBM [18].

1.2.1.2 Radiotherapy

Due to the infiltrative nature of the disease, most GBMs recur following surgery. This invasive nature necessitates the use of adjuvant radiotherapy that is either combined with or subsequently followed by chemotherapy. Postoperative radiotherapy was considered as the mainstay of treatment for GBM patients in early 1980s, since it extended the median survival from 3-4 months to a range of 7-12 months [19-22]. The standard of care for radiotherapy for GBM is focal, fractionated external beam radiotherapy (EBRT), but new techniques and technologies continue to evolve. There are no prospective, randomised studies that compare ERBT with intensity-modulated radiotherapy (IMRT), which is becoming widely used and appears to be comparable to more traditional 3-dimensional EBRT [23]. A growing number of dose-intensification techniques, such as brachytherapy, hyperfractionation, and the combination of EBRT with stereotactic radiation boosts, have been investigated, but none of them has been clearly shown to be superior to standard EBRT.

1.2.1.3 Chemotherapy

The efficacy of chemotherapy for GBM continues to be generally disappointing. For several decades, nitrosoureas, particularly carmustine (BCNU) and lomustine (CCNU), were the most common chemotherapeutics used alone or in combination with radiotherapy [24, 25]. Nitrosureas constitute alkylating agents that induce strong DNA damage. Due to their lipophilic property, nitrosureas show substantial penetration into 11 the brain tissue, which is in contrast to most conventional chemotherapeutic drugs used in cancer treatment. Nitrosoureas has been proved to increase the median survival, at least in patients with intermediate grade glioma [26].

Because of the limited success of the treatment with nitrosureas, a second generation alkylating drug, TMZ, entered clinical trials for the treatment of malignant gliomas [27,

28]. A major advance in the therapy with TMZ was the opportunity to administer an oral medication with optimal bioavailability of almost 100% [27, 29]. Its ability to cross the blood-brain-barrier to achieve effective concentrations in the CNS was also attractive to its use in malignant brain tumours. The cytotoxicity of TMZ is thought to be mainly exerted by DNA damage via alkylation of the O6 position of guanine and the production of DNA interstrand crosslinks.

Concurrent TMZ and radiotherapy was tested in an international Phase III trial run by the European Organization for Research and Treatment of Cancer (EORTC) and the

National Cancer Institute of Canada (NCIC) and demonstrated significant survival benefit to the patient when compared to radiotherapy alone [22]. Shortly after publication in 2005, the chemoradiotherapy treatment was quickly established as the standard of care for GBM. In the EORTC/NCIC study, a total of 573 patients with newly diagnosed GBM were enrolled. This study reported the combination therapy of

TMZ and radiotherapy increased median survival time when compared with radiotherapy alone (14.6 months versus 12.1 months, p<0.001). At the 5-year analysis of this study, the 5-year overall survival rate was 9.8% for the combination therapy group versus 1.9% for the radiotherapy alone group (p<0.001), with a median follow-up 12 of 61 months [14]. With this strong evidence, TMZ in combination with radiotherapy is widely prescribed and currently considered the standard of care for patients with newly diagnosed GBM. Unfortunately, there is still no clearly established standard of care for recurrent GBM.

Early evidence was suggestive that the epigenetic silencing of a DNA repair gene, O-6- methylguanine- DNA methyltransferase (MGMT) by promoter methylation was associated with good prognosis for patients with GBM treated with TMZ [30, 31]. A subgroup analysis was conducted as part of the EORTC/NCIC Phase III trial. Patients positive for MGMT promoter methylation showed a significantly prolonged median survival time when compared with patients with an unmethylated MGMT promoter

(21.7 months versus 15.3 months, P <0.001) [32]. MGMT promoter methylation status is widely used to predict the efficacy for combination therapy of TMZ and radiotherapy for newly diagnosed GBM, although in many pathology departments around the world, this test is not routinely performed.

Although the combination therapy of TMZ and radiotherapy has become the standard of care, most GBMs will eventually relapse. In contrast to the current standard of care for newly diagnosed GBM, there is still no clearly established standard treatment for recurrent GBM. Thus, better understanding of resistant mechanism and the development of new treatment strategies are urgently needed in order to overcome the resistance of

GBM to current therapy as well as improve the effectiveness of treatment for patients with an unmethylated MGMT promoter.

1.2.2 Novel therapeutic approaches

Treatment of GBM can be divided into two major categories: ‘up-front’ treatment of newly diagnosed GBM and ‘salvage’ treatment at disease recurrence. Novel agents or treatment delivery techniques are normally first tested in the recurrent disease setting, where there are few approved treatment alternatives. Promising agents may then be combined with EBRT and TMZ in the initial treatment setting because this is the established standard of care.

The increasing understanding of signalling pathways involved in the initiation of malignant gliomas has motivated the development of novel targeted therapies. Although the first generation targeted agents, such as anti-EGFR compounds, have generally not been as effective in GBM as would have been expected based on pre-clinical findings

[33, 34], recent improvements in target identification, drug development, clinical trial design, and patient stratification to specific therapies have generated some optimism for the next generation of targeted therapies. Moreover, anti-angiogenic therapy with bevacizumab, a humanised monoclonal antibody against the VEGF-A ligand, has now been used in the management of patients with recurrent GBM. Novel treatment strategies, such as targeting glioma stem cells and the use of immunotherapies, in combination with advanced imaging and tissue biomarkers promise to further improve the management of patient and outcome in GBM.

1.2.2.1 EGFR-targeted therapy

The most common genetic aberration associated with malignant glioma is the dysregulation of the EGFR, also referred to as ERBB1 or HER1, with a frequency of about 50% [35]. Dysregulation of EGFR has been shown to enhance tumour growth, migration, invasion, angiogenesis and metastasis [36]. In addition, it is a poor prognostic factor that correlates with shortened overall survival in GBM patients [37].

Several possible mechanisms are attributed to the dysregulation of this receptor. These include gene amplification and intrinsic alterations of the receptor structure as a result of mutation, of which the most common mutant form associated with GBM is EGFRvIII

[38].

Blocking ligand binding to its cognate receptor could normalise growth rates, induce apoptosis, and increase tumour susceptibility to chemotherapeutic agents. Monoclonal antibodies, both unconjugated and conjugated, directly target wild type EGFR (wt

EGFR) and EGFRvIII have been developed for therapeutic use in GBM. Erlotinib and gefitinib were the first generation of EGFR-targeted agents to be investigated in newly diagnosed and recurrent malignant gliomas, either as monotherapy, or in combination with other cytotoxic agents. These agents failed to demonstrate any significant treatment benefit in phase I and II trials [34, 39, 40]. Limited activity was also observed with cetuximab, the most developed unconjugated monoclonal antibody that functions to prevent EGFR-mediated signal transduction by interfering with ligand binding and

EGFR extracellular dimerisation [41]. New efforts have been made to test new second- generation irreversible EGFR inhibitors. These inhibitors have shown good efficacy in non-small cell lung cancer and erlotinib-resistant tumours [42].

1.2.2.2 Inhibition of PI3K/Akt/mTOR signalling pathway

The phosphatidylinositol 3-kinase (PI3K) pathway is frequently dysregulated in many human cancers, including GBM [43]. Amplification of EGFR leads to activation of downstream kinases, including PI3K, Akt, and mammalian target of rapamycin (mTOR)

[44]. Inactivation of PTEN and activating mutations in PI3K itself collectively occur in a majority of GBM tumours, effectively uncoupling PI3K from upstream control by

EGFR [45].

The PI3K/AKT/mTOR signalling pathway plays a central role in cell proliferation, survival, motility, metabolism, and angiogenesis. Therefore, it is not surprising that dysregulation of this pathway through genetic alterations in several proteins, including p85, p110, PTEN, and Akt, has been demonstrated to play a pivotal role in the pathogenesis of cancer [46, 47]. Tyrosine kinase expression and subsequent signalling, particularly EGFR signalling, have long been implicated in the pathogenesis of GBM.

Amplification of EGFR in GBM leads to activation of the PI3K pathway and has been noted in approximately 45% of GBM cases [33]. Similarly, loss of function mutations, chromosomal deletions, or epigenetic gene silencing of PTEN have been found in approximately 40% of GBM cases and have been shown to lead to poor survival [48].

Taken together, alterations of at least one of the EGFR, PTEN, or PIK3CA genes have been detected in 63~86% of primary and 31% of secondary GBM. These data therefore highlight the significance of this pathway in the pathophysiology of this disease [48].

Due to the aberrant hyperactivation of the PI3K pathway, inhibition of its components presents an attractive target for cancer therapeutics. There has been a tremendous effort to develop PI3K pathway inhibitors for the treatment of cancer. In patients with GBM,

16 inhibitors of PI3K pathway as single agents or in combination with other agents and/or irradiation have yielded mostly infrequent and short-lived responses. However, the results of these studies have led to a more profound understanding of the PI3K pathway in GBM and the development of potentially more efficacious and better tolerated agents.

1.2.2.2.1 PI3K inhibitors

BKM120 is a pan-class I PI3K inhibitor without mTOR and Vps34 activity. BKM120 demonstrated a dose-dependent growth inhibition in immortalised GBM cell lines, including U87, U251, LN229, LN18, and D54. Furthermore, administration of

BKM120 via oral gavage was well-tolerated by mice harbouring intracranial U87 xenografts and prolonged their median survival time [49]. BKM120 can readily penetrate the blood-brain barrier, making it an attractive option for the treatment of

GBM. There is an ongoing phase II trial of BKM120 among GBM patients in first or second recurrence with evidence of activation of the PI3K pathway (NCT01339052). In addition, a phase I trial of BKM120 with radiotherapy and temozolomide in patients with newly diagnosed GBM (NCT01473901) and a phase I/II trial of BKM120 with bevacizumab in patients with recurrent GBM (NCT01349660) are underway.

Another pan-PI3K inhibitor, PX-886, is a semi-synthetic derivative of wortmannin and irreversibly inhibits PI3K through the formation of a covalent bond with PI3K. In glioma cells, PX-866 dramatically inhibited proliferation in a variety of cell lines, with greater sensitivity seen in PTEN-negative cell lines, where IC50 values were 3-fold lower (low micro-molar range) than in PTEN-positive cell lines. PX-866 also resulted in increased autophagy and decreased the invasive and angiogenic potential of cells. In 17 human U87 mouse xenograft models, PX-866 inhibited subcutaneous tumour growth and increased the median survival time of animals with intracranial tumours [50].

1.2.2.2.2 Akt inhibitors

The principal role of Akt is to facilitate cellular survival and suppress apoptotic cell death. Because activation of Akt has been demonstrated to be a key event in tumourigenesis, the inhibition of AKT is a promising therapeutic target for GBM.

Several AKT inhibitors are being tested in clinical trials, of which KRX-0401 is the most advanced. KRX-0401 is an allosteric inhibitor of AKT and has been shown to block phosphorylation of AKT but does not have an effect on PI3K activation [51].

Based on the promising pre-clinical data, a phase II trial of KRX-0401 in recurrent

GBM was conducted but unfortunately ended with minimal single-agent activity

(NCT00590954). These data suggesting that the combined inhibition of AKT and mTOR may be more effective than inhibition of these targets alone led to an ongoing phase I/II trial of KRX-0401 with temsirolimus (mTOR inhibitor) in recurrent high- grade gliomas (NCT01051557).

1.2.2.2.3 mTOR inhibitors mTOR regulates signalling through the canonical PI3K pathway using 2 distinct functional complexes, mTORC1 and mTORC2. mTORC1 (rapamycin-sensitive) positively regulates cell proliferation through phosphorylation of the translational regulators S6K1 and 4EBP1, whereas mTORC2 (rapamycin-insensitive) regulates AKT signalling [52]. Clinical trials with the first generation mTOR inhibitor rapamycin and its analogues have failed to achieve a significant therapeutic outcome. In addition, 18 increased proliferation of tumour cells was observed in some malignancies following treatment with rapamycin, an inhibitor of mTORC1 but not mTORC2. The mechanism was postulated to be related to the negative-feedback regulation of mTORC2 on PI3K signalling. For this reason, simultaneous inhibition of both mTORC1 and mTORC2 was proposed to be of a therapeutic advantage [53-57]. Current dual mTORC1/2 inhibitors including AZD8055 and AZD2014 and dual mTOR/PI3K inhibitor (XL765) are being evaluated in clinical trials with recurrent GBM patients (Table 1.2).

Table 1.2 PI3K/AKT/mTOR pathway inhibitors currently in clinical trials for GBM.

ClinicalTrials.gov Agent Name Target Clinical Trial Identifier:

pan-PI3K BKM120 Phase II NCT01339052 inhibitor

pan-PI3K XL147 Phase II NCT01240460 inhibitor

pan-PI3K PX866 Phase II NCT01259869 inhibitor

XL765 PI3K/mTOR Phase Ib/II NCT01240460

KRX-0401 Akt Phase III NCT00590954

KRX- Akt+mTOR Phase I/II NCT01051557 0401+temsirolimus

AZD8055 mTORC1/2 Phase I/II NCT01316809

1.2.2.3 Targeting glioma stem cells

Cancer stem cells (CSCs) are generally characterised as the population of cells within a tumour niche that have the ability to self-renew and abnormally differentiate, initiate tumourigenesis, and regenerate a phenocopy of the original patient's tumour when inoculated into immuno-compromised mice in vivo [58]. In line with the general definition of CSCs, glioma stem cells (GSCs) also demonstrate a capacity for self- renewal, multipotency, and induction of tumourigenesis [59]. Growing evidence implicates GSCs play an important role in the mechanism of resistance to chemoradiotherapy, and indicates that GSCs are critical in initiating GBM and mediating tumour recurrence [60, 61]. Thus, targeting GSCs has been proposed as an attractive treatment strategy.

Therapies that induce the differentiation of GSCs into non-GSCs with lower tumourigenic potential might be a promising and particularly non-cytotoxic strategy for targeting GSCs. Several potential targets have been proposed from the previous studies.

Bone morphogenetic proteins (BMPs) are a group of cancer signalling molecules that bind to BMP surface receptors in order to mediate the differentiation of GSCs [62].

BMPs have been tested as pro-differentiating factors for GBM treatment. Treatment of

GSCs with BMPs, by direct implantation of BMP-bearing beads, induced their differentiation and reduced their tumorigenic potential [62]. Nonetheless, a follow-up study demonstrated that GSCs might modulate BMP receptor expression epigenetically to a more resistant phenotype that could escape from BMP-induced differentiation [63].

Another differentiation-inducing agent under investigation is all-trans retinoic acid

(ATRA). ATRA is a vitamin A derivative that binds to the nuclear retinoic acid receptor and has made acute promyelocytic leukaemia a curable disease [64]. Treatment of GSCs with ATRA induced pro-differentiation effects with subsequent functional

20 consequences, such as decreased GSC tumourigenicity, motility and impaired cytokine secretion [64]. Similar effects were reported by using 15-deoxy-Δ12,14-prostaglandin

J2, although with different mechanism of action [65]. Recent studies also have identified several microRNAs as post-translational regulators of GSCs differentiation.

For instance, overexpression of miR-124 and miR-137 induced the differentiation of

CD133+ GSCs, but both of them also force normal neural stem cells to differentiate, making specific delivery to GSC a critical concern [66].

Targeting GSC pathways is another potential therapeutic strategy to block the function of GSCs. Several targets have been investigated, including growth and intracellular signalling factors and immune pathways. Of these targets being tested, only epidermal growth factor (EGF) was able to promote neural sphere formation and enhance the self- renewal capabilities of GSCs that express the EGFR [67]. Although inhibition of EGFR signalling demonstrated to induced proliferation arrest and apoptosis of GSCs [67, 68], and there are also many available EGFR inhibitors including the clinically approved drugs such as erlotinib and gefitinib, we should always bear in mind that neither of them demonstrate any significant benefit in Phase I and Phase II trials as we discussed above.

Other approaches to target GSCs include targeting the microenvironment (peri-vascular niches) which is highly hypoxic, overcoming GSCs resistance to standard chemoradiotherapy based on DNA damage and so on [69]. All in all, regardless of the speciﬁc target, a major obstacle of any GSCs direct targeting strategy is the requirement to spare normal stem cells. Similar to the selectivity for anti-tumour agents to induce cell death in tumour cells but not normal cells, agents targeting GSCs have to 21 demonstrate that they selectively target GSCs while sparing normal stem cells in order for their clinical potential to be realised. From this perspective, it is possible that some of the pathways identiﬁed in GSCs are too essential to the development and/or maintenance of normal stem cells. It would imply that targeting these pathways would affect, and potentially have serious adverse effects, on normal stem cells. Therefore, the need of improving selectivity is a critical factor to address prior to consideration for translation to the clinical setting.

1.2.2.4 Anti-angiogenic therapy

GBM is highly vascularised brain tumour and its growth has been shown to be angiogenesis-dependent, thus sparking an interest in developing anti-angiogenic therapeutic strategies. Increased vascular permeability leading to cerebral oedema and microvascular proliferation are hallmarks of GBM. This is due to high expression of pro-angiogenic cytokines, particularly the vascular endothelial growth factor (VEGF) and its signalling via endothelial tyrosine kinase receptor VEGF receptor (VEGFR)-2

[70]. The VEGF family is comprised of five different proteins encoded by different genes including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor

(PGF). VEGFs mediate angiogenic signals to the vascular endothelium via high-affinity receptor tyrosine kinases, designated VEGFR-1 (Flt1), VEGFR-2 (Flk1/KDR) and

VEGFR-3 (Flt4). These receptors are expressed almost exclusively on endothelial cells.

The direct actions of VEGF include stimulation of endothelial mitogenesis, promotion of endothelial survival via Akt-dependent pathway and regulation of vascular permeability. VEGFs also stimulate expression of tissue plasminogen activator, collagenases and matrix metalloproteinases (MMPs) (Hicklin et al. 2005).

Several mechanisms have been proposed for the efficacy of anti-angiogenic agents. The early ‘starvation hypothesis’ of anti-angiogenic therapy was considered to destroy the tumour vasculature, by means of depriving the oxygen and nutrients supply for the tumour. However, increasing evidence shows that mechanisms of anti-angiogenic therapy are more intricate and may also rely on tumour types [71]. As to GBM, it has been noticed that anti-angiogenic therapy not only blocks its vascularisation but also has anti-tumour effects [72]. More recently, a hypothesis raised by Jain et al. postulated that certain anti-angiogenic agents were able to transiently ‘normalise’ the aberrant tumour vasculature of solid tumours accompanied by increased blood flow, such that the normalised vasculature facilitated efficient delivery of oxygen and chemotherapeutic agents [73]. However, the vessel normalisation appears to be transient and it occurs only in the first month of anti-angiogenic therapy, after which the degradation and destruction of tumour vessel leads to tumour hypoxia.

There are quite a few anti-angiogenic agents under intensive investigation in the treatment of GBM due to its highly vascularised nature (Table 1.3). However, results from these trials are generally disappointing as none of them to date has substantially changed overall survival. The benefits from anti-angiogenic therapy are at best transitory and then followed by a restoration of tumour growth and progression, indicating the rapid establishment of resistance occurs. To date, the molecular and cellular basis of the progression following anti-angiogenic therapy remains unclear.

Several putative mechanisms of resistance to anti-angiogenic therapy have been explored, such as angiogenic redundancy [74, 75], anti-angiogenesis-induced intratumoural hypoxia [73, 76], and hypoxia-induced recruitment of various bone marrow-derived cells (BMDCs). 23

Table 1.3 Anti-angiogenic agents in trials for GBM.

Anti-angiogenic Mode of action Targets References agents

Fibronectin-based Adnectin (CT- VEGFR-2 [77] inhibitor 322)

Lenalidomide [78] Immunomodulatory Thalidomide [79, 80]

Integrin inhibitor Cilengitide αVβ3/αVβ5 [81, 82]

Bevacizumab VEGF [83] Monoclonal Rilotumumab antibody HGF/SF [84, 85] (AMG 102)

Recombinant Aflibercept VEGF, PDGF [86] fusion protein

Thrombospondin-1 ABT-510 [87] mimetic peptide

Serine/threonine Enzastaurin PKC-β [88-90] kinase inhibitor

VEGFR-2, Brivanib [91] FGFR-1 and -2

Cediranib VEGFRs, PDGF-β and c-KIT [92-94]

BCR–ABL, c-KIT, EPHA2 and Tyrosine kinase Dasatinib [95, 96] and PDGFR-β inhibitor Imatinib PDGFR [97-99]

Pazopanib VEGFRs, PDGFR-α/β, and c- [100] (GW786034) KIT

Anti-angiogenic Mode of action Targets References agents

VEGFR-2, Flt3, PDGFR, Sorafenib [101-103] FGFR-1, RAF and c-KIT

c-KIT, VEGFR-1–3, PDGFR- Sunitinib [104] α, PDGFR-β, and Fit3 kinase

Tandutinib Flt3 kinase, PDGFR-β and c- Tyrosine kinase [105, 106] (MLN518) KIT inhibitor Vandetanib VEGFR, [107-109] (ZD6474) RET and EGFR

Vatalanib VEGFR-2, all VEGFRs, [96, 110] (PTK787) PDGFR-b and c-KIT

XL-184 MET, VEGF-2 and RET [111]

VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; HGF/SF, hepatocyte growth factor/scatter factor; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factors receptor; PKC-β, Protein kinase C-β; FGFR, fibroblast growth factor receptor.

A recent pre-clinical study working with GBM xenograft models derived from patient tumour spheroids suggested that the ‘side effects’ of anti-angiogenic agents including increased hypoxia, increased invasive potential and anaerobic tumour metabolism reflected by elevated metabolites associated with glycolysis appeared to play a critical role in the progression of recurrent GBM following anti-angiogenic therapy failure

[112]. Specifically, an increase in lactate and alanine metabolites together with an induction of hypoxia-inducible factor 1-α (HIF-1α) and an up-regulation of the

PI3K/Akt pathway were observed as a result of anti-VEGF treatment. These results indicate that anti-angiogenic therapy led to a more hypoxic microenvironment that switched glucose metabolism from oxidative phosphorylation to glycolysis. This glycolytic phenotype further resulted in the enhanced tumour invasion into normal brain

[112]. Furthermore, a study using cutting-edge multivoxel 1H MR spectroscopy further pointed out that anti-VEGF-induced hypoxia and its corresponding metabolic change were only detected within the compact tumour area rather than the area with diffuse infiltrative growth [113].

Regardless of the mechanism of resistance being exploited by tumours, progressed tumours following the failure of anti-angiogenic therapy normally do not respond to any further chemotherapeutic approaches. In this regard, novel therapeutic strategies are urgently needed not only for patients with newly diagnosed GBM but also for those with recurrent GBM following prior treatments, particularly for the anti-angiogenic treatment.

1.3 The Warburg effect and metabolic remodelling of GBM

A striking contrast between normal differentiated cells and tumour cells is the different modes of metabolism. Tumour cells undergoing metabolic remodelling acquire the capabilities of utilisation and/or synthesis of important metabolites including glucose, glycogen, fatty acids, amino acids and glutamine. In the early 1920s, Otto Warburg was the first to describe a metabolic adaptation that occurs within solid tumours [114].

Warburg noticed that normal tissues generally used glycolysis to generate about 10% of the total cellular ATP and the mitochondria to generate the remaining 90% via oxidative phosphorylation. However, in many tumour tissues, more than 50% of cellular ATP can be generated from glycolysis, even in the presence of adequate oxygen, which is termed aerobic glycolysis. Based on this phenomenon, an advanced imaging tool, Positron

Emission Tomography (PET) with (18F)-deoxyglucose was designed for cancer diagnosis.

Compared to oxidative phosphorylation, glycolysis is less efficient in terms of ATP production, as it generates only two ATP molecules per molecule of glucose, whereas complete oxidation of one glucose molecule by oxidative phosphorylation can generate up to 36 ATP molecules [115]. Despite its low efficiency in ATP yield per molecule of glucose, aerobic glycolysis can generate ATP faster than oxidative phosphorylation, which is more preferable for rapidly dividing tumour cells [116]. In addition, another major function of aerobic glycolysis is to support macromolecular synthesis. Generating new daughter cells requires the replication of all cellular contents, including DNA,

RNA, proteins and lipids. Glucose can provide the precursors for the chemical constituents (e.g., nucleotides, amino acids, and lipids) that are used to build

27 macromolecules essential for cell division. Therefore, a major function of up-regulated glycolysis in proliferating cells may be to maintain the levels of glycolytic intermediates required to support biosynthesis [117, 118]. By using aerobic glycolysis, tumour cells divert more pyruvate to lactate in cytosol, which may have several benefits for tumour cells. Excreting high levels of lactate may support their survival, growth, invasion and metastasis by conditioning the tumour microenvironment [119].

GBM is characterised by pathological heterogeneity with regions of pseudopalisading necrotic cells under hypoxia (pO2=2.5-5%) and infiltrating tumour cells into normal brain under normal oxygen conditions (pO2=10%) [120]. Under such a diverse microenvironment, GBM cells up-regulate glycolysis three times more than that of normal brain tissue, measured by an increased lactate to pyruvate ratio [121]. This metabolic phenotype was also confirmed by using stereotactic microdialysis of tumour and normal brain tissue in GBM patients. The lactate to pyruvate ratio was higher in the tumour tissue compared to its adjacent brain [122].

It also has been validated on molecular basis that altered metabolism in GBM is highly likely to be multi-factorial. The role of the tumour microenvironment, stabilisation of

HIF-1α [123], and growth factors/PI3K/Akt pathway [124] are all involved in metabolic remodelling. Moreover, specific metabolic enzymes have been implicated downstream in establishing the Warburg effect, such as hexokinase 2 (HK2) [125, 126], pyruvate kinase isoform M2 (PKM2) [127], and pyruvate dehydrogenase kinase (PDK) [128] and lactate dehydrogenase type A (LDH-A) [129]. These alterations in GBM may offer

28 multiple potential targets for treatment strategies designed to target aberrant glucose metabolism.

1.4 Inhibition of aerobic glycolysis, a potential therapeutic strategy to

target aberrant glucose metabolism in GBM

The metabolic distinction between cancer cells and normal cells may offer a very selective therapeutic target to be exploited, as aerobic glycolysis is not typically seen in normal tissues apart from skeletal muscle during strenuous exercise. Therefore, targeting glycolysis is a potential therapeutic strategy to eliminate malignancy while sparing healthy cells. The complex pathways and altered metabolic enzymes that lead to increased glycolytic metabolism in the majority of human cancers provide multiple potential targets for therapeutic strategies designed to be selectively eradicating cancer cells [130].

1.4.1 Tumour microenvironment and HIF-1

Necrosis and vascular proliferation are the pathological features that distinguish GBM from anaplastic and low grade gliomas. In GBM, hypercellular zones called pseudopalisades typically surrounding necrotic foci are exposed to moderate levels of hypoxia (pO2=2.5-5%) [120]. The critical role of the HIF system in up-regulating glycolysis as an adaptation to hypoxia and non-adaptively in cancer (producing aerobic glycolysis) has resulted in considerable interest in the development of inhibiting strategies. A key player is the transcription factor HIF-1, which is an αβ-heterodimer:

HIF-1β is constitutively expressed and its mRNA and protein are maintained at a constant level regardless of oxygen availability [131], whereas HIF-1α protein has a 29 short half-life (∼5 min) and is highly regulated by oxygen [132]. The transcription and synthesis of HIF-1α are constitutive and appear unaffected by oxygen [131]. However, in normoxic conditions, the HIF-1α proteins are rapidly degraded, resulting in essentially non-detectable HIF-1α protein [133]. During hypoxia, HIF-1α is stabilised and translocates from the cytoplasm to the nucleus, where it dimerises with HIF-1β to form the HIF complex, which is transcriptionally active [131]. Hypoxia and HIF-1 increase almost all the enzymes involved in the glycolytic pathway and the glucose transporters 1 and 3 (GLUT1, 3) [130]. Furthermore, the metabolic products of glycolysis, such as lactate and pyruvate, have been reported to regulate the expression of hypoxia-inducible genes independently of hypoxia by stimulating the accumulation of HIF-1α in human gliomas, hence establishing a potential positive feedback loop

[134].

The efficacy of many novel anti-cancer agents that target signal transduction pathways may be due in part to their indirect inhibition of HIF-1. Although a large number of novel compounds have been shown to inhibit HIF-1, no compound has been demonstrated to directly and specifically inhibit HIF-1 activity so far. BAY87-2243, an inhibitor of HIF-1 activity and a stabiliser of HIF-1α, is now being tested in a Phase I clinical trial (ClinicalTrials.gov identifier: NCT01297530) and could be the first compound of the class. Patients are also currently recruited for a Phase I trial testing

EZN-2968, an antisense oligonucleotide targeting HIF-1α [135]. Given HIF-1 not only regulates the glycolytic pathway, but also regulates the transcription of many genes involved in critical aspects of cancer biology, including immortalisation, maintenance of stem cells, genetic instability, vascularisation, metabolic reprogramming, and

30 invasion/metastasis [136], small molecule of HIF-1 inhibitor would be a very promising therapeutic agent to target malignancies.

1.4.2 PI3K/Akt pathway

HIF-1 is also regulated via the PI3K/Akt signalling pathway. Activation of Akt can induce transcription of HIF-1 target genes, such as VEGF, COX-1, PGK-1 and PFK

[137]. On the contrary, transfection of wild-type PTEN into human GBM cell lines inhibited transcription of these HIF-1 target genes [137]. Treatment of prostate cancer cells with the PI3K inhibitor LY294002 has been shown to inhibit both basal expression of HIF-1α and expression induced by growth factors and hypoxia. Transfection with dominant-negative Akt also prevented transcription of HIF-1 target genes [138]. Similar results have been validated in GBM cell lines [139].

In addition, previous studies also support a role of Akt in the remodelling of glucose metabolism of cancer cells. In particular, activation of Akt has been associated with enhanced glucose uptake and aerobic glycolysis. For example, Akt promotes the translocation of GLUT1 to the cytoplasmic membrane to facilitate glucose uptake and activates HK2 to trap glucose within the cell [140]. Moreover, activated Akt stabilises

HIF-1 during normoxic conditions in order to affect transcription of glycolytic enzymes mediated by HIF-1α, and aerobic glycolysis [141]. Therefore, it is likely that aerobic glycolysis facilitates neoplastic transformation as well as the promotion of angiogenesis.

Therapeutically, it has been suggested that inhibitors of the PI3K/Akt pathway could lead to effective killing of cancer cells via glucose metabolism inhibition or anti- angiogenesis. Numerous pre-clinical studies and clinical trials have shown potent efficacy of PI3K/Akt pathway drugs, such as Buparlisib (BKM 120) and XL765

(SAR245409), used alone or in combination with other therapeutic agents in GBM [50,

142] (inhibitors of PI3K/Akt/mTOR pathway evaluated in clinical trials were reviewed in section 1.2.2.2). However, this strategy has limitations because by targeting the

PI3K/Akt pathway, a proliferation arrest is induced rather than cell death [143].

1.4.3 Hexokinase 2 (HK2)

HK2 is a member of the HK family of enzymes that control the first rate-limiting step of glycolysis: the phosphorylation of glucose to glucose-6-phosphate (G-6-P). Once phosphorylated, negatively charged G-6-P is trapped inside the cell where it fuels both glycolysis and the pentose phosphate pathway (PPP). Compared to other HK isoforms,

HK2 is a product of HIF-1 target gene [144] and exists in a phosphorylated form bound to the mitochondrial outer membrane where it interacts with the voltage-dependent anion channel (VDAC) [145, 146]. This localisation promotes its stability and reduces feedback inhibition from its product G-6-P [145], thereby ensuring glucose can be trapped by tumour cells expressing mitochondria-bound HK2 (Figure 1.3).

Figure 1.3 Delivery of glucose and ATP to hexokinase 2 (HK2) bound to the mitochondrial outer membrane within a malignant cell.

ATP is delivered via mitochondrial adenine nucleotide translocase (ANT) to the voltage-dependent anion channel (VDAC)-bound HK2 in tumour cells. This schematic was kindly provided by Prof. Philip Hogg.

HK2 is expressed at basal levels in the skeletal and adipose tissue, but rarely in normal brain, where HK1 is predominantly expressed. Several studies have confirmed that HK2 is overexpressed in GBM [125, 147], which is also confirmed by TCGA data. HK2 plays a critical role in perturbed GBM glycolysis [126]. As a result of stable inhibition of HK2 in GBM cells, aerobic glycolysis was inhibited and normal oxidative glucose metabolism was promoted. In addition, reduced HK2 expression impacted significantly on reduced tumourigenicity in both subcutaneous and intracranial xenograft models

[126]. On the flip side, overexpression of HK2 in GBM cells promoted lactate formation and proliferation [126].

Although the fundamental role of HK2 expression in GBM for maintaining glycolytic phenotype has been demonstrated, there is still a lack of small molecule inhibitors targeting HK2 specifically. A structural analogue of glucose, 2-deoxy-D-glucose (2-

DG), inhibited glycolysis via competitive inhibition of HK [148-150]. Although 2-DG has shown promising results in a number of cancer models, most clinical trials have been terminated due to its intolerable systematic toxicity (ClinicalTrials.gov identifier:

NCT00247403, NCT00633087). Lonidamine, an orally administered small molecule that inhibits glycolysis by the inactivating the mitochondria-bounded HK, has been described since the early 1980’s [151]. Lonidamine was tested in a Phase II clinical trial for GBM in combination with diazepam, but failed to show therapeutic benefit in terms of time-to-progression and overall survival [152]. Another leading compound 3- bromopyruvate (3-BP), a pyruvate analogue, is both an alkylating agent and an inhibitor of HK2. 3-BP was shown to inhibit tumour growth in a dose-dependent fashion in vivo, however, toxicity and an unknown mechanism of action prevented its further development [153].

1.4.4 Lactate dehydrogenase type A (LDH-A)

Pyruvate is a hub of different metabolic pathways. It is produced by glycolysis and malate oxidation in proliferating cells. Pyruvate is also the major fuel of the TCA cycle, the precursor of alanine in a reversible transamination reaction involving glutamate as the nitrogen donor, and acts as a substrate of a redox reaction generating lactate [154]. 34

The conversion of pyruvate into lactate also allows glycolytic cells to maintain the levels of pyruvate low enough to avoid cell death [155]. This reversible reaction is catalysed by the lactate dehydrogenase (LDH) family encoded by LDH-A and LDH-B genes, which can be combined to generate five isoforms LDH-1 (B4), LDH-2 (B3A1),

LDH-3 (B2A2), LDH-4 (B1A3), and LDH-5 (A4) [156]. LDH-A is the key glycolytic enzyme that catalyses the formation of lactate from pyruvate, with LDH-B favouring the backward conversion of pyruvate from lactate. LDH-A is a HIF-1-target gene and is therefore induced by hypoxia transcriptionally [130].

In proliferating cancer cells, the majority of the pyruvate generated from glucose (>90%) is converted to lactate by LDH-A, which is readily secreted into the extracellular environment rather than being oxidised. By converting pyruvate to lactate, LDH-A recovers the NAD+ needed to maintain glycolysis. This step is critical for the maintenance of tumour proliferation in vivo [157]. LDH-A is frequently up-regulated in many tumour types, and high expression is often linked to poor prognosis. Increased serum LDH levels have been shown to negatively correlate with survival in GBM [158].

As a number of studies have demonstrated that reduction of LHD-A expression either by siRNA or small molecules inhibits tumour progression [159, 160], the development of specific small molecule LDH-A inhibitor is underway [161].

1.4.5 Pyruvate kinase isoform M2 (PKM2)

Pyruvate kinase (PK) is a key glycolytic enzyme which catalyses another rate-limiting step of glycolysis: dephosphoryting phosphoenolpyruvate (PEP) into pyruvate to produce ATP. PK has four isoforms, of which PKM1 and PKM2 are produced by 35 alternative splicing of the PKM2 gene transcripts which is another target gene of HIF-1

[162]. PKM1 is primarily expressed in the brain and muscle while PKM2 is expressed in proliferating tissues including embryonic tissue and tumour cells [163]. The tumour specific PKM2 gene is expressed either as an active tetramer or as a dimer with a low affinity for PEP. PKM2 in its highly active tetrameric conformation (Km PEP=0.03 mM) provides high yield ATP production from glycolysis, whereas in its nearly inactive dimeric conformation (Km PEP=0.46 mM) PKM2 provides a metabolic bottleneck allowing glycolytic intermediates to be redirected toward biosynthesis, notably fuelling through the PPP for DNA synthesis [164]. The balance between tetrameric and dimeric

PKM2 is an oscillating phenomenon subject to allosteric regulation [164]. GBM cells express high levels of PKM2 as well as PKM1 supporting an incomplete switch in splice isoforms. Knockdown of total PKM2 does not lead to a switch in GBM metabolism from aerobic glycolysis to oxidative phosphorylation, unlike knockdown of

HK2 [126], suggestive that PKM2 inhibitors might not be as potent as HK2 inhibitors to target aerobic glycolysis in GBM.

1.4.6 Pyruvate dehydrogenase kinase (PDK)

Pyruvate dehydrogenase (PDH) is a mitochondrial multi-enzyme complex that catalyses the oxidative decarboxylation of pyruvate. The enzymatic activity of PDH is regulated by the phosphorylation/dephosphorylation cycle. The mitochondrial matrix protein, pyruvate dehydrogenase kinase (PDK), is an important inhibitor of oxidative phosphorylation via its phosphorylation of the E1 alpha subunit of PDH. PDK has four isoforms (PDK1-4). PDK1 is another HIF-1 target gene product. HIF-1 transactivates the PDK1 gene resulting in inactivation of PDH, which converts pyruvate to acetyl-CoA,

36 thereby decreasing conversion of pyruvate to acetyl-CoA and compromising oxidative phosphorylation [165].

1.4.6.1 Inhibition of PDK by dichloroacetate

Dichloroacetate (DCA) is a small molecule (150 Da) that can activate PDH by inhibiting PDK at a concentration of 10~250 μM, in a dose-dependent manner [166].

DCA inhibits all the PDK isoforms, of which PDK2 is the most sensitive to DCA [167].

In the past several decades, DCA has been used extensively for the treatment of mitochondrial diseases. As first described in 1969 by Stacpoole [168], DCA could lower lactate levels and alleviate the symptoms associated with lactic acidosis in patients with mitochondrial diseases by inhibiting PDK and coupling glycolysis to mitochondrial oxidative phosphorylation. Since then, DCA has been tested in clinical trials in both mitochondria and non-mitochondrial diseases in order to limit lactic acidosis [168]. In the first study, 30 patients with MELAS syndrome were treated with

25 mg/kg/day of oral DCA [169]. Most of these patients had developed symptomatic peripheral neuropathy, compared to the placebo group, which resulted in early termination of the study. 17 out of the 19 patients who had reported peripheral neuropathy had recovery of the neuropathy following 9 months discontinuation of DCA.

No other toxicities were reported in that study.

1.4.6.2 DCA in cancer: pre-clinical work and clinical trials

In cancer treatment, DCA was found to activate oxidative phosphorylation, and trigger apoptosis in cancer cells [170]. The mechanism by which DCA induces apoptosis of cancer cells is via an enhancement of a flux of electrons through the electron transport 37 chain (ETC) resulting in greater depolarisation of the mitochondrial membrane potential

(which is generally hyperpolarised in tumour cells) and release of cytochrome c followed by subsequent activation of apoptosis. Restoration of the oxidative phosphorylation function generates reactive oxygen species (ROS) which leads to an up-regulation of the voltage-dependent K+ channel resulting in an efflux of K+ and activation of caspase [170]. Although there are some reports describing controversies for DCA in its anti-tumour efficacy in vitro and in vivo, evidence supports the effective role of DCA in killing several types of cancer cells [170-173]. Michelakis et al. [128] have recently tested the efficacy of DCA to reverse cancer-specific metabolic and mitochondrial remodelling in a small cohort of GBM patients, suggesting metabolic modulation through PDK inhibition as a novel therapeutic strategy for the treatment of this deadly brain tumour. DCA treatment rapidly reversed mitochondrial hyperpolarisation in GBM cells, which in turn induced apoptosis and increased mitochondrial reactive oxygen species by inhibiting HIF-1α, promoting p53 activation and suppressing angiogenesis [174]. Another recent study investigated the impact of

DCA on the apoptotic pathway in a rat model, suggesting the dependence of early tumourigenesis on glycolysis, and providing the rationale for the combination of DCA with conventional treatments to eradicate cancer stem cells. These findings suggest that

PDK could be a promising therapeutic target for the treatment of malignant gliomas. A recent phase I trial of DCA with in adults with recurrent malignant brain tumours

(including recurrent WHO grade III - IV gliomas or metastases from a primary cancer outside the central nervous system), which concluded that chronic administration of

DCA is feasible and well-tolerated in these patients [175]. Notably, another open- labelled phase II trial of oral DCA (NCT01029925) in patients with metastatic breast cancer and advanced stage non-small cell lung cancer (NSCLC) reported patients with

38 previously treated advanced NSCLC did not benefit from oral DCA, thus suggesting

DCA should be considered with platinum-based chemotherapy in hypoxic tumours rather than as a single agent in advanced NSCLC [176].

1.5 Targeting mitochondria as a therapeutic strategy in cancer treatment

Although the mitochondrion is considered to be the energy powerhouse of the cell, there is sufficient evidence that mitochondria are also central integration sites for biological signals that promote cell life or cell death. It is well established that the mitochondria play a crucial role in mediating intrinsic pathways of apoptosis [177]. Mitochondria activate apoptosis via regulation of pro-apoptotic protein translocation from the mitochondrial inter-membrane space to the cytosol. As mitochondria are critical regulators of cell death and given mitochondrial functions are often altered in tumour cells as opposed to healthy cells, targeting of mitochondria represent a promising therapeutic strategy to eradicate chemotherapy-refractory cancer cells.

Cancer cell mitochondria are structurally and functionally different from their normal cell counterparts [178, 179]. For instance, the inner membrane lipid composition of various tumour mitochondria has been observed with higher levels of cholesterol, different total phospholipid content, and changes in the quantity of individual phospholipids compared to normal cells [180]. Additionally, comparison of polypeptide profiles between mitochondria within normal and cancer cells shows quite a few differences in the appearance and relative abundance of several proteins as well [181].

Functionally, aberrations of mitochondrial bioenergetic function in cancer cells have been observed long time ago, including disparities between normal and cancer cells 39 with regard to favourite for respiratory substrates, rates of electron and anion transport, and the capacity to retain calcium [182]. The activities of certain enzymes essential to oxidative phosphorylation are known to be lower in cancer as opposed to normal cells.

For example, the maximal velocity of ATPase activity in mitochondria isolated from hepatocellular carcinoma is greatly decreased compared to that in normal liver [183].

Moreover, the oxidase activity of cytochrome c , a critical intermediate in apoptosis, in total cellular homogenate and the mitochondria isolated from human cancer cell lines in vitro is also considerably decreased compared with that measured in the non-cancerous cell line [179].

Despite the great number of metabolic aberrations so far being identified, apparently there is no such one common to all cancer cells. In other words, it is highly likely that the alteration of metabolic states in cancer cells is not the cause of malignancy but a secondary adaptation which is indispensable to support the activities of cancer cells.

However, transformed malignant cells exhibit an extensive metabolic reprogramming that renders them more susceptible to mitochondrial perturbations than non- immortalised cells [184, 185]. Specifically, elevated ROS levels primarily stemming from the mitochondria are a marked feature of cancer cells that are variously attributed to inefficient electron transport in the respiratory chain, up-regulated metabolic demand, and decreased ROS scavenging etc. [186-188]. Increased ROS levels in cancer cells compared to normal cells has been exploited to eliminate cancer cells selectively by chemically boosting ROS levels over a critical homeostatic threshold that is intolerable for either growth or survival of tumour cells but tolerable by normal cells [189]. From this point of view, mitochondria-targeted agents have emerged as a means of selectively targeting tumours. 40

The correction of cancer-associated mitochondrial dysfunctions and the activation of apoptosis by pharmacological agents that induce or facilitate mitochondrial membrane permeabilisation represent attractive strategies for cancer therapy. Mitochondrial membrane permeabilisation is a central event involved in apoptosis. Some anti-cancer drugs are designed to conjugate to cell surface death receptors and activate apoptosis through a TP53-independent signalling mechanism known as the extrinsic apoptotic pathway. However, most chemotherapeutic agents as well as radiotherapy trigger apoptosis of tumour cells through the intrinsic pathway via the activation of the pro- apoptotic members of the B-cell lymphoma protein-2 (Bcl-2) superfamily. Both extrinsic and intrinsic apoptotic signalling pathways involve mitochondrial membrane permeabilisation [190, 191]. During this process, the mitochondrial outer and inner membranes are both permeabilised, resulting in the release of soluble proteins from the inter membrane space. As a consequence, a number of proteases and nucleases are activated to begin the process of breaking down the cell [192].

A number of therapeutic strategies have been developed based on targeting tumour mitochondrial proteins and functions (Table 1.4), including compounds that target the mitochondrial permeability transition pore and induce the production of ROS. As mitochondria are the most prominent source of intracellular ROS and low levels of ROS have been implicated in the stemness of cancer cells [193], selective targeting of cancer stem cells with mitochondria-targeted compounds is likely to attract great interest.

Table 1.4 Examples of metabolism targeting compounds.

Class Compound Target or mode of action References

A-385358 BCL-XL [194]

ABT-263, ABT- BCL-2, BCL-XL, BCL-W [195] 737

Modulators of the BCL-2, BCL-XL, BCL-W, AT-101 [196] BCL-2 protein MCL1

family GX15-070 BCL-2, BCL-XL, BCL-W, [197] (obatoclax) MCL1

HA14-1 BCL-2 [198]

Oblimersen BCL-2 mRNA antisense [199]

2-Deoxy-d- HK [200] glucose

3- HK2–VDAC interaction [201] Bromopyruvate

Dichloroacetate PDK inhibitor [170]

HK2 peptide HK2–VDAC interaction [202] Metabolic LDH-A shRNA LDH-A [157] inhibitors Methyl HK2–VDAC interaction [203] jasmonate

Orlistat Fatty acid synthase [204]

SB-204990 ATP citrate lyase [205]

Acetyl-CoA carboxylase Soraphen A [205] inhibitor

Class Compound Target or mode of action References

Arsenite trioxide ANT ligand, ROS production [206]

VDAC-targeting Clodronate ANT inhibitor [207]

and/or ANT- GSAO ANT cross linker [208] targeting agents Lonidamine ANT ligand [152]

PK11195 PBR ligand [209]

2- SOD inhibition [210, 211] Methoxyestradiol

ATN-224 SOD inhibition [212]

β-lapachone ROS production [213]

Buthionine GSH synthesis inhibitor [214] sulphoximine

Imexon GSH depletion [215] ROS regulators Mangafodipir SOD mimic [216]

Menadione ROS production [217]

Motexafin ROS production [218] gadolinium

GSH depletion, GPX PEITCs [219] inhibition

STA-4783 ROS production [220, 221]

All-trans-retinoic ANT ligand [222] acid

Permeability transition pore Retinoids CD437 [206, 223] complex

Perturbation of Ca2+ ST1926 [224] homeostasis

Class Compound Target or mode of action References

Mitochondrial HSP90 Gamitrinibs [199] ATPase inhibitors

PU24FCI, PU- HSP90 inhibitors HSP90 inhibitors [225] H58, PU-H71

Inhibitor of the HSP90– Shepherdin [226] survivin interaction

α- tocopheryl Ubiquinone-binding sites in [227] succinate respiratory complex II

Natural Permeability transition pore Betulinic acid [228] compounds and complex

derivatives DMAPT ROS production [229]

Parthenolide ROS production [230]

Resveratrol F1-ATPase [231]

ANT, adenine nucleotide translocase; ATN-224, tetrathiomolybdate; BCL-2, B-cell lymphoma protein 2; BCL-W, also known as BCL2-like protein 2 (BCL2L2); BCL-XL, also known as BCL2-like protein 1 (BCL2L1); CD437, 6-[3-(1-adamantyl)-4- hydroxyphenyl]-2-naphthalene carboxylic acid; DMAPT, dimethylamino-parthenolide; GSAO, 4-(N-(S-glutathionylacetyl)amino) phenylarsenoxide; HA14-1, 2-amino-6- bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; GPX, glutathione peroxidase; GSH, reduced glutathione; HK, hexokinase; HSP90, heat-shock protein, 90 kDa; LDH-A, lactate dehydrogenase A; MCL1, myeloid cell leukaemia sequence 1; PBR, peripheral benzodiazepine receptor; PDK, pyruvate dehydrogenase kinase; PEITCs, phenyl ethyl isothiocyanates; PU24FCl, 8-(2-chloro-3,4,5-trimethoxybenzyl)-2- ﬂuoro-9-(pent-4-ynyl)-9H-purin-6-amine; PU-H58 (8-(6-bromobenzo[d][1,3]dioxol-5- ylthio)-9-(pent-4-ynyl)-9H-purin-6-amine; PU-H71, 8-(6-iodobenzo[d][1,3]dioxol-5- ylthio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine; ROS, reactive oxygen species; shRNA, short hairpin RNA; SOD, superoxide dismutase; ST1926, (E)-3-(4′-hydroxy-3′- adamantylbiphenyl-4-yl)acrylic acid; STA-4783, elesclomol; VDAC, voltage-dependent anion channel. 44

Anti-cancer agents that directly target mitochondria also have the potential to bypass the resistance mechanisms that generated from conventional chemotherapeutics. Most conventional anti-tumour drugs target signalling pathways (such as DNA damage and endoplasmic reticulum stress) that lie upstream and converge on mitochondria due to their role as integrators of pro-death and pro-survival signals [232]. In this scenario, drugs that directly target mitochondria may provide a unique tool to circumvent the upstream processes, and may therefore be effective in some other resistant forms of cancer.

1.6 PENAO: a novel synthetic mitochondrial toxin with both anti-

angiogenic and anti-tumour effects

A promising mitochondrial target is the HK2-VDAC-adenine nucleotide translocase

(ANT) complex that spans the mitochondrial outer and inner membranes (see Figure

1.3). This complex links glycolysis, oxidative phosphorylation and mitochondria- mediated apoptosis in cancer cells [233].

The first step in glycolysis is converting glucose and ATP to G-6-P and ADP, which is catalysed by HK (see section 1.4.3). Cancer cells mostly employ an isoform (HK2) that is bound to mitochondria via interaction with the VDAC (Figure 1.3) [234-236]. VDAC is associated with inner membrane ANT, which is the ADP/ATP translocator that exports ATP from the mitochondrial matrix and imports ADP into the matrix across the inner membrane [237]. ANT is thought to exert two functions in cancer cells: it provides ATP to HK2, to phosphorylate and trap glucose in the cell [238], and is a component of the mitochondrial permeability transition pore (MPTP) [237], which is 45 involved in the permeability of the mitochondrial inner membrane. Opening of this pore by inactivating ANT allows the equilibration of solutes <1500 Da in size to move across the inner membrane. This leads to uncoupling of oxidative phosphorylation and increase in superoxide levels, loss of mitochondrial trans-membrane potential and decrease in oxygen consumption. These effects of ANT blockade result in proliferation arrest and mitochondria-mediated apoptosis [208].

The first generation of ANT inhibitor GSAO (4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid) is a tripeptide trivalent arsenical [208]. GSAO is a pro-drug which is activated by γ-glutamyl transpeptidase (γ-GT) at the cell surface to produce

GCAO (4-(N-(S-cysteinylglycylacetyl) amino) phenylarsonous acid) (Figure 1.4) [239].

GCAO gets into the cell through an organic ion transporter (OATP) and is further processed by dipeptidases to CAO (4-(N-(S-cysteinylacetyl)amino) phenylarsonous acid) in the cytosol. CAO enters the mitochondrial matrix where the arsenical moiety cross- links Cys160 and Cys257 on the matrix face of ANT, which inactivates the transporter.

This leads to partial uncoupling of oxidative phosphorylation, elevation in superoxide production and arrest of cell proliferation [208]. CAO reacts with ANT only when cells are proliferating, so GSAO has little effect on growth quiescent endothelial cells [208].

Cytosolic levels of GCAO and CAO are controlled via being exported by the multidrug- resistance associated protein isoforms 1 and 2 (MRP1/2) [240]. GSAO is currently being tested in a Phase I/IIa clinical trial in adults with solid tumours refractory to standard therapy (NCT01147029). To date, 27 patients have been treated with GSAO at

7 dose levels. GSAO has been well tolerated and there is preliminary evidence of anti- tumour activity, particularly in ovarian cancer. 4 of 6 ovarian cancer patients have had

46 stable disease for up to 16 cycles of treatment. The trial is being performed by Cancer

Research UK (CRUK) at hospitals in Manchester and Oxford. GSAO is the first

Australian compound trialled by CRUK. The target population for Phase II studies will possibly be platinum-refractory ovarian cancer patients.

GCAO

GSAO OATP γGT GCAO MRP1/2 dipeptidase

plasma membrane

CAO

ANT

mitochondria

Figure 1.4 Mechanism of action of GSAO.

GSAO is activated by γ-GT at the cell surface to produce GCAO, which gets into the cell through an OATP and is further processed by dipeptidases to CAO. CAO then enters the mitochondrial matrix where the arsenical moiety cross-links Cys160 and Cys257 on the matrix face of ANT, which inactivates this transporter. GSAO, 4-(N-(S- glutathionylacetyl)amino) phenylarsonous acid; γ-GT, γ-glutamyl transpeptidase; GCAO, 4-(N-(S-cysteinylglycylacetyl) amino) phenylarsonous acid; OATP, organic ion transporter; CAO, 4-(N-(S-cysteinylacetyl)amino) phenylarsonous acid; MRP, multidrug-resistance associated protein; ANT, adenine nucleotide translocase. This schematic was extracted from ‘Optimization of the antitumor efficacy of a synthetic mitochondrial toxin by increasing the residence time in the cytosol’ [241].

The second generation ANT inhibitor, PENAO (4-(N-(S-penicillaminylacetyl) amino) phenylarsonous acid), is a derivative of its first generation GSAO. PENAO has been designed to bypass the pro-drug processing and metabolism of GSAO to enhance its 47 efficacy [241]. As a cysteine mimetic of CAO, PENAO does not require processing by

γ-GT at the cell surface, which was predicted to enhance its efficacy in vitro and in vivo.

PENAO accumulates in cells much faster than GSAO and CAO, which translates to more potent effects on both endothelial and tumour cells in culture. The faster rate of accumulation of PENAO compared to CAO is mostly due to decreased rate of export by

MRP1/2 (Figure 1.5). PENAO, therefore, is an at least a 20-fold better inhibitor of cell proliferation and cell killing agent than GSAO (Dilda et al., 2009). PENAO currently is being tested in a Phase I clinical trial in adults with solid tumours refractory to standard therapy in Australia.

PENAO

OATP MRP1/2

plasma membrane

ANT

mitochondria

Figure 1.5 Mechanism of action of PENAO.

PENAO enters the cell through an OATP, and then gets into the mitochondrial matrix where the arsenical moiety cross-links Cys160 and Cys257 on the matrix face of ANT, which inactivates this transporter. PENAO, 4-(N-(S-penicillaminylacetyl) amino) phenylarsonous acid; OATP, organic ion transporter; MRP, multidrug-resistance associated protein; ANT, adenine nucleotide translocase. This schematic was extracted from ‘Optimization of the antitumor efficacy of a synthetic mitochondrial toxin by increasing the residence time in the cytosol’ [241].

Like GSAO and CAO, PENAO triggered swelling of isolated rat liver mitochondria in a time- and concentration-dependent manner. Comparison of the time for maximal swelling as a function of CAO or PENAO concentration indicates that the two compounds are comparable in their effects on mitochondrial integrity. PENAO is also a

20-fold more effective inhibitor of tumour growth in mice than GSAO. Comparable

49 anti-tumour activity was observed with administration of 0.25 mg/kg/day PENAO and 5 mg/kg/day GSAO. No systemic toxicity of either PENAO or GSAO was apparent at these testing doses. Analysis of PENAO treated tumours showed a marked reduction in the endothelial marker CD31 expression as well as significant inhibition of BxPC-3 tumour cell proliferation [241].

In summary, there is clearly biological heterogeneity within GBM this most common and malignant form of primary brain cancer. Its behaviour is extremely aggressive and despite decades of effort, median survival is just beginning to improve. As GBM is highly resistant to conventional anti-tumour therapy and has a high recurrence rate, it is a crucial goal of brain cancer research to develop alternative anti-tumour therapies.

Recent analysis of tumour metabolism in situ reveals that the GBM cells rely on both glycolysis and mitochondrial oxidation for the glucose metabolism [242]. Meanwhile,

GBM cells demonstrate a striking ability to rewire their circuitry to maintain the flux of bioenergetic metabolism, particularly when one of the metabolic pathways is blocked therapeutically [243]. All these observations suggest that the homeostasis of bioenergetic metabolism of GBM cells might be disturbed maximally by targeting glycolysis and mitochondrial metabolism simultaneously.

1.7 Aims of this thesis

The overall aim of this thesis is to improve treatment of GBM by exploring the efficacy of dual-targeting of glucose metabolism in GBM cells. This study will also improve the fundamental understanding of the interaction between glycolytic pathway and mitochondrial glucose oxidative pathway in GBM cells under therapeutic inhibitions. 50

By investigating the mechanism of action of a novel arsenic-based mitochondrial toxin

PENAO and assessing its therapeutic potency alone and in combination with a glycolytic inhibitor DCA on GBM cells, this thesis will test the hypothesis that a dual- targeting of glucose metabolism has potential to perturb the homeostasis of energy metabolism in GBM cells maximally, thereby inducing the inhibition of unstoppable tumour growth.

The specific aims of the thesis are:

Aim 1: To investigate the anti-tumour efficacy and to study the mechanism of action of

PENAO on GBM cells;

Aim 2: To determine the efficacy of DCA as a monotherapy and in combination with

PENAO in vitro, and to further investigate the mechanism of action of the combined therapy;

Aim 3: To test the efficacy of PENAO and DCA as monotherapies in a heterotopic

(subcutaneous) GBM mouse model and as a combination therapy in an orthotopic

(intracranial) GBM mouse model.

2 CHAPTER 2: Materials and Methods

2.1 Introduction

This chapter describes the general materials and methods used within this thesis.

Specific methods are detailed in the relevant chapters and the suppliers of materials are listed when being first mentioned in the text.

2.2 In vitro methods

2.2.1 Cell culture

2.2.1.1 Maintenance of cell lines

Immortalised GBM cell lines (U87, U251) were purchased from American Tissue

Culture Collection (ATCC) and validated for authenticity by CellBank Australia. The cells were maintained in Minimum Essential Media (MEM; Gibco® Life Technologies,

CA, USA) supplemented with 10% Fetal Bovine Serum (FBS) and 2 mM L-glutamine.

Cells were cultured in a humidified, 37°C incubator in a 5% CO2 environment (standard incubation conditions). Cells were cultured on plastic culture plates (6-well, 96-well) and flasks (T25, T75, T175) (NuncTM, Thermo Scientific, Waltham, MA, USA). Cells were detached from the plates or flasks for the purpose of experimentation with pre- warmed 0.05% trypsin, 0.53 mM ethylenediaminetetraacetic acid (EDTA) solution

(Gibco® Life Technologies, CA, USA) following washes with pre-warmed, sterile

Dulbecco’s Phosphate Buffered Saline (PBS; Gibco® Life Technologies, CA, USA).

Trypsin-EDTA was inactivated by two volumes of media containing 10% FBS.

Patient-derived GBM cell lines (HW1, RN1, WK1 and BAH1) were kindly provided by our collaborative researchers at Queensland Institute of Medical Research. The patient- derived GBM cell line G13 was cultured “in-house” in the Cure Brain Cancer Neuro-

Oncology Laboratory. G13 cell line was derived from the tumour tissue of a patient with recurrent GBM. All patient-derived GBM cell lines were cultured in RHB-A medium (Stem Cell Sciences, UK) supplemented with 20 ng/ml of epidermal growth factor (EGF) (Sigma-Aldrich, St. Louis, MO, USA) and fibroblast growth factor (FGF)

(Sigma-Aldrich, St. Louis, MO, USA) to avoid differentiation. To avoid the formation of neurosphere, all the containers for the growth of patient-derived cells were coated with diluted Matrigel (BD bioscience) solution for at least 3 hr in the standard incubation conditions. Matrigel was diluted at 1:100 with sterile PBS. Cells were incubated as per described above. Cells were detached from the plates or flasks for the purpose of experimentation with Accutase (Sigma-Aldrich, St. Louis, MO, USA) following washes with pre-warmed, sterile PBS. Accutase was inactivated by 1 mg/ml of trypsin inhibitors (Sigma-Aldrich, St. Louis, MO, USA).

2.2.1.2 Cryopreservation of Cell Lines

Cells were collected as described in Section 2.2.1.1 and resuspended in media containing 20% FBS and 10% dimethylsulfoxide (DMSO; Sigma-Aldrich, St. Louis,

MO, USA). The cell suspension was dispensed into 2 mL cryovials and frozen in an isopropanol container and stored in -80°C for 24 hr before being transferred to the 53 liquid nitrogen tanks for long-term storage. For recovery of frozen cells, vials were rapidly thawed at 37°C and cell suspension was transferred into pre-warmed media containing 10% FBS. Cells were centrifuged and the cell pellet was resuspended in fresh media and incubated under standard conditions.

2.2.2 Preparation of PENAO and dichloroacetate (DCA)

PENAO is a water-soluble powder and was kindly provided by Professor Phillip Hogg

(UNSW Australia) and Dr Pierre Dilda (UNSW Australia) as part of an ongoing collaboration. PENAO powder was dissolved with titration buffer (140 mM NaCl, 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ), 20 mM glycine, 1 mM EDTA, pH 7.0), to make the stock solution and sterilised by passing through a

0.22 µm filter. The stock solution was titrated each time for concentration determination and was stored at 4°C for up to 2 weeks. The working solution of PENAO was made by further dilution with corresponding cell culture media.

DCA powder (Sigma-Aldrich Cat. # 347795) is water-soluble and was dissolved in cell culture media (for in vitro experiment) or sterile water (for in vivo experiment) to make a stock solution.

2.2.3 Trypan blue cell counting

Cells were counted using the Countess™ automated cell counter (Life Technologies,

CA, USA). The counter distinguishes non-viable cells by their uptake of trypan blue stain. Specifically, cell suspension (10 µL) was mixed with 4% trypan blue (10 µL) and

54 was loaded onto a Countess™ cell counting chamber slide (Invitrogen, Life

Technologies, CA, USA). Cell images from the sample on the slide were acquired and the cell count and viability after staining with trypan blue stain were automatically calculated.

2.2.4 Cell proliferation assay

To assess cell proliferation, CellTiter 96® AQueous One Solution Cell Proliferation

Assay (Promega Corporation, Madison, WI, USA) was used according to the manufacturer’s instruction. This assay is a colourimetry-based method for determining the number of viable cells in proliferation or cytotoxicity assays. The CellTiter 96®

AQueous One Solution Reagent contains a novel tetrazolium compound [3-(4, 5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES). PES has enhanced chemical stability, which allows it to be combined with MTS to form a stable complex. The MTS tetrazolium compound is bioreduced by viable cells to a coloured formazan product, which is detectable by a spectrophotometer at a wavelength of 490 nm.

Specifically, cells were seeded at optimised densities (Table 2.1) in 200 µL medium per well into 96-well, flat-bottomed culture plates. Cells were allowed to adhere for 24 hr under standard incubation conditions and then treated with multiple dilutions of the drugs for 72 hr. In all experiments, ‘medium only’ and ‘medium with cells’ control groups were included and assayed in parallel with the treatment wells to account for baseline absorbance and drug-free cell viability, respectively (Figure 2.1). By using a 55 multi-channel pipette, pre-warmed MTS reagent (20 µL) was added into each well of

96-well plates containing medium (100 µL). Plates were incubated under standard incubation conditions for approximately 30-60 min. The absorbance was measured at

490 nm using the SpectraMax® M2 Multi-Mode Microplate Readers and analysed with the SoftMax®Pro Data Acquisition & Analysis Software. Cell numbers in the untreated control (‘medium with cells’) was normalised to 100%, and the viable cell numbers for all treatment groups were expressed as percentage of control.

Figure 2.1 A schematic design of MTS assays.

36 peripheral wells were filled with sterile Dulbecco’s Phosphate Buffered Saline (PBS) to prevent the effect of evaporation on cell viability. Each experiment included ‘medium- only’ and ‘medium with cells’ control wells. 6 wells containing ‘medium-only’ were used to blank absorbance measured at 490 nm for MTS assay. ‘Medium with cells’ control was seeded to monitor drug-free proliferation rates of cells.

Table 2.1 Optimised seeding density for 72 hr proliferation assay.

Cell lines Cell seeding density (cells/cm2)

U87 6000

U251 3000

RN1 15000

HW1 15000

BAH1 15000

WK1 10000

2.2.5 Cell cycle analysis

Cells were seeded in 6-well plates at optimised seeding densities and subsequently treated. Post-treatment, cells were harvested and fixed in cold (-20°C) ethanol (80% v/v in PBS) for at least 2 hr. Fixed cells were washed with PBS and stained in the dark with a solution containing propidium iodide (PI) (10 µg/mL), Triton X-100 (0.1%) and

RNase (100 µg/mL) for 20 min at room temperature. DNA content was analysed using a

BD FACSCanto II flow cytometer and 10000 events were acquired. Data analysis was performed using FlowJo software (TreeStar Inc, Ashland, OR, USA).

2.2.6 Invasion assay

The invasion assay was performed using the xCELLigence System Real-Time Cell

Analyser (RTCA) DP instrument (Roche, Indianapolis, IN, USA). This instrument provides kinetic information about cell movement by dynamically recording the entire invasion process in real-time without labelling cells, considerably improving invasion

57 assay quality. The Real-Time Cell Analyser (RTCA) dual-plate (DP) instrument utilises the cell invasion/migration (CIM)-Plate 16, which features microelectronic sensors integrated onto the underside of the microporous polyethylene terephthalate (PET) membrane of a Boyden-like chamber (Figure 2.2). As cells move from the upper chamber through the membrane into the bottom chamber in response to chemoattractant, they contact and adhere to the electronic sensors, resulting in increased impedance. The impedance positively correlates to increasing numbers of migrated cells on the underside of the membrane. The cell-index values (that reflect the impedance changes) are automatically and continuously recorded by the RTCA DP instrument. Therefore, cell invasion activity can be monitored via the cell-index profile.

Figure 2.2 Analysis of cell invasion in real-time with the cell invasion/migration (CIM)-Plate 16.

The plate features two separable sections for ease of experimental setup. Cells seeded in the upper chamber move through the microporous membrane into the lower chamber containing a chemoattractant. Cells adhering to the microelectrode sensors lead to an increase in impedance, which is measured in real-time by the Real-Time Cell Analyser (RTCA) dual-plate (DP) Instrument. This graph was extracted from ‘Real-time, label-free monitoring of cellular invasion and migration with the xCELLigence system [244]’.

Briefly, cells were seeded at an optimised density in T25 flasks and allowed to adhere for 24 hr. The cells were treated with PENAO for 24 hr, trypsinised and transferred to a

CIM-Plate 16 to monitor real-time invasion. In this system, cells were seeded in the upper chamber of the CIM-Plate pre-coated with medium containing 1% FBS and 1:30 dilution of Matrigel for 4 hr. The upper chamber was then assembled with the lower chamber of the CIM-Plate containing medium with 10% FBS as an attractant. Cell invasion through the Matrigel was monitored over 16-24 hr. In a separate plate, identically treated cells were transferred to an E-Plate 96 to monitor real-time attachment and proliferation using the xCELLigence instrument. This was to ensure that the cells remained viable during the course of the invasion assay.

2.2.7 Oxidative stress detection assays

2.2.7.1 Cytosolic production of reactive oxygen species (ROS)

Cytosol levels of ROS were detected by dihydroethidium (DHE) (Life Technologies,

CA, USA) staining. DHE is an oxidative fluorescent probe which allows the detection of the cytosolic ROS generation in intact tissues. DHE is the sodium borohydride- reduced derivative of ethidium bromide. Being a lipophilic cell-permeable dye, DHE can be rapidly oxidised to a fluorescent product or ethidium bromide by the ROS produced within the cells. In the cytosol, DHE emits blue fluorescence (excitation max

365 nm, emission max 420 nm). Once this probe is oxidised to ethidium bromide by

ROS, it binds to DNA and stains the cell nucleus red.

In brief, cells were seeded at an optimised density per well into 6-well plates. Cells were allowed to adhere for 24 hr and then treated with drugs for 16 hr. DHE was added to the medium at a final concentration of 2.5 μM and the cells were stained for 30 min in 59 the dark at 37°C. After staining, cells were detached (see section 2.2.1.1) and resuspended in 1 mL of PBS for flow analysis. To gate out the dead cells during analysis, Sytox Blue (Life Technologies, CA, USA) was added at a final concentration of 1 μM for counter-staining of dead cells. DHE and Sytox Blue fluorescences were analysed using a BD FACSCantoTM II and 10,000 events were acquired. After data collection, the flow cytometry analysis software FlowJo was used to analyse and graph the dot plots. Cells treated with 40 mM menadione (Sigma-Aldrich, St. Louis, MO,

USA) and 2 mM diethyldithiocarbamate (DDC) (Sigma-Aldrich, St. Louis, MO, USA) were used as positive control.

2.2.7.2 Mitochondrial superoxide production

Mitochondrial superoxide production was measured using the fluorogenic dye,

MitoSOXTM Red (Life Technologies, CA, USA). MitoSOXTM Red permeates viable cells and selectively localises to the mitochondria. Once in the mitochondria,

MitoSOXTM Red reagent is oxidised by superoxide and exhibits red fluorescence.

MitoSOXTM Red reagent is readily oxidised by superoxide but not by other ROS or reactive nitrogen species (RNS),.

Cells were prepared and treated as per section 2.2.7.2. MitoSOXTM Red was added to the medium at a final concentration of 5 μM and incubated for 30 min in the dark at

37°C. After staining, cells were detached (see section 2.2.1.1) and resuspended in 1 mL of Hank’s Balanced Salt Solution with calcium and magnesium (HBSS/Ca/Mg)

(Gibco® Life Technologies, CA, USA) for flow analysis. Samples were analysed using flow cytometry and dead cells were gated out as per described above (see section 60

2.2.7.1). Cells treated with 100 µM antimycin (Sigma-Aldrich, St. Louis, MO, USA) were used as positive control.

2.2.8 Mitochondrial trans-membrane potential detection assay

Loss of mitochondrial trans-membrane potential, an indicator of apoptosis, was detected by the fluorescent cationic dye, JC-1 (5, 5’, 6, 6’-tetrachloro-1, 1’, 3, 3’-tetraethyl- imidacarbocyanine iodide, MitoPTTM). The structure of JC-1 reagent allows it to easily penetrate healthy cells and mitochondria. Once inside a healthy cell, the lipophilic JC-1 carrying a delocalised positive charge enters the negatively charged mitochondria where it aggregates and fluoresces red. When the mitochondrial membrane potential collapses in apoptotic cells, JC-1 can no longer accumulate inside the mitochondria. Instead, it is dispersed throughout the cell in a monomeric form, which fluoresces green.

Briefly, cells were prepared and treated with indicated drugs as per described above (see section 2.2.7.1). JC-1 was added to the medium at a final concentration of 2 μM. After

15 min of staining in the dark at 37°C, cells were detached and resuspended with 1 mL of PBS for flow analysis (see section 2.2.7.1). Cells with depolarised mitochondria are represented by loss of red fluorescence (PE) and gain of green fluorescence (FITC).

Cells treated with 50 µM carbonylcyanide m-chlorophenylhydrazone (CCCP) (Sigma-

Aldrich, St. Louis, MO, USA) were used as positive control.

2.2.9 Apoptotic assays

2.2.9.1 Annexin V and PI staining

An early apoptotic event is the loss of plasma membrane asymmetry. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kDa Ca2+-dependent phospholipid-binding protein that has a high affinity for PS and binds to cells with exposed PS. When labelled with a fluorescent tag, such as FITC, Annexin V can be used with flow cytometry to measure this event. Since necrotic cells also expose PS as a result of lost membrane integrity, PI is utilised concurrently as a DNA stain to distinguish necrotic cells from Annexin V- labelled cell clusters. Viable and early apoptotic cells with intact membranes exclude PI, whereas the membranes of late apoptotic and necrotic cells are permeable to PI. As such, early apoptotic cells are Annexin V positive and PI negative.

The Annexin-V-FLUOS Staining Kit (Roche® Applied Science, Indianapolis, IN, USA) was used in a flow cytometry platform. In brief, cells were cultured and seeded as described in section 2.2.7.1. After 24 hr drug treatment, cell pellets were collected and resuspended in staining buffer (100 μL) supplemented with equal amounts (2 uL) of

Annexin-V-FLUOS solution and PI solution as described by the manufacturer. After 10 min of staining in the dark at room temperature, 500 μL staining buffer were added to each cell suspension and Annexin-V-FITC and PI fluorescence were acquired and plotted as per 2.2.7.1. Cells treated with 5 µM staurosporine were used as positive control.

2.2.9.2 Poly ADP-ribose polymerase (PARP) cleavage detection

Another well-established apoptotic marker is cleaved PARP (c-PARP). During apoptosis, PARP, a 116 kDa nuclear protein that normally functions in DNA damage detection and repair, is cleaved by caspase-3 and caspase-7 between Asp214 and

Gly215 to yield the p85 and p25 fragments. This cleavage effectively neutralises the ability of PARP to participate in DNA damage repair, and contributes to the cells’ commitment to undergo apoptosis. Sensitive detection of c-PARP therefore is able to distinguish different types of cell death and quantify apoptotic cells in a total cell population. In contrast to the Annexin-V and PI staining (which measures early-stage apoptosis), this methodology specifically looks at late-stage apoptosis after the activation of caspase-3.

Cells were prepared and treated as per section 2.2.9.1 to induce apoptosis. Cell lysates were prepared from harvested cells and the level of c-PARP was detected by western blotting (see 2.2.10).

2.2.10 Preparation of cell lysates and western blotting

2.2.10.1 Preparation of cell lysates

Treated cells were collected and centrifuged at 1000 rpm for 5 min at room temperature.

After discarding the supernatant, the cell pellet was washed in 1 mL of PBS and transferred to 1.5 mL microfuge tube followed by centrifugation at 1000 rpm for 5 min at room temperature. After removing the supernatant, the cell pellet was lysed in three pellet volumes (e.g. 60 ul for cells collected from 3 wells of 6-well plate) of cell lysis

63 buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 5 mM EDTA, 0.1% sodium dodecyl sulphate (SDS), 0.01% Triton X-100, 0.01% NP-40) with freshly added reducing agent

(Dithiothreitol; DTT) and protease and phosphatase inhibitors (2 µg/mL Aprotinin, 5

µg/mL Leupeptin, 1 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM Na3OV4).

The cells were lysed on ice for 1 hr and then sonicated in ice cold water for 5 min. Cell lysates were centrifuged at 14000 rpm for 10 min at 4 °C to remove debris, and the final cell extracts were transferred to fresh microfuge tubes and stored at -20°C.

2.2.10.2 Protein concentration quantification

Protein concentration of the cell extracts was determined with the Protein Assay Kit

(Bio-Rad, Hercules, CA, USA). This colourimetry-based assay measures total protein concentrations, based on the Bradford dye-binding method (Bradford 1976), which measures the colour change of Coomassie Brilliant Blue G-250 dye in response to various concentrations of protein. This assay involves the addition of the dye to protein solutions and subsequent measurement at 595 nm with a spectrophotometer.

Comparison to a standard curve generated from Bovine Serum Albumin (BSA; Sigma-

Aldrich, St. Louis, MO, USA) with known concentrations provides a relative measurement of unknown protein concentrations.

2.2.10.3 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting

6×Laemmli sample buffer was added to the cell lysates to a final concentration of 1×.

The proteins were denatured by boiling at 95°C for 5 min. 30-50 µg of protein was loaded per well along with molecular weight marker (Precision Plus Protein™

Kaleidoscope™ Standards, Bio-Rad, Hercules, CA, USA) onto precast polyacrylamide

64 gel (4–15% Mini-PROTEAN® TGXTM, Bio-Rad, Hercules, CA, USA) placed in the

Mini-PROTEAN® system which was pre-filled with running buffer (1×

Tris/Glycine/SDS).The gel was running at 120 V for 65 min.

Following the electrophoretic separation, the proteins were transferred to nitrocellulose membrane (Amersham Hybond ECL Nitrocellulose Membrane, GE Healthcare,

Buckinghamshire, UK) using the Mini Trans-Blot® (Bio-Rad, Hercules, CA, USA) system (100V, 150 min). The membrane was blocked for 1 hr at room temperature in

Tris-buffered saline with (0.05%) Tween (0.05% TBST) containing 5% skim milk.

After the blocking step, the membrane was incubated overnight at 4°C with the primary antibody (anti-c-PARP, Cell Signaling Technology, Danvers, MA, USA) at 1:1000 dilution and the loading control (anti-β-actin, Cell Signaling Technology, Danvers, MA,

USA) at 1:4000 dilution in the blocking solution (0.05% TBST containing 5% skim milk). After three washes with 0.05% TBST (5 min each), the membrane was incubated with 1:2000 dilution of secondary antibody (Immun-Star Goat Anti-Rabbit-Horseradish peroxidase(HRP) Conjugate, Bio-Rad, Hercules, CA, USA) in the same block solution at room temperature for 2 hr. After another three washes with TBST (5 min each), the

Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare,

Buckinghamshire, UK) were added for signal development. Images were acquired by using the ImageQuant LAS 4000 system (GE Healthcare, Buckinghamshire, UK).

2.2.11 PENAO-DCA combination treatment

Briefly, cells were seeded in 96-well plates as described in section 2.2.4. After 24 hr of incubation, test wells were set up as per outlined in Figure 2.3. Each row contained 65 increasing concentrations of PENAO or DCA. The first two rows contained PENAO only and the second two rows contained DCA only. The last two rows contained a combination of PENAO and DCA at the intersecting concentrations (see Table 2.2). 50

μL of media containing each drug was added to the existing 100 μL media in each well, giving a final volume of 200 μL. For the single drug-treated wells, 50 μL of drug-free medium was added on top of the 50 μL drug-containing medium to keep the final volume consistent at 200 μL. A column of background control wells (no seeded cells) as well as a second column of untreated control wells were included. Each experiment was repeated three times on different days.

Figure 2.3 A schematic design of combination cytotoxicity assays.

Each row contained untreated control and increasing concentrations of PENAO or/and DCA. The first two rows contained PENAO only and the second two rows contained DCA only. The last two rows contained a combination of PENAO and DCA at the intersecting concentrations.

Table 2.2 List of tested drug concentrations.

Drug Concentrations (×IC50)

Drug 1 (D1) 0.5 0.625 0.75 0.875 1.0 1.25 1.5 2.0

Drug 2 (D2) 0.5 0.625 0.75 0.875 1.0 1.25 1.5 2.0

0.5×(D1)+ 0.625×(D1)+ 0.75×(D1)+ 0.875×(D1)+ 1.0×(D1)+ 1.25×(D1)+ 1.5×(D1)+ 2.0×(D1)+ Drug 1+Drug 2 0.5×(D2) 0.625× (D2) 0.75×(D2) 0.875×(D2) 1.0×(D2) 1.25×(D2) 1.5×(D2) 2.0×(D2)

Constant-ratio combinations (range from 0.5 to 2.0×IC50) of drugs were tested.

The cytotoxic interaction between PENAO and DCA was evaluated using the median drug effect analysis method of Chou and Talalay [245], processed by the computer program CalcuSyn 2.1 (Biosoft, GB, UK). Cell viability was converted to fraction affected before being entered into CalcuSyn (e.g. fraction affected of 0.75 is equivalent to cell viability of 25%). The program was unable to evaluate fraction affected of 0, therefore any cases of 100% cell viability was entered as 0.01.

2.2.12 Oxygen consumption and acid production assays

The XF24 Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, MA, USA) measures in real-time the oxygen consumption and the acid production by cultured adherent cells. The array of biosensors was each independently calibrated using a reagent of known pH and oxygen concentration prior to commencing an experiment.

Briefly, cells were seeded in Matrigel-coated XF 24-well cell culture microplates at an optimised cell density in 100 µL of MEM with 10% FBS (for U87) or Dulbecco’s

Modified Eagle’s Medium (DMEM)/F12 (for RN1) and then incubated for 24 hr to adhere. After incubation, media was added to 1 mL in the presence of tested drugs at indicated concentrations for another 24 hr treatment. Assays were initiated by removing the medium from each well and replacing it with 800 µL of phenol red-free and bicarbonate-free DMEM (for U87) or DMEM/F12 (for RN1) containing 2 mM L- glutamine and 5 mM glucose. The cells were incubated at 37°C for 1 hr to allow equilibration of media temperature and pH before the first rate measurement. Each rate measurement cycle consisted of 3 min mixing to allow equilibration of oxygen partial pressure, 2 min wait and then 3 min simultaneous measurements of oxygen consumption rate (OCR) and extracellular and acidification rate (ECAR) . In the end, cells were harvested and the number and percentage of viable cells were determined by 68 flow cytometry with Annexin V and PI staining. The measurements of acid production and oxygen consumption rate were normalised using the viable cell count.

2.3 In vivo methods

2.3.1 Mice

For different experimental purposes, 6-8 weeks old female BALB/c nude mice or non- obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were purchased from the Animal Resources Centre (ARC), Western Australia. All mice were maintained at the Biological Resources Centre (BRC), UNSW Australia. All studies were conducted in accordance with the guidelines of the Animal Care and Ethics

Committee (ACEC) of UNSW Australia.

2.3.2 Heterotopic tumour implantation (subcutaneous xenograft)

The skin of the flank was cleaned with 70% ethanol and 100 µL of cell suspension in

PBS containing 3×106 U87 cells was injected subcutaneously into the left flank of mice using a 1 mL 25 gauge insulin syringe. Animal monitoring and measurement of tumour size were carried out twice weekly after tumour induction. Once the average tumour size reached a volume of 50~70 mm3, animals were randomly assigned into groups of 8-

9 for the treatment arms and control arms. In the instances when necrosis/ulceration of the skin over the tumour site occurred or the tumour volume reached 1 cm3, the mice were euthanised.

2.3.3 Orthotopic tumour implantation (intracranial xenograft)

2.3.3.1 Preoperative preparation

On the day of surgery, the mice were weighed and recorded. The mice were injected intraperitoneally with 5 mg/kg Rimadyl (carprofen, Zoetis, New York, NY, USA) in 0.1 mL saline 30 min prior to procedure initiation.

2.3.3.2 Anaesthesia induction

The mice were anaesthetised with isoflurane (2.5%, 1 L/min oxygen, with 3% maintenance dose) in a sealed knock-down box. Once the mouse was completely anaesthetised, it was transferred onto the pre-heated pad (37°C) on the surgery platform.

During the whole surgical process, the mouse was anaesthetised by continuous mask inhalation of isoflurane (2.5%, 1 L/min oxygen, with 1-3% maintenance dose). Depth of anaesthesia was assessed by respiration frequency and toe pinch reflex every 2-5 min.

2.3.3.3 Intracranial injection

The skin of the epicranium was disinfected with 70% ethanol. A 1 cm long incision was made right above the sagittal suture of the skull. The surgical field was exposed by using the ear pieces to retract the skin and each side of the incision was fixed. A 2 mm drill hole was then made and 2 µL of cell suspension in PBS containing 5x104 U87 cells was delivered into the right striatum (0.2 μL/min, total 10 min) by stereotactic injection through a Hamilton syringe (Hamilton Company, Reno, NV, USA) connected to the manipulating arm of the stereotactic device (David KOPF® Instruments, Tujunga, CA,

USA) (Figure 2.4). The following stereotactic coordinates (mm from bregma) were used:

70 anterio-posterior (AP) +1.0, medio-lateral (ML) +1.5, dorso-ventral (DV) -3.0 (Figure

2.5, 2.6).

Figure 2.4 The procedure of tumour cell implantation.

Figure 2.5 The site of injection point on the skull surface of the mouse head.

Figure 2.6 Stereotactic coordinates of mouse brain for intracranial injection.

The coordinates were extracted from ‘The mouse brain in stereotactic coordinates, the coronal plates and diagrams’.

All injections were made over a period of 10 min followed by another 5 min of dispersing time to ensure optimal parenchymal compliance. The micro-injector needle was then retracted slowly to prevent reflux of cell suspension. The burr hole in the skull was closed with bone wax (Johnson & Johnson, New Brunswick, NJ, USA) and the incision was closed with tissue glue (3MTM VetbondTM Tissue Adhesive, North Ryde,

NSW, Australia). The total duration of the implantation procedure was approximately

30 min, throughout which the mouse received sterile eye drops to avoid eye dryness.

2.3.3.4 Post-operative recovery

Post-surgical mice were moved to the recovery area, placed on the pre-heated pad and monitored until they woke up. Mice were returned to their cages, given free access to food and water and monitored daily for the first seven post-operative days for any signs of neurological dysfunction.

2.3.4 Drug administration

2.3.4.1 Administration of PENAO

PENAO was continuously administered to the mice via Alzet Osmotic Pumps (model

1004, 2002; ALZET® Cupertino, CA, USA), which are miniature, infusion pumps used for continuous dosing of unrestrained laboratory mice. Continuous administration over

28 days was achieved by loading an osmotic mini-pump with 100 μL of PENAO for dosage of 1 mg/kg/day or 200 μL of PENAO for dosage of 3 mg/kg/day, respectively.

The pump was implanted subcutaneously in the right flank of the anesthetised mouse.

Briefly, once the animal was anesthetised, the skin was cleaned with 70% ethanol over the implantation site. A suitable incision adjacent to the site chosen for pump placement was made. A haemostat was inserted into the incision and, by opening and closing the jaws of the haemostat, the subcutaneous tissue was separated to create a pocket for the pump. A loaded pump was inserted into the pocket with the delivery portal first and away from incision. This minimised interaction between the compound delivered and the healing of the incision. Once the pump was inserted, the incision was closed with wound clips (ALZET®, Cupertino, CA, USA).

2.3.4.2 Administration of DCA

DCA was administered twice daily by intragastric gavage at varying doses between 25 and 75 mg/kg/dose for different experimental purposes. The route of administration

(intragastric gavage) was chosen because a previous study had proven that it was more effective than intraperitoneal administration in tumour growth inhibition [173].

2.3.5 Tumour volume measurements

The tumour volume was measured using a calliper twice a week after tumour cells injection. Subcutaneous tumour volume was calculated from three linear dimensions

using equations of ‘ ’.

2.3.6 Assessment of neurological dysfunction

Post-surgical animals were monitored at least twice a week for any signs of neurological dysfunction, including hunched posture, lack of movement, lack of grooming, abnormal feeding patterns and vocalisation. Any animal that developed signs or symptoms of toxicity, and/or had weight loss equal to or greater than 20% of its original body weight, was immediately euthanised by inhalation of an overdose of carbon dioxide.

2.3.7 Measurement of arsenic levels in the plasma and brain

Blood was collected by cardiac puncture into 4% acid citrate dextrose and plasma was prepared by centrifugation at 2000 g for 10 min at 4°C. Whole brains of animals were harvested at the final endpoints: neurological decline or at the time of moribund. The brain was separated into three parts: healthy (left) hemisphere, tumour part (right), and

74 the remaining part of the right hemisphere. The weight of each part of the brain was recorded for subsequent calculation of arsenic concentrations. The plasma and tumour samples were diluted 2 and 5 fold, respectively, with 70% w/w nitric acid. The tumour samples were rotated at room temperature for 2 hr and the supernatant was collected by centrifugation at 58 g for 5 min. The samples were analysed for arsenic using an Elan

6100 Inductively Coupled Plasma (ICP) Mass Spectrometer (Perkin Elmer Sciex

Instruments, Shelton, CT, USA) by ICP-Elementary Analysis Laboratory, UNSW

Australia.

2.3.8 Immunohistochemistry (IHC)

Tumour cell morphology and proliferation were assessed by Haematoxylin and eosin

(H&E) staining and immunostaining for Ki-67, respectively. In brief, excised tumours were fixed in formalin solution (neutral buffered, 10%) (Sigma-Aldrich, St. Louis, MO,

USA), embedded in paraffin, cut into 5 μm thick sections and mounted on Superfrost slides (work performed by the Histology and Microscopy Unit, UNSW Australia).

Mounted sections were deparaffinised in xylene and rehydrated in PBS with an ethanol gradient. A heat-mediated antigen retrieval step was performed using 10 mM citrate buffer (pH 6.0) at 95°C for 15 min. Sections were incubated with anti-Ki67 antibody

(rabbit anti-human Ki-67; Abcam, Cambridge, MA, USA) at 1:100 dilution for 1 hr at room temperature and with secondary HRP-conjugated goat anti-rabbit antibody for 30 min at room temperature. Tumour cell proliferative index was determined by the proportion of Ki-67-positive cells under ten 40× magnification fields.

2.4 Statistical analysis

Results were presented as means ± standard deviation (SD). All statistical analyses were performed using the GraphPad Prism software (GraphPad, San Diego, CA, USA). Cell cycle analysis, JC‐1 and Annexin V/PI results were subjected to Mann-Whitney test or

Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks corrected by Dunn’s test to evaluate the significance of the results within groups. The relative tumour volume (RTV) growth curves were compared using repeated measures two-way

ANOVA. The comparison of Kaplan-Meier survival curves was performed using Log- rank (Mantel-Cox) test. Statistical analysis for significance between the vehicle and

PENAO-treated tumour sections was performed using Mann-Whitney test. All tests of statistical significance were two-sided and p values < 0.05 were considered statistically significant.

3 CHAPTER 3: PENAO, a Mitochondrial Toxin

Targeting Glucose Metabolism of GBM Cells In Vitro

3.1 Introduction

3.1.1 The role of mitochondria in gliomas

It has long been recognised that malignant tumours, including gliomas, preferentially use aerobic glycolysis to generate ATP and show an inherent resistance to apoptosis [35,

246]. This indicates the underlying involvement of dysfunctional mitochondria in tumour pathophysiology, which is not well understood. The most recognised function of mitochondria is the production of ATP, the chemical form of cellular energy, via oxidative phosphorylation. This is the principal energy-producing pathway of normal glial cells and the ensuing step following glycolysis in the presence of oxygen [247,

248]. Mitochondria also play pivotal roles in the regulation of cell proliferation and apoptosis, two other critical areas of dysfunction in gliomas.

Normal ATP production via oxidative phosphorylation generates reactive oxygen species (ROS) in the mitochondria. ROS are non-lethal at physiological levels and function as intracellular messengers. However, overproduction of ROS results in irreversible cellular damage and apoptotic cell death [249, 250]. In glial cells, there is a pool of glutathione (GSH) and its oxidised form glutathione disulphide (GSSG) working as a redox buffer system that prevents the build-up of ROS to harmful levels

[251]. In glioma cells, the level of GSH/GSSG buffer is substantially lower in the highly proliferative tumour periphery compared to the necrotic centre [252]. This is important because it has been shown that glucose withdrawal increased ROS production and induced apoptosis in glioma cells due to insufficient redox buffering, but this did not occur in normal human astrocytes (NHA) [252]. In these glioma cells, an increased breakdown of fatty acid in the mitochondria for ATP generation was also observed after glucose withdrawal [253]. However, loss of normal mitochondrial structure, stability and function in glioma cells led to abnormalities of the fatty acid metabolism, which resulted in the overproduction of ROS and lethal oxidative stress [254]. Additionally, studies of cardiolipin, a unique structure specific to mitochondria, highlighted increased levels of ROS as a direct cause of cardiolipin dysfunction through oxidation and resulted in loss of mitochondrial function [255-257]. This deleterious cycle may contribute to further mitochondrial damage and uncoupling from normal cellular functions in glioma cells (Figure 3.1). Therefore, the rapidly proliferating glioma cells, with their high energy demands and low GSH/GSSG buffer levels, is highly vulnerable to apoptosis induced by the metabolic effects of insufficient glucose or inhibitors of glucose metabolism, once again highlighting the potential of targeting glucose metabolism to impede glioma progression.

Figure 3.1 Effects of glucose withdrawal on dysfunctional mitochondria in glioma cells.

ROS, reactive oxygen species; GSH, glutathione; GSSG, glutathione disulphide. This schematic was redrawn from ‘The role of mitochondria in glioma pathophysiology’ [254].

3.1.2 Targeting mitochondria with arsenic-based compounds

Treatments with arsenic-based compounds have resulted in high rates of remission of some cancers such as acute promyelocytic leukaemia [258]. The mechanism of action and cancer cell selectivity by arsenic-based compounds have only recently been unravelled [259]. Increased understanding and awareness of the importance of intracellular redox systems and regulation of ROS production by controlling mitochondrial function were attributable. Tumour cells have elevated ROS levels and oxidative damage to macromolecules, which most likely contribute to their oncogenic transformation and increased metabolic activity [260, 261]. In addition, the intrinsic 79 elevated level of oxidative stress renders tumour cells more susceptible than normal cells to the action of ROS-inducing cytotoxic agents, a property which may be exploited in anti-tumour therapies [262, 263]. Many of the cellular targets for the arsenic-based compounds are mitochondrial proteins involved in regulating the production of ROS

[264]. Inhibition of these proteins by disulphide linkage of vicinal thiol groups can lead to increased production of ROS and induction of apoptotic pathways [265]. Adenine nucleotide translocase (ANT) is the most abundant protein in the mitochondrial inner membrane and catalyses the exchange of mitochondrial ATP for cytosolic ADP across the mitochondrial inner membrane [266]. The link of ANT with another protein, the voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane, forms a complex involved in the early stages of apoptosis activated via the mitochondrial pathway [267] (Figure 3.2). The two components of this complex form part of the mitochondrial permeability transition pore (MPTP), a channel mediating the release of molecules from the mitochondria to activate apoptosis. In cancer cells, ANT has two functions: it transfers ATP to mitochondria-bound hexokinase 2 (HK2) to phosphorylate and trap glucose in the cell (see section 1.4.3, Figure 1.3), and it is a regulator of the

MPTP, which controls the permeability of the mitochondrial inner membrane [237].

Opening of MPTP by inactivating ANT allows free passage of molecules < 1500 Da in size through the mitochondrial inner membrane [267], which leads to uncoupling of oxidative phosphorylation and increase in superoxide levels, loss of trans-membrane potential and decrease in oxygen consumption [233]. These effects of ANT blockade result in proliferation arrest and mitochondria-mediated apoptotic cell death [208].

Figure 3.2 Proposed mitochondrial permeability transition pore (MPTP) complex architecture.

A hypothetical schematic outlines the role of MPTP opening in apoptosis. A channel formed by the link of the voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane and the adenine nucleotide translocase (ANT) in the mitochondrial inner membrane, is a complex involved in the early stages of the intrinsic apoptotic pathway. MPTP opening through mitochondrial uncoupling, ROS production and mitochondrial depolarisation allows free diffusion of solutes across the membrane. The opening of the MPTP ultimately results in mitochondrial swelling, mitochondrial Ca2+ efflux and the release of pro-apoptotic proteins from the mitochondrial inter membrane space, such as cytochrome c and second mitochondria-derived activator of caspases/ direct IAP-binding protein with low pI (Smac/DIABLO). This schematic was adapted and redrawn from ‘Transient mitochondrial permeability transition pore opening mediates pre-conditioning-induced protection [268] ’.

3.2 Hypothesis and aims of this chapter

Working hypothesis of this chapter:

PENAO is a novel organo-arsenoxide compound that targets ANT in mitochondria. The rapidly proliferating glioma cells are highly susceptible to apoptosis induced by glucose withdrawal due to its high energy demands and low GSH/GSSG buffer levels. I thus hypothesise that PENAO will effectively inhibit the growth of GBM cells in vitro as well as induce apoptotic cell death by targeting mitochondrial glucose oxidation in

GBM cells.

The specific aims of this chapter are:

1. To investigate the efficacy of PENAO on GBM cells;

a) To establish the dose-response curves for PENAO tested on a panel GBM

cells. Half maximal inhibitory concentrations (IC50) will be determined;

b) To measure cell cycle distribution in GBM cells treated with PENAO;

c) To measure the invasion potential of GBM cells in the presence of PENAO;

2. To understand the mechanism of action of PENAO;

a) To measure the oxidative stress (superoxide production), mitochondrial trans-

membrane potential loss and apoptosis in GBM cells treated with PENAO;

b) To determine if there is a direct effect on glucose metabolism of GBM cells

after treatment with PENAO.

3.3 Results

3.3.1 PENAO triggered proliferation arrest of GBM cells

Work from colleagues previously showed significant efficacy of PENAO in pancreatic and ovarian cancer cell lines in vitro [241]. Working with another PhD student in the

Cure Brain Cancer group, Sylvia Chung, we investigated the cell killing potential of

PENAO on the growth of immortalised GBM cells (U87 and U251), patient-derived primary/recurrent GBM cells (RN1, HW1, WK1, BAH1 and G13) and non-cancerous cells (lung fibroblast cell line MRC-5 and normal human astrocytes (NHA)). After 72 hr of exposure to PENAO at a range between 0.1 µM to 20 µM, a significant reduction in cell viability was observed in both immortalised GBM cell lines (Figure 3.3). The proliferation arrest effect was further confirmed in patient-derived primary GBM cells

(RN1, HW1, WK1 and BAH1) and a recurrent GBM cell line (G13) (Figure 3.3 and

Table 3.1). The IC50 of PENAO was determined for the cell lines and summarised in

Table 3.1. Variable response to PENAO was observed among the GBM cell lines (IC50 ranges from 0.7 ± 0.1 µM to 4.5 ± 0.7 µM), and in general, the patient-derived GBM cells were more sensitive to PENAO treatment (lower IC50 values) compared to immortalised GBM cell lines (See Table 3.1 for IC50 summary). The IC50 values of

PENAO for non-cancerous cells were 1.8-14.5 times higher than those for GBM cells

(Table 3.1).

Figure 3.3 Response of various cells to PENAO treatment.

Dose-response curves showing the viabilities of immortalised GBM cell lines (U87 and U251), patient-derived primary GBM cell lines (RN1, HW1, WK1 and BAH1), patient- derived recurrent GBM cell line (G13) and non-cancerous cell lines (NHA, MRC-5) after 72 hr PENAO treatment. Measurements were collected using MTS assay. The data points and error bars are expressed as mean ± SD for triplicate measurements. The

IC50 of PENAO on NHA and MRC-5 was tested by Sylvia Chung.

Table 3.1 Summary of IC50 values of PENAO on all tested GBM and non- cancerous cell lines.

Cell types Cell lines IC50 (µM)

U87 4.5±0.7 Immortalised GBM cell lines U251 3.1±0.4

RN1 1.2±0.3

HW1 0.6±0.1 Patient-derived primary GBM cells WK1 1.4±0.3

BAH1 0.7±0.1

Patient-derived recurrent GBM cells G13 2.0±0.1

MRC5 8.7±0.7 Non-cancerous cell lines NHA 7.9±1.2

Cell viabilities of immortalised GBM cell lines (U87 and U251), patient-derived primary and recurrent GBM cells (RN1, HW1, WK1, BAH1 and G13) and non-cancerous cell lines (MRC5 and normal human astrocyte (NHA)) were measured by MTS assay after

PENAO treatment. The half maximal inhibitory concentration (IC50) values were determined at 72 hr time point for PENAO treatments. Values are expressed as mean ± SD of three independent experiments.

3.3.2 PENAO induced cell cycle arrest of GBM cells

To determine whether the proliferative inhibition of PENAO on GBM cells involved cell cycle changes, I examined cell cycle phase distributions of the treated cells by flow cytometry. When the immortalised GBM cells (U87 and U251) were treated with increasing doses of PENAO for 24 hr, significant G2/M phase arrest was observed. As shown in Figure 3.4 A, compared to the untreated control, treatment of cells with

PENAO resulted in a dose-dependent increase in the proportion of cells in the G2/M phase of the cell cycle and corresponding decrease in the S phase. Specifically, the addition of 6 µM PENAO significantly increased the percentage of U87 cells in the

G2/M phase to 25.1% compared to the untreated control (15.7%) (p<0.001), while decreasing the percentage of U87 cells in the S phase to 17.2% as opposed to the untreated control (26.3%) (p<0.01) (Figure 3.4 A). The cell cycle arrest effect was also confirmed in the U251 cell line (Figure 3.4 B). In the case of U251 cells, treatment with

4.5 µM PENAO significantly (p<0.001) increased the proportion of cells in the G2/M phase (27.3%) compared to the untreated control (17.9%). Meanwhile, it caused a significant decreased (p<0.001) of cell proportion in the S phase (35.8%) compared to the control sample (43.4%) (Figure 3.4 B). No significant change was observed in the cell proportions in the G1 phase upon PENAO treatment (Figure 3.4 A, B).

Figure 3.4 PENAO induced cell cycle arrest of GBM cells at G2/M phase.

The proportions of (A) U87 cells and (B) U251 cells in the G2/M phase were significantly increased in a dose-dependent manner while the cell proportions in the S phase were decreased after 24 hr PENAO treatment. No significant changes occurred in the G1 phase (A, B). The data points and error bars are expressed as mean ± SD for triplicate measurements. Statistical significance was determined with ANOVA corrected by Dunn’s test, comparing drug-treated samples with untreated control. **p-value <0.01, ***p-value <0.001.

3.3.3 PENAO inhibited invasion of GBM cells

To determine if PENAO had any effect on cell’s capacity to invade, cell invasion was measured using the xCELLigence DP system. This system based on impedance is able to monitor invasion of free-labelled cells in real time. The U251 cell line was selected as our model for this invasion assay, as U251 tumours in orthotopic mouse model show a diffusely infiltrative pattern of invasion into normal brain parenchyma [269]. U251 cells were pre-treated with low doses of PENAO (1-2.5 µM) for 24 hr prior to being monitored with the xCELLigence DP system. Cell invasion was monitored for up to 16 hr and a dose-dependent decrease in cell invasion was observed in the PENAO-treated groups (Figure 3.5 A, B). To ensure the decrease in invasion was not a result of cell viability loss, a ‘mirror plate’ was used to monitor cell viability simultaneously.

Treatments with PENAO up to a concentration of 2.5 µM did not affect the viability of

U251 cells (Figure 3.5 B).

Figure 3.5 Inhibition of U251 cell invasion by PENAO.

U251 cells were pre-treated with PENAO for 24 hr and invasion through Matrigel was measured in real-time using the xCELLigence DP system. In a parallel experiment, identically treated cells were monitored with the xCELLigence MP system to confirm viability. (A) A representative graph of invasion over time; (B) The invasion rate and cell viability as a function of PENAO concentration. The data points and error bars are expressed as mean ± SD for triplicate measurements. Statistical significance was determined with ANOVA corrected by Dunn’s test, comparing drug-treated samples with untreated control. ****p-value <0.0001. Note: Experiment was done by Peter Luk.

3.3.4 PENAO induced apoptosis in GBM cells

To further examine whether PENAO was competent to induce apoptotic cell death, I measured apoptotic markers, Annexin V/ propidium iodide (PI) staining and poly ADP- ribose polymerase (PARP) cleavage (c-PARP) post-PENAO treatment (24 hr) in U87 and U251 cells. Compared to the untreated control, a significant increase in Annexin V positive cells was observed for both cell lines (6µM PENAO) (p<0.05) (Figure 3.6 A).

Further increasing PENAO dose up to 12 µM boosted the proportion of apoptotic cells

88 to 50~70% of the total cell populations (Figure 3.6 A). The dose-dependent apoptotic effect induced by PENAO in both U87 and U251 cell lines was further confirmed by western blotting to detect the level of (c-PARP), a hallmark of apoptosis and caspase activation (Figure 3.6 B).

Figure 3.6 Apoptosis was induced in GBM cells dose-dependently after 24 hr PENAO treatment.

(A) Cells were stained with the Annexin-V-FLUOS Staining kit after 24 hr of PENAO treatment. The proportion of apoptotic cells increased with PENAO treatment in a dose- dependent manner. (B) Apoptotic cell death was further confirmed by the poly ADP- ribose polymerase (PARP) cleavage (c-PARP). The data points and error bars are expressed as mean ± SD for triplicate measurements. The western blot shown is the representative of three independent experiments. Statistical significance was determined with ANOVA corrected by Dunn’s test, comparing drug-treated samples with untreated control. *p-value <0.05, ***p-value <0.001.

3.3.5 Mechanism of action of PENAO on GBM cells

3.3.5.1 Inhibition of mitochondrial function in GBM cells by PENAO triggered

production of ROS

To specifically address the mechanism of action of PENAO in GBM cells, I sought to determine whether PENAO had a direct effect on the mitochondria of GBM cells. I first 89 examined whether the effect of PENAO on GBM cells was resulted from oxidative stress by using dihydroethidium (DHE) staining. DHE is a measure of ROS release which is an indicator of cytosolic ROS generation in intact tissues [270]. Immortalised

GBM cell lines (U87 and U251) were incubated with increasing concentrations of

PENAO for 16 hr and the cytosolic production of ROS was measured using DHE fluorescence and flow cytometry. As shown in Figure 3.7, cellular levels of ROS increased dose-dependently with PENAO treatment in both cell lines.

Figure 3.7 Cytosolic ROS production was induced in GBM cells after 16 hr PENAO treatment.

(A) Immortalised GBM cell lines (U87 and U251) were stained with dihydroethidium (DHE) dye after 16 hr of PENAO treatment. PENAO induced cytosolic production of superoxide dose-dependently. (B) Representative histogram overlays (U87) acquired from flow cytometry. The data points and error bars are expressed as mean ± SD for triplicate measurements. Statistical significance was determined with ANOVA corrected by Dunn’s test, comparing drug-treated samples with untreated control. *p-value<0.05, **p-value<0.01.

To detect the mitochondrial superoxide production, MitoSOX Red was used to stain

U87 cells treated with PENAO. Approximately 16 hr post-PENAO treatment, the mitochondrial superoxide in U87 cells increased dose-dependently (Figure 3.8 A, B).

Figure 3.8 Mitochondrial superoxide production was induced in U87 cells after 16 hr PENAO treatment.

U87 cells were stained with MitoSOX Red after 16 hr of PENAO treatment. (A) PENAO induced mitochondrial superoxide production dose-dependently. (B) Representative flow cytometry histogram overlays showed 6 µM PENAO shifted the mean reading of MitoSOX Red fluorescence, indicating increased level of mitochondrial superoxide. The data points and error bars are expressed as mean ± SD for triplicate measurements. Statistical significance was determined with ANOVA corrected by Dunn’s test, comparing drug-treated samples with untreated control. *p-value<0.05, **p-value<0.01.

3.3.5.2 The ROS production triggered by PENAO in GBM cells was quenched by

addition of small peptide thiols

ROS react with cysteine thiolates, which in turn quenches ROS in cells [271].

Glutathione ethyl ester (GSH-EE) and N-acetyl cysteine (NAC) are plasma membrane- permeable thiols that can increase intracellular levels of GSH [272, 273]. Therefore, it was hypothesised that these compounds would quench the superoxide anion generated by PENAO and inhibit the anti-proliferative effect of this compound. To corroborate this hypothesis, the efficacy of PENAO in combination with GSH-EE or NAC was examined. In accordance with the hypothesis, both GSH-EE and NAC inhibited the anti-proliferative effect of PENAO in GBM cells. The PENAO IC50 for proliferation arrest of U87 cells increased from 4 µM to 9 µM in the presence of 500 µM GSH-EE and to >10 µM in the presence of 500 µM NAC (Figure 3.9 A). The PENAO IC50 for proliferation arrest of U251 cells was doubled (from 2.5 µM to 5 µM) in the presence of

500 µM GSH-EE and it further increased to 9 µM in the presence of 500 µM NAC

(Figure 3.9 B). Mechanistically, incubation of NAC with PENAO-treated U87 cells largely ablated the ROS produced in these cells (Figure 3.9 C).

Figure 3.9 The anti-proliferative effect of PENAO was attenuated by addition of small peptide thiols glutathione ethyl ester (GSH-EE) and N- acetyl cysteine (NAC).

(A) U87 and (B) U251 cells were incubated with increasing concentrations of PENAO in the absence or presence of GSH-EE (500 µM) or NAC (500 µM) for 72 hr and cell viabilities were measured by MTS assays. The anti-proliferative effect of PENAO was blunted by the addition of GSH-EE or NAC. (C) U87 cells were incubated with PENAO (6 µM) in the absence or presence of NAC (500 µM) for 16 hr and cytosolic production of ROS was measured from DHE fluorescence by flow cytometry. The representative histogram overlays from flow cytometry shows incubation of NAC with PENAO-treated U87 cells largely ablated the superoxide production in these cells. The data points and error bars are expressed as mean ± SD for triplicate measurements.

3.3.5.3 PENAO disrupted the mitochondrial trans-membrane potential in GBM

cells

Opening of the MPTP is accompanied by a change in the trans-membrane potential, which can be measured using the JC-1 dye. The ratio of distribution of JC-1 between the cytosol (green fluorescence) and mitochondria (red fluorescence) reflects mitochondrial trans-membrane potential (Smiley et al., 1991). PENAO triggered a concentration-dependent loss of mitochondrial trans-membrane potential in both U87 and U251 cells (Figure 3.10 A). The loss in trans-membrane potential as a function of

PENAO concentration was comparable to that of the PENAO-induced ROS production

(Figure 3.7). Specifically, after 16 hr of treatment, a significant increase in depolarisation of mitochondrial trans-membrane potential was achieved in U87 (Figure

3.10 A, B) and U251 (Figure 3.10 A) cells with 6 µM (p<0.01) and 3 µM (p<0.05) of

PENAO respectively.

Figure 3.10 Loss of mitochondrial trans-membrane potential was induced in GBM cells by PENAO treatment.

(A) Immortalised GBM cell lines (U87 and U251) were treated with increasing doses of PENAO for 16 hr followed by JC-1 staining. (B) Representative 2-D dot plots from flow cytometry demonstrated mitochondrial depolarisation of U87 cells occurred after PENAO treatment in a dose-dependent manner. The data points and error bars are expressed as mean ± SD for triplicate measurements. Statistical significance was determined with ANOVA corrected by Dunn’s test, comparing drug-treated samples with untreated control. *p-value<0.05, **p-value<0.01, ***p-value<0.001, ****p- value<0.0001.

3.3.5.4 PENAO-induced mitochondrial superoxide production preceded loss of

mitochondrial trans-membrane potential

As a mitochondrial toxin, PENAO is supposed to first induce production of mitochondrial superoxide, which then leads to ensuing loss of mitochondrial trans- membrane potential. To validate this hypothesis, a time points-based assay was conducted to determine the initial event after PENAO treatment. U87 cells were treated with PENAO (6 µM) for 4 hr and mitochondrial superoxide production and mitochondrial trans-membrane potential were measured. A significant increase of mitochondrial superoxide production (p<0.01) was observed (Figure 3.11 A), but no significant change in trans-membrane potential was detected (Figure 3.11 B).

Figure 3.11 PENAO-induced mitochondrial superoxide production preceded loss of mitochondrial trans-membrane potential.

U87 cells were stained with (A) MitoSOX Red or (B) JC-1 after 4 hr exposure to PENAO (6 µM). PENAO-induced mitochondrial superoxide production significantly increased (p<0.01) after 4 hr PENAO treatment (A), but no significant change in mitochondrial depolarisation was observed (B). The data points and error bars are expressed as mean ± SD for triplicate measurements. Statistical significance was determined with ANOVA corrected by Dunn’s test, comparing drug-treated samples with untreated control. **p-value<0.01. Note: experiment performed in conjunction with Sylvia Chung.

3.3.6 PENAO inhibited the mitochondrial glucose oxidation in GBM cells

Loss of mitochondrial trans-membrane potential leads to uncoupling of the electron transportation chain (ETC) and impaired oxidative phosphorylation [274]. To further investigate the anti-metabolic effect of PENAO as a mitochondrial toxin on GBM cells, oxygen consumption of U87 cells post-PENAO treatment was measured using the XF24 extracellular flux analyser. This platform simultaneously measures the two major energy producing pathways of the cell in real-time: mitochondrial respiration (measured by oxygen consumption rate (OCR)) and glycolysis (measured by extracellular acidification rate (ECAR)). Treatment of U87 cells with PENAO for 24 hr resulted in a concentration-dependent decrease in the OCR (Figure 3.12 A, C) and corresponding increase in the ECAR (Figure 3.12 B, C). Specifically, OCR was significantly inhibited by 4 µM PENAO (p<0.0001) and was further inhibited by increasing the concentration of PENAO to 7 µM (Figure 3.12 A, C). Simultaneously, ECAR was significantly elevated by PENAO at the concentration of 4 µM (p<0.0001). However, higher doses of

PENAO did not induce a dose-dependent increase in ECAR which appeared to be saturated at approximately 1200 mpH/min/106 cells (Figure 3.12 B, C). This result was further confirmed by treating patient-derived GBM cell line RN1 with PENAO (Figure

3.12 D).

Figure 3.12 Blockade of mitochondrial function inhibited oxygen consumption and induced an increase in acid production in GBM cells.

U87 cells were incubated with increasing concentrations of PENAO and (A) oxygen consumption rate (OCR) and (B) extracellular acidification rate (ECAR) were measured five times over 35 min. (C) The average change in OCR and ECAR in U87 cells and (D) in RN1 cells as a function of PENAO concentration. 4 µM and 1 µM PENAO significantly inhibited the OCR (p<0.0001) whereas increased the ECAR (p<0.0001) in U87 and RN1 cells, respectively. The line plots are the representatives of three independent experiments. The data points and error bars are expressed as mean ± SD for triplicate measurements. Statistical significance was determined with ANOVA corrected by Dunn’s test, comparing drug-treated samples with untreated control. ***p- value<0.001, ****p-value<0.0001.

3.4 Discussion

In this chapter, the mitochondria of GBM cells were targeted using the small synthetic organo-arsenical, PENAO, which demonstrated potent anti-tumour activity across multiple GBM cell lines including immortalised GBM cell lines and patient-derived

GBM cell lines. Compared to non-cancerous cell lines, GBM cells were at least 2-fold more susceptible to mitochondrial perturbation by PENAO, which confirmed previous hypothesis that arsenic-based compounds selectively targeted highly proliferative cancer cells [262, 263]. It is well known that prolonged tissue culturing can alter cellular genetic and morphological characteristics, and as a result, immortalised cancer cell lines normally do not accurately reflect the key features of human tumour. From this point of view, patient-derived GBM cells may represent a more valuable model for basic and therapeutic researches. In contrast to immortalised GBM cell lines, the IC50 values of

PENAO for patient-derived GBM cell lines are relative lower, indicating PENAO is likely to be more effective in the clinical setting. A recent study that has examined the metabolic status of patient-derived GBM cells indicates these cells preferentially rely on mitochondrial oxidative phosphorylation for glucose metabolism [242]. This finding further supports our in vitro results that patient-derived GBM cells are more sensitive to the inhibition of mitochondrial oxidative phosphorylation as opposed to immortalised

GBM cells that rely on aerobic glycolysis to generate the energy needed for cellular processes.

Low micro-molar concentrations of PENAO induced proliferation arrest in GBM cells, which was further confirmed with cell cycle analysis showing PENAO treatment arrested GBM cells at the G2/M phase, the most radiosensitive phase [275, 276], and

100 decreased the cell proportion in S phase of the cell cycle, the most radioresistant phase

[275, 276]. These findings suggest that PENAO treatment may have the potential in sensitising GBM cells to ionising irradiation. Due to this finding, PENAO in combination with irradiation is being tested in our group. The invasion of glioma cells into normal brain tissue is a key aspect of brain tumour pathology and the main cause of mortality in patients with GBM [277]. As a potential candidate for GBM treatment, inhibition of invasion is of potential clinical significant given the invasive nature of this disease. We therefore tested the anti-invasive effect of PENAO and found the invasion of GBM cells was attenuated by low concentrations of PENAO in a dose-dependent manner. Accumulating evidence demonstrates that arsenic compound, such as arsenic trioxide, inhibits invasion/migration in cancer cells in vitro by activating the ROS- dependent matrix metalloproteinase-2 (MMP) pathway [278]. Although the MMP expression was not examined after PENAO treatment, our data confirmed that the invasion of GBM cells could be effectively hindered by PENAO treatment modulating

ROS production. An increasing number of evidence demonstrates that the invasiveness of glioma cells could be enhanced by current therapeutic modalities, including anti- angiogenic therapy [112, 279] and radiotherapy [280, 281]. In this regard, the invasiveness of glioma cells induced by anti-angiogenic or radiation therapy may be blocked by combining PENAO or other arsenic compounds.

The pathways that control apoptosis are altered in GBM cells leading to resistance towards apoptotic stimuli in general [282]. By increasing the concentration of PENAO and treatment time, apoptosis of GBM cells was noted. It is well documented that apoptotic cell death could be induced by arsenic compounds in several cancer types, including human leukaemia [283], pancreatic [284], and colorectal cancer cells [285]. 101

Herein, we proved apoptotic pathway in GBM cells was activated by PENAO treatment.

Like most of the chemotherapeutics, PENAO triggered apoptotic pathway via intrinsic pathway (mitochondrial-mediated apoptotic pathway). This finding further reaffirms that GBM cells are sensitive to reactive oxygen species (ROS)-inducing agent due to its low level of GSH/GSSH buffer and high level of ROS [252]. Taken together, all of the effects observed above (anti-proliferative, anti-invasive, and apoptotic effects) correlated with levels of PENAO that induced the elevation of ROS, the active molecules that participate in relaying cellular signals [286]. In cancer cells, increased generation of ROS often overwhelms the antioxidant systems, leading to oxidative stress. Different levels of oxidative stress appear to confer different outcomes in cancer cells: mild oxidative stress at sub-lethal levels triggers activation of cell signalling mechanisms such as increased proliferation, migration and invasion, whereas high oxidative stress can lead to cell death [287, 288]. To validate that ROS played a role in the cytotoxicity of PENAO, I demonstrated that blocking PENAO-mediated production of ROS with exogenously applied small thiols impeded the anti-proliferative effect of

PENAO, further confirming that ROS-mediated the anti-proliferative effect of this compound. Another distinctive event of the early stages of apoptosis which occurred following ROS elevation is the loss of mitochondrial trans-membrane potential [286].

Thus, I then demonstrated that the mitochondria in GBM cells were depolarised by

PENAO treatment dose-dependently and in a timely manner following the elevation of mitochondrial superoxide. These results further verified PENAO triggered mitochondria-mediated (intrinsic) apoptosis pathway. Metabolically, inhibition of mitochondrial functions by PENAO decreased oxygen consumption of GBM cells, indicating PENAO treatment led to uncoupling of oxidative phosphorylation. However, treating GBM cells with PENAO not only resulted in a significant decrease in oxygen

102 consumption, but also induced acid production, signalling an impaired mitochondrial respiration could be compensated by the intact glycolytic pathway to balance and maintain the bioenergetic metabolism. This finding was in agreement with a recent report stating that GBM cells were capable of tolerating inhibition of glycolysis or oxidative phosphorylation, and only combinatorial inhibition of both pathways would result in a substantial depletion of intracellular ATP [289]. It has been well known that proliferating cancer cells preferentially utilise aerobic glycolysis to support growth, a metabolic alteration commonly referred to as the ‘Warburg effect’[290]. GBM, like most cancers, also presents this unique metabolic state to utilise aerobic glycolysis as the primary supplier of ATP [121]. Therefore, it may not be an ideal therapeutic strategy if forcing GBM cells to aerobic glycolysis by targeting the mitochondria. In this regard, combining PENAO with a glycolytic inhibitor may enhance the tumour cell killing by further disturbing the homeostasis of bioenergetic metabolism in GBM cells.

In conclusion, in this chapter, I presented the pre-clinical testing of PENAO, a potential drug for GBM treatment by targeting mitochondrial metabolism. Compared to non- cancerous cells, GBM cells showed high sensitivity to the pharmacological inhibition of mitochondrial function. Although mitochondrial oxidative phosphorylation may be an

Achilles heel for this tumour type, it should never be overlooked that cancer cells are capable of using multiple pathways to produce energy, which renders them resistant to therapies if only targeting individual metabolic pathways.

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4 CHAPTER 4: Dual-Targeting of Glucose Metabolism

in GBM cells In Vitro

4.1 Introduction

As discussed in the previous chapter, proliferating cancer cells preferentially use aerobic glycolysis to support their growth, a metabolic alteration commonly referred to as the

‘Warburg effect’ [114]. However, the finding of a recent elegant study that measured the metabolic flux using 13C-labelled nutrients in patient-derived xenograft mouse models ruled out the possibility that tumour metabolism was confined to aerobic glycolysis during aggressive growth in vivo, particularly in GBMs with diffuse infiltration, which ensures access to the nutrient- and oxygen-rich environment of the brain. [242]. These results supported that the mitochondrial oxidative phosphorylation could also be used by infiltrative GBM cells for the disposition of glucose carbon. Since cancer cells possess a striking ability of rewiring their circuitry to maintain flux to critical downstream signalling nodes [291], similar plasticity may apply to their metabolic circuitry. It has been proposed that tumour cells could utilise either glycolysis or oxidative phosphorylation if one or the other pathway is blocked therapeutically

[243]. This hypothesis was confirmed by a study demonstrating that ATP was mainly generated by both aerobic glycolysis and oxidative phosphorylation and that inhibition of glycolysis by the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) could be compensated through increased oxidative phosphorylation, and vice versa [289]. In this regard, future studies are needed to better understand how cancer cells balance

104 difference pathways of bioenergetic metabolism. Moreover, therapeutics with dual- blockade of aerobic glycolysis and oxidative phosphorylation would theoretically be more efficient in depriving GBM cells of the demanded energy.

4.1.1 Dual-targeting of glycolysis and mitochondrial oxidative phosphorylation in

cancer cells as a promising therapeutic strategy

That both aerobic glycolysis and oxidative phosphorylation play key roles in the metabolism of cancer cells has led to new research focus–drugs that dually inhibit both pathways [292, 293]. Several agents including 2-DG, 3-bromopyruvate (3-BP) or dichloroacetate (DCA) inhibit glycolysis and have been reported to be effective anticancer agents in vitro and in vivo [294, 295]. This approach unfortunately has yielded few positive results in human clinical trials. One of the most frequently used anti- glycolytic agents is 2-DG, which is phosphorylated by hexokinase (HK) and subsequently inhibits ATP generation via the glycolytic pathway [296, 297]. However, the high concentration of 2-DG required for glycolytic inhibition (> 20 mM) would inevitably induce intolerable hypoglycaemia [298]. Hence, the efficacy of 2-DG is limited by its systemic toxicity in patients undergoing clinical trials for glioma treatment [299]. DCA, a pyruvate dehydrogenase kinase (PDK) inhibitor that blocks the glycolytic pathway, has been found to have anti-tumour properties by inducing cell- cycle arrest and apoptosis in small-cell lung cancer [170], prostate [300], colorectal cancer [301], breast cancer , and head and neck squamous cell carcinoma models [302].

In a small cohort study, although the effective therapeutic dose of DCA was tolerated by patients with GBM, a dose-dependent peripheral neuropathy was still observed, which restricted its maximum effect upon dose escalation [128].

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A recent strategy has shown improved sensitisation of tumour cells to glycolysis inhibition by the combination of glycolytic inhibitors and mitochondrial toxins [303,

304]. Investigators have trialled rhodamine-123, rotenone, antimycin and oligomycin, which all inhibited oxidative phosphorylation in combination with glycolytic inhibitors

[305]. The rationale behind this approach is that compromised oxidative phosphorylation in tumour cells leads to stimulation of glycolysis for the maintenance of ATP generation. Studies have demonstrated that cells with defective mitochondrial electron transport chain (ETC) are more susceptible to DCA [173]. Derived from this strategy, mitochondria-targeted drugs (Mito-CP and Mito-Q) were employed to synergise with the glycolytic inhibitor 2-DG to trigger apoptotic cell death of breast and hepatocellular cancers in vitro [306, 307]. Furthermore, arsenic trioxide in combination with DCA has been found to an effective anti-cancer strategy by strongly suppressing c-

Myc and HIF-1α in breast cancer cells [308]. Taken together, simultaneous targeting of glycolysis and mitochondrial oxidative phosphorylation has been proposed as an attractive experimental chemotherapeutic strategy to eradicate immortalised cancer cells by maximally disturbing their bioenergetic metabolism.

4.2 Hypothesis and aims of this chapter

Working hypothesis of this chapter:

In chapter 3, I demonstrated that PENAO induced proliferation arrest, impeded invasion and triggered apoptotic cell death in GBM cells. The mechanism of action was via inhibition of mitochondrial respiration (oxidative phosphorylation). However, an increase in acid production (glycolysis) was also observed post-PENAO treatment, indicating an alternative source of energy production had been sought by the cells. I

106 herein hypothesise that the use of dual-agents targeting both glycolysis and oxidative phosphorylation would be more effective in cancer cell eradication. In this chapter, the in vitro efficacy of DCA as a monotherapy and in combination with PENAO will be assessed.

The specific aims of this chapter are:

1. To test the efficacy of DCA on a panel of GBM cells and calculate the half

maximal inhibitory concentrations (IC50);

2. To determine if the combination of PENAO and DCA is more effective than

PENAO or DCA monotherapy. Cell viability, shift in cell cycle distribution,

apoptosis, mitochondrial superoxide level and mitochondrial trans-membrane

potential will be measured;

3. To measure the oxygen consumption rate (OCR) and extracellular acidification

rate (ECAR) in GBM cells treated with PENAO-DCA combination.

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4.3 Results

4.3.1 The effect of DCA as a single agent on GBM cells in vitro

Immortalised GBM cell lines (U87 and U251), patient-derived GBM cell lines (RN1 and HW1) and non-cancerous cell lines (lung fibroblast cell line MRC5 and normal human astrocytes (NHA)) were treated with DCA at a dose range (5-100 mM). The efficacy of DCA for tumour cells was tested under either normoxic (21% O2) or hypoxic condition (1% O2). The reason for testing in both conditions assumed glycolytic inhibition would be more efficacious under hypoxia compared to normoxia.

The IC50 of DCA was determined for each cell line (Figure 4.1) and summarised in

Table 4.1. After 72 hr of exposure to DCA in normoxia, impeded cell proliferation was observed in all of the cell lines (Figure 4.1 A-D). The anti-proliferative effect of DCA was modest. The IC50 values of DCA for U87 and U251 cells were 40.6 ± 6.2 mM and

29.3 ± 5.7 mM respectively (Table 4.1). When treated in hypoxic condition (1% O2), the

IC50 values of DCA for U87 and U251 cells were slightly lower(36.5 ± 4.6 mM and

24.3 ± 2.4mM respectively) (Table 4.1). Thus, DCA is effective but only at suprapharmacological concentrations rather than clinical relevant concentrations (0.5-1 mM). Similar IC50 values were also observed in patient-derived GBM cell lines under normoxia and hypoxia, further confirming the modest efficacy of DCA on GBM cells in vitro (Table 4.1). To compare the sensitivity to DCA between GBM cells and non- cancerous cells, MRC-5 and NHA were treated with DCA. The IC50 values of DCA for non-cancerous cells were 1.2-2.7 fold higher than those for GBM cells (Table 4.1).

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Figure 4.1 Response of GBM cells to DCA treatment.

Representative dose-response curves showing the cell proliferation of immortalised GBM cell lines (A, B)and patient-derived GBM cell lines (C, D) after 72 hr DCA treatment under either normoxic (21% O2) or hypoxic (1% O2) conditions. Cell proliferation was measured using MTS assay. The data points and error bars are expressed as mean ± SD for triplicate measurements.

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Table 4.1 Summary of half maximal inhibitory concentrations (IC50) of DCA under different conditions.

DCA, IC (mM) Cell type Cell lines 50 Normoxia(21% O2) Hypoxia (1% O2) Immortalised GBM cell U87 40.6 ± 6.2 36.5 ± 4.6 lines U251 29.3 ± 5.7 24.3 ± 2.4 Patient-derived GBM RN1 53.4 ± 4.6 62.5 ± 2.5 cells HW1 49.2 ± 9.3 30.6 ± 7.1 Non-cancerous cell MRC5 80.1 ± 5.5 N.A. lines NHA 66.7 ± 6.4 N.A.

GBM cell lines, patient-derived GBM cells and non-cancerous cell lines were treated with DCA for 72 hr. Cell proliferation was measured with MTS assay. IC50 values were derived from the dose-response curves. Values are expressed as mean ± SD of three independent experiments. N.A., not available.

4.3.2 Synergistic anti-proliferative effect of PENAO and DCA on GBM cells

To evaluate the anti-proliferative effect of the combined therapy, combinations of

PENAO and DCA were tested and cell proliferation was measured with MTS assay.

Anti-proliferative interaction between PENAO and DCA was evaluated using the median-effect analysis method generated by Chou and Talalay [245] and processed by the computer program CalcuSyn 2.1 (Biosoft®). The Chou-Talalay method for assessing drug combination is based on the median-effect equation derived from the mass-action law principle, which is the unified theory that provides the common link between single entity and multiple entities [245]. The resulting combination index (CI) theorem of

Chou-Talalay offers quantitative definition for additivism (CI = 1), synergism (CI < 1), and antagonism (CI > 1) in drug combinations [245]. Using the CI-isobologram calculation, a synergistic effect between PENAO and DCA over a range of concentrations (0.625-0.875×IC50) was observed for U87 cells (CI=0.67±0.09) (Figure

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4.2 A and B) while additive effects were observed for U251 (CI=1.00 ± 0.15), RN1

(CI=1.04 ± 0.17) and HW1 (CI= 1.09 ± 0.11) cells (Figure 4.2 A).

Figure 4.2 PENAO and DCA additively/synergistically induced proliferation arrest in GBM cell lines.

The interaction between PENAO and DCA was quantified by the combination index (CI). (A) PENAO-DCA combination induced synergistic proliferation arrest (CI<1) in U87 cells and additive proliferation arrest (CI≈1) in U251, RN1 and HW1 cells over a range of concentrations (0.625-0.875×IC50). (B) Representative dose-response curve of U87 cells treated with PENAO, DCA or the combination. The final dosage was a combination of the indicated IC50 of each agent, e.g. 0.5×IC50 was 0.5×IC50 of PENAO ® combined with 0.5×IC50 of DCA. The CI was calculated using Calcusyn 2.1 (Biosoft ). CI<1, CI=1 and CI>1 are defined as synergistic, additive and antagonistic effects, respectively. Data shown are expressed as mean ± SD of three independent experiments.

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To evaluate the selective proliferation arrest of cancer cells by PENAO-DCA combination, proliferation of GBM cells and non-cancerous cells were measured after

72 hr treatment of PENAO (5 µM) in combination with DCA (10 mM) (Figure 4.3). No apparent proliferation arrest from the combined treatment was observed in the non- cancerous cells, in contrast to GBM cells where significant cell proliferation arrest was observed (>90% of the cell population). To further investigate the synergistic mechanism of PENAO-DCA combination, U87 cells were used for subsequent experiments.

Figure 4.3 Different sensitivities to PENAO-DCA combination of GBM cells and non-cancerous cells.

Immortalised GBM cell lines (U87 and U251), patient-derived GBM cell lines (RN1 and HW1) and non-cancerous cell lines (MRC5 and normal human astrocytes (NHA)) were treated with PENAO (5 µM) in combination with DCA (10 mM) for 72 hr and cell proliferation was determined using MTS assay. Data shown are representative of three independent experiments and expressed as mean ± SD for triplicate measurements.

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4.3.3 PENAO and DCA synergised to induce cell cycle arrest of GBM cells at the

G2/M phase

In section 4.3.2, PENAO synergised with DCA to induce proliferation arrest in U87 cells. The effect of the combined treatment on cell cycle distribution in U87 cells was then conducted. Compared to the untreated control (15% of cells in G2/M phase), cells treated with either PENAO (4.5 µM) or DCA (25 mM) exhibited a small but significant increase in the proportion of cells residing in the G2/M phase of the cell cycle (23% and

22% respectively) (p<0.01) (Figure 4.4 A). A combination of both PENAO (4.5 µM) and DCA (25 mM) significantly affected the cell cycle dynamics with 37% cells in the

G2/M phase (p<0.001) (Figure 4.4 A, B). Coinciding with the G2/M arrest, the percentage population of cells in the S phase significantly decreased from 30% in the untreated control to 15% in the combination-treated group (p<0.01) (Figure 4.4 A). No significant shift in cell cycle was observed in the G1 phase (Figure 4.4 A).

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Figure 4.4 Cell cycle distribution of U87 cells was shifted by PENAO-DCA combination treatment.

(A) PENAO in combination with DCA significantly induced U87 cell cycle redistribution towards the G2/M phase and reduced cell proportion in the S phase. (B) Representative histogram overlays from flow cytometry show increased U87 cell proportion in the G2/M phase after treatment with PENAO-DCA combination. Data shown are expressed as mean ± SD of three independent experiments. Statistical significance values were determined with ANOVA corrected by Dunn’s test, comparing drug-treated samples with untreated control. **p-value<0.01, ***p-value<0.001.

4.3.4 Combination of PENAO and DCA led to increased apoptosis

I earlier demonstrated apoptotic cell death was triggered in GBM cells treated with

PENAO (see section 3.3.4). To analyse whether the PENAO-DCA combination could further increase the rate of apoptosis in U87 cells compared with single agent treatment, cells were stained with Annexin V and acquired by flow cytometry. As a monotherapy,

DCA did not induce apoptosis in U87 cells at concentrations of up to 50 mM (Figure

4.5 A). However, treating cells with the combination (PENAO 5 µM +DCA 10 mM) led to a doubling in the proportion of apoptotic cells (12-15%) compared to the treatment with PENAO alone (5-7%) (p<0.01) (Figure 4.5 B). This enhanced apoptotic cell death was further confirmed by western blotting to detect the level of poly (ADP-ribose) 114 polymerase cleavage (c-PARP), and the pattern was in agreement with the Annexin V staining (Figure 4.5 C).

Figure 4.5 DCA enhanced PENAO-induced apoptosis in U87 cells.

(A) The proportion of apoptotic U87 cells induced by DCA as a single agent. The proportion of apoptotic cells (B) and poly (ADP-ribose) polymerase cleavage (c-PARP) expression (C) of U87 cells induced by PENAO (5 µM) was further increased by addition of DCA (10 mM). Data shown are expressed as mean ± SD of three independent experiments (A, B) and representative of three independent experiments (C). Statistical significance values were measured using the Student’s t-test. *p- value<0.05, **p-value<0.01.

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4.3.5 PENAO in combination with DCA increased mitochondrial superoxide

production and loss of mitochondrial trans-membrane potential

In chapter 3, I demonstrated that PENAO triggered apoptosis via inducing oxidative stress (mitochondrial superoxide production) and depolarising mitochondria (loss of mitochondrial trans-membrane potential in GBM cells. In addition, the mode of action of DCA is also linked with enhanced oxidative activity as well as loss of mitochondrial trans-membrane potential [128, 170, 301]. To investigate whether the combination of

PENAO and DCA triggered apoptosis by the same mechanism, U87 cells were treated with PENAO (5 µM) alone, DCA (10 mM) alone and the combination of PENAO (5

µM) and DCA (10 mM) followed by mitochondrial superoxide detection and mitochondrial trans-membrane potential measurements. After 16 hr treatment, PENAO alone induced a marked increase in mitochondrial superoxide production (p<0.01)

(Figure 4.6 A). In contrast, no observable elevation of superoxide was detected when the cells were treated with DCA alone (Figure 4.6 A). However, the combined treatment significantly increased mitochondrial superoxide production (p<0.01), up to 1.25-fold more than that measured with PENAO alone (Figure 4.6 A). Moreover, measurements of the mitochondrial trans-membrane potential revealed that compared to PENAO alone

(6% of cells with depolarised mitochondria), treatment with PENAO-DCA combination significantly increased the proportion of cells with depolarised mitochondria to 15%

(p<0.001) (Figure 4.6 B).

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Figure 4.6 DCA enhanced the apoptotic activity of PENAO by elevating the production of mitochondrial superoxide and boosting the depolarisation of mitochondria.

(A) U87 cells were treated with PENAO (5 µM), DCA (10 mM) and a combination of both agents for 16 hr followed by MitoSOX Red staining to detect mitochondrial superoxide levels. DCA (10 mM) enhanced PENAO-induced mitochondrial superoxide production in U87 cells. (B) Same treatment groups of U87 cells were stained by JC-1 to measure the mitochondrial trans-membrane potential. DCA (10 mM) enhanced PENAO-induced mitochondrial depolarisation in U87 cells. Data shown are expressed as mean ± SD of three independent experiments. Statistical significance values were measured using the Student’s t-test. *p-value<0.05, **p-value<0.01, ****p- value<0.0001.

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4.3.6 PENAO in combination with DCA inhibited oxygen consumption and acid

production in GBM cells simultaneously

As mentioned previously (see section 3.3.6), PENAO has a direct inhibitory effect on

OCR, an indicator of mitochondrial glucose oxidation. Simultaneously, ECAR, an indicator of glycolysis, was elevated as compensation to balance and maintain the energy generation. Recognising that treatment with PENAO was not sufficiently competent to shut down the energy supply to GBM cells thoroughly, I then asked if concurrent inhibition of glycolysis would be an ideal combination for PENAO to further disturb the bioenergetic metabolism of GBM cells. To test this hypothesis, I assessed the anti-metabolic effect of PENAO-DCA combination on U87 cells and RN1 cells in vitro. The OCR and ECAR were simultaneously measured after 24 hr PENAO or/and

DCA treatments. As shown in Figure 4.7, treatment of U87 cells with PENAO (5 µM) alone resulted in a significant decrease in OCR (p<0.05) and a slight increase in ECAR levels. In contrast, DCA treatment (10 mM) prompted a slight increase in OCR and a significant decrease in ECAR levels (p<0.01) (Figure 4.7 A, B, C). The combination treatment triggered a significant and sharp decrease in both OCR (Figure 4.7 A, C) and

ECAR (Figure 4.7 B, C) compared to the untreated control (p<0.01). This result was further confirmed by treating patient-derived GBM cell line RN1 with PENAO (Figure

4.7 D).

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Figure 4.7 Combination treatment of PENAO and DCA suppressed both oxygen consumption and acid production in GBM cells.

U87 cells were incubated with PENAO (5 µM), DCA (10 mM) or the combination of PENAO (5 µM) and DCA (10 mM) for 24 hr followed by measurements of oxygen consumption rate (OCR) (A) and extracellular acidification rate (ECAR) (B) over 40 min. (C) Summary of mean changes in OCR and ECAR from (A) and (B). Same experiment was conducted by treating patient-derived GBM cells RN1 with PENAO (2 µM), DCA (10mM) or the combination of PENAO (2 µM) and DCA (10 mM) (D). Data shown are representative of three independent experiments and expressed as mean ± SD for triplicate measurements. Statistical significance values were measured using the Student’s t-test. *p-value <0.05, **p-value <0.01, ***p-value <0.001.

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4.4 Discussion

Herein, I show that combining the mitochondrial toxin PENAO with the glycolytic inhibitor DCA engendered significant synergism in U87 tumour cell killing. Using in vitro system, I demonstrated (i) PENAO acted together with DCA on U87 cells to induce cell cycle arrest in the G2/M phase while decreasing cell proportions in the S phase, boost mitochondrial superoxide production, depolarise the mitochondria and induce apoptosis; (ii) DCA shifted the glucose metabolism of U87 cells away from glycolysis to oxidative phosphorylation; and (iii) metabolically, oxidative phosphorylation and glycolysis were simultaneously blocked by this dual-targeting strategy.

In general terms, it is thought that cancer cells reorganise the metabolic steps in the process of glucose utilisation and energy production [309]. A classic biochemical adaptation of cancer cells is the metabolic shift from mitochondrial oxidative phosphorylation to aerobic glycolysis regardless of oxygen availability, a phenomenon termed the ‘Warburg Effect’ [114]. The highly glycolytic metabolic status of GBM makes it an attractive target of anti-glycolytic therapy. Several agents including 2-DG,

3-BP, lonidamine, and DCA that inhibit glycolytic metabolism have been used as effective anti-cancer agents in both in vitro and in vivo models [294, 295]. Nevertheless, the outcome of this approach in human clinical trials has not been promising. The efficacy of 2-DG is limited by its systemic toxicity in patients undergoing clinical trials for glioma treatment [299]. Another glycolytic inhibitor lonidamine has completed phase III trial, however, its clinical success has so far been impaired by significant pancreatic and hepatic toxicities [310]. Michelakis et al. have recently studied the potency of DCA to reverse cancer-specific metabolic and reactivate hyperpolarised

120 mitochondria in GBM. They first studied 49 freshly excised GBM tumour tissue samples and compared them to normal brain tissues obtained during epilepsy surgery using functional confocal microscopy. They found GBM tumours had increased mitochondrial membrane potential compared to normal brain tissue. A small cohort of

GBM patients (n=5) then were administered with DCA at a dose of 6.25 mg/kg orally twice a day, which led to clinically stable at 18 months of DCA therapy in four of five patients. These evidence indicates DCA treatment rapidly reversed hyperpolarised mitochondrial membrane potential and induced apoptosis and increased mitochondrial reactive oxygen species in GBM by inhibiting HIF-1α, promoting p53 activation and suppressing angiogenesis [128]. Several glycolytic inhibitors are currently being tested in clinical trials based on these pre-clinical data, however it is worth noting that recent studies of human orthotopic gliomas in mice and human gliomas in situ suggested the

‘Warburg Effect’ may not necessarily be the case in brain tumours, where the mitochondrial oxidative phosphorylation appeared to be more indispensable for their glucose metabolism and chemoresistance [242, 311]. Although it still remains unresolved as to whether cancer cells in the brain preferentially catabolise glucose via aerobic glycolysis or mitochondrial oxidative phosphorylation, it has been proposed that tumour cells can utilise either bioenergetic pathway due to the plasticity in their metabolic circuit, particularly when one or the other pathway is blocked pharmacologically [243, 289].

Emerging research has focused on drugs that inhibit both pathways concurrently [306,

307]. PENAO in conjunction with DCA exerted a synergistic or additive cytotoxicity on

GBM cells and spared toxicity on non-cancerous cells. The mode of cytotoxic action of

PENAO is correlated with enhanced mitochondrial production of superoxide, loss of

121 mitochondrial trans-membrane potential and inhibition of oxidative phosphorylation, all of which can lead to the mitochondria-mediated apoptotic cell death. The general principles of combining DCA with PENAO are supported by previous literatures which have demonstrated that cells with mitochondrial defects displayed higher sensitivity to the cytostatic effects of DCA [173, 312].

DCA is a pyruvate mimetic compound, which stimulates mitochondrial function by inhibiting PDKs. Inhibition of PDKs by DCA reactivates PDH and allows pyruvate to be oxidised in the mitochondria. It has been reported that PDK2 is the most sensitive

(apparent Ki values≈ 0.2mM) and PDK3 the most resistant (apparent Ki values≈8mM), while PDK1 and PDK4 are relatively sensitive to DCA inhibition [313]. DCA has been reported to have cytotoxic effects in vitro [170, 173, 301], with some responses at clinically relevant concentrations (0.5~1 mM), while others require suprapharmacologic levels (10–100 mM) and still other groups have found no direct toxicity in vitro [302,

314, 315]. Our in vitro data showed that the IC50 values of DCA as monotherapy for immortalised GBM cell lines and patient-derived GBM cell lines were at suprapharmacological levels (25-60 mM), which is consistent with part of previous reports. This finding supports the idea that clinically relevant concentrations of DCA

(less than 1 mM) are not directly cytotoxic in vitro. The reason for this apparent cellular resistance in vitro is not an inactivation of DCA under tissue culture conditions [316]or an inability to inactivate PDKs, because 0.2~8 mM is adequate to inhibit all of the

PDKs according to the apparent Ki values of DCA [313]. Therefore, the basis for the limited anticancer effect of DCA in culture likely lies in the complex cellular physiology and the enormous excess of metabolites present in culture media. However, when a low concentration of DCA (10 mM) was combined with PENAO, the cytotoxic

122 effect of PENAO was enhanced, indicating DCA could sensitise GBM cells to PENAO treatment. 10 mM was selected as testing dosage because this level of DCA is capable of inhibiting all the PDKs to maximally block the glycolysis of tumour cells. My results are also supported by another study, which reported that DCA induced a small but significant increase in caspase 3/7 activity without activating apoptosis [317].

In addition, I showed that low concentrations of DCA worked synergistically with

PENAO and significant increases in apoptotic cells compared to either treatment alone were measured. Increased G2/M cell cycle arrest, elevated mitochondrial superoxide production and mitochondrial depolarisation were also observed when GBM cells were treated with the combination compared to either drug alone. Most importantly, this dual therapy resulted in significant metabolic disturbance of the bioenergetic homeostasis in

GBM cells by inhibiting both oxidative phosphorylation and glycolysis, which further confirmed the underlying mechanism of PENAO-DCA combination that maximally blocked the bioenergetic metabolism of cancer cells. Interestingly, a recent study reported DCA treatment led to an alteration in the multidrug resistance protein (MDR) phenotype of tumour cells (inhibition of multidrug resistant protein 1, MRP1), thereby enhancing the effectiveness of cisplain in a Dalton's lymphoma mouse model [318].

MRP1/2 is known to blunt the effect of PENAO by exporting it from cytosol [241].

This finding also indicates another underlying mechanism of synergy achieved by the combination of PENAO and DCA that the efficacy of PENAO could be enhanced through the inhibition of MRP1 expression by DCA treatment. Notably, recent findings have indicated that a metabolic shift to glycolysis occurred in GBM cells during anti- angiogenic therapy with bevacizumab, which was thought to be associated with resistance to anti-angiogenic therapy and enhanced tumour cell invasion [112]. Most

123 importantly, thus far there is no effective treatment for recurrent GBM patients who progress following bevacizumab treatment. Reversal of the bevacizumab-induced shift in glucose metabolism using DCA has been shown to effectively inhibit the neoplastic growth of GBM in vivo [319]. Therefore, adjuvant therapy with drugs targeting both glycolytic and mitochondrial glucose metabolism could be more beneficial in the anti- angiogenic therapy for GBM. Taken together, the in vitro data in this chapter provide the proof of concept that dual-targeting of glucose metabolism might serve as a novel therapy for GBM.

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5 CHAPTER 5: Targeting Glucose Metabolism in GBM

In Vivo

5.1 Introduction

5.1.1 GBM rodent models

A number of animal models have been developed, involving the subcutaneous or intracranial implantation of human GBM cell lines into rodents to test novel therapeutic agents that target different processes and pathways of human GBM such as angiogenesis [279] and invasion [320]. Athymic mice injected with human U251 and

U87 GBM xenografts are the most commonly used animal models of GBM to test therapeutic approaches [279, 320, 321]. Both models recapitulate some features of the human GBM, such as tumour formation, necrosis, or angiogenesis. The U87 model has been proved useful for studying GBM angiogenesis and evaluating the efficacy of anti- angiogenic therapeutics [322-324]. The advantages of these glioma models are attributed to their highly efficient tumourigenesis, reproducible growth rates, and an accurate knowledge of the location of tumour engraftment [325]. Although athymic glioma mouse models have been used in pre-clinical glioma research for over 30 years, their use remains controversial, particularly for their lack of infiltration [326].

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5.1.1.1 U251 GBM xenograft model

The U251 cell line was originally obtained from a 75-year-old male with GBM and established as an immortalised cell line by Ponten and colleagues [327]. It is known to mimic the salient features of human GBM and as such has received significant attention over the past decades in xenograft mouse models [269]. The U251 cell line has been used in both subcutaneous and intracranial models in immunodeficient mice [328].

However, differences in gene expression profile between subcutaneous and intracranial tumour models have been reported [328], noting the importance of different in vivo growth conditions and the role of the microenvironment in U251 xenografts. At the histopathological level, U251 tumours show a diffusely infiltrative pattern of invasion into normal brain parenchyma, significant foci of palisading necrosis, a tortuous pattern of microvascular proliferation, hypertrophic endothelium, cellular pleomorphism, cellular atypia, mitotic figures and foci of oedema and haemorrhage [269].

Immunohistochemical analysis of U251 tumours from orthotopic xenografts shows striking similarities to human GBM, with neoplastic cells positive for glial fibrilliary acid protein (GFAP) [269]. Tumour cells show atypically high level of proliferation with over 50% of tumour cell nuclei staining positive for Ki-67. The U251 xenograft model also displays similarities to human GBM at the genetic level, with presence of non-functional mutant tumour suppressor p53 as well as mutant PTEN [5, 9, 35, 269,

329, 330].

In summary, U251 tumour recapitulates many prominent histological and immunohistochemical features of human GBM. In addition, it carries a number of genetic alterations with similarities to human GBM, including alterations in the key tumour suppressors and oncogenic pathways. Furthermore, magnetic resonance imaging 126

(MRI) features of the U251 mouse model correlate with human GBM, including a poorly demarcated, infiltrative tumour border on the T2-weighted images and a necrotic centre with an intense rim observed on post-contrast T1-weighted images [269].

5.1.1.2 U87 GBM xenograft model

The U87 cell line was originally established with tumour cells from a female GBM patient [327]. Histologically, U87 tumours are highly cellular with atypia including mitotic figures and irregular nucleoli, and profuse neovascularisation [331-333]. Unlike the U251 model and human GBM, these tumours show minimal parenchymal infiltration with a well-demarcated tumour mass surrounded by reactive astrocytes [269].

Additionally, U87 tumour vasculature exhibits significantly more homogeneous and leaky vessels, which provides greater access for systemic drugs [334].

Immunohistochemical analysis of U87 tumours from orthotopic xenografts displays negative stain for GFAP and over 40% positive nuclei stained for Ki-67. Compared to

U251 cells and human GBM at the genetic level, U87 cells carry wild-type tumour suppressor p53 [269], PTEN mutant [5, 9], and show an overexpression members of the

PI3K/Akt pathway as a result of high Akt expression [35, 335]. Notably, although the disparities between U87 and U251 were observed when grown as monolayer in vitro or as subcutaneous xenografts in vivo, both cell lines displayed similar gene expression patterns when they were grown intracranially [328], indicating an in vivo growth environment induced a set of modifications in gene expression profiles.

In summary, U87 shows less similarity to human GBM when compared to U251, and as such, caution must be taken when extrapolating conclusions from studies using the U87 127 cell line. U87 model lacks a key feature of human GBM, the diffusely invasive infiltration of tumour cells into normal brain parenchyma, which significantly contributes to the resistance of this tumour type to chemoradiotherapy and high recurrence rate [330]. MRI features of the U87 orthotopic mouse model show a homogeneous and enhanced, well-demarcated tumour nodule on the T2-weighted imaging, which does not correlate well with human GBM [269]. Overall, this tumour model has been used to test anti-angiogenic therapies, but limitations include the aforementioned dissimilarities to human GBM and the usual concerns with xenograft models.

Due to incomplete recapitulation of key pathological/genetic features of human GBM, these models bearing immortalised GBM cell lines have made it difficult to predict the outcome of novel therapies. Even though novel therapies have been demonstrated effective to reduce the tumour mass in these GBM models, the translation of these treatments, from the pre-clinical model to the patients, is extremely low. Therefore, there is a compelling need for more accurate and reproducible intracranial tumour models, which recapitulate key features of the human GBM, particularly the extremely invasive nature of this cancer.

5.1.1.3 Patient-derived orthotopic GBM xenograft models

The tumour microenvironment critically affects the biological behaviours of xenograft tumours (Charles et al., 2011; Langley and Fidler, 2011; Lathia et al., 2011). Orthotopic xenografts bearing patient-derived GBM cells have been suggested to recapitulate the biology of the disease more precisely compared to immortalised GBM cell lines (Lee et 128 al., 2006; Xie et al., 2008). In particular, transplanting patient-derived GBM cells into immunodeficient mice yielded tumours sharing similar histology and global gene expression patterns with their parental tumours [336]. Patient-derived tumours exhibit a highly invasive nature, as opposed to subcutaneous or intracranial tumours with immortalised GBM cell lines [326]. These translational models bearing patient-derived

GBM cells were further evaluated functionally, which provided evidence that these pre- clinical models recapitulated the biology of human GBM in situ [336].

5.2 Hypothesis and aims of this chapter

Working hypothesis of this chapter:

In chapter 4, I demonstrated that PENAO synergised with DCA to inhibit the growth of

GBM cells in vitro via dual-targeting of their glucose metabolism. To further evaluate this combination, in this chapter I will test the hypothesis that PENAO in combination with DCA will inhibit tumour growth more efficaciously than either drug alone using in vivo models. When beginning this work, Dr McDonald’s laboratory had no animal models established. As part of my body of work, I established the orthotopic models that had been used for multiple experiments. This model will also be discussed.

The specific aims of this chapter are:

1. To fully establish an orthotopic mouse model of GBM (intracranial xenograft)

using an immortalised GBM cell line and a patient-derived primary GBM cell

line and to characterise their individual features in situ;

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2. To test the efficacy of PENAO and DCA as single agents on heterotopic

(subcutaneous implantation) mouse model of GBM.

3. To test the maximum tolerated dose (MTD) of PENAO in combination with

DCA using tumour-free mice;

4. To determine the efficacy of PENAO, DCA and their combinations using an

orthotopic mouse model of GBM.

5.3 Results

5.3.1 Discrepant characteristics between GBM cell line (U87) and patient-derived

primary GBM cells (RN1) in orthotopic mouse models

To characterise immortalised GBM cell line and patient-derived GBM cell line in situ, orthotopic mouse models bearing U87 and RN1 cells were developed. BALB/c nude mice and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice received injections of 1×105 U87 cells and 2×105 RN1 cells in the right caudate putamen, respectively (selections of mouse strains and cell numbers referred to relevant literatures [279, 336, 337]). Mice were euthanised when symptoms of neurological decline associated with tumour burden (difficulties in eating and drinking, losing >20% of their body weight or having trouble ambulating) were evident. Consistent with previous reports [338], U87 cells formed a well-demarcated tumour mass barely with noticeable parenchymal infiltration (Figure 5.1 A, B). In contrast to U87, RN1 formed a tumour resembling what is typically observed in the patient, whereby the GBM was infiltrative (Figure 5.1 C, D). The median survival time of BALB/c nude mice bearing

U87 tumours was 30 days after intracranial inoculation with tumour cells compared to a median survival time of 74 days for mice bearing RN1 tumours (Figure 5.2).

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Figure 5.1 Hematoxylin and eosin (H&E) stain of U87 and RN1 tumours from orthotopic xenografts.

Histological H&E staining of excised tumours is presented. b, normal brain; n, necrotic area; t, tumour. (A) U87 tumour appears spherical (darker staining regions circled by blue dotted line) and shows a clearly demarcated tumour profile (30 days after tumour inoculation). (B) Closer view of U87 tumour shows distinct boundaries (indicated with arrow) and very little infiltration into adjacent normal brain tissue. (C) RN1, a patient- derived primary GBM tumour, shows a highly scattered and invasive phenotype with irregular borders (darker staining areas) (73 days after tumour inoculation) (D) Closer view of RN1 reveals that tumour cells infiltrate into the brain parenchyma with no clear brain-tumour boundary. Note: Experiment performed in conjunction with Sylvia Chung and Hayley Franklin. Intracranial injection, animal monitoring and sample collection were performed by Han Shen and Sylvia Chung. H&E staining was performed by Hayley Franklin.

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Figure 5.2 Survival times for mice bearing intracranial U87 or RN1 tumours.

Kaplan-Meier survival curves were plotted for athymic BALB/c nude mice bearing U87 cells (n=6) and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice bearing RN1 cells (n=5). The median survival time was 30 days and 74 days for U87 and RN1 models, respectively (95% confidence interval). Note: Experiment performed in conjunction with Sylvia Chung. Intracranial injection, animal monitoring and sample collection were performed by Han Shen and Sylvia Chung.

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5.3.2 PENAO and DCA inhibited heterotopic U87 tumour growth as single

agents

The effect of PENAO was tested as a monotherapy in a heterotopic U87 model

(subcutaneous implantation). Approximately 7-9 weeks old female BALB/c nude mice were injected with 3×106 U87 cells in 100 µL PBS subcutaneously in the proximal midline. Tumours were given approximately 2 weeks to establish and grow to 50-70 mm3 in size, after which the mice were randomised into groups of 8-9. Because of its short half-life, PENAO needed to be administered continuously. The mice were implanted with micro-osmotic pumps (Model 2002) (ALZET Corporation, Palo Alto,

CA) subcutaneously in the flank, which delivered vehicle control (saline), 1 mg/kg/day or 3 mg/kg/day of PENAO (dose selection was determined by previous experiments conducted by Sylvia Chung). To determine the drug efficacy, tumour volume

(calculated as ‘ ’) and animal

weight were measured every 3-5 days. Significant reductions in tumour volume were observed after 14 days in mice treated with both 1 and 3 mg/kg/day PENAO compared to the vehicle control (Fig 5.3A).

DCA (50, 100 or 150 mg/kg/day) or distilled water (vehicle) was administered to mice by intragastic gavage for 17 days (dose and route determined by previous studies [319,

339]). Tumour-bearing mice treated with 50 mg/kg/day DCA demonstrated significant reductions in tumour volume at day 17 compared to the vehicle control. Interestingly, tumour suppression did not improve with increased DCA dosage (Figure 5.3 B).

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Figure 5.3 Anti-tumour activities of PENAO and DCA as single agents on subcutaneous U87 xenografts.

(A) PENAO inhibited the growth of subcutaneous U87 tumours significantly and dose- dependently. (B) Three tested dosages of DCA (50, 100, 150 mg/kg/day) significantly hindered the growth of subcutaneous U87 tumours to a similar extent without a dose- dependent manner. The results are plotted using the relative tumour volume (RTV) as a function of treatment duration. The data are presented as the mean ± SD of 8-9 tumours. The RTV growth curves were compared using two-way repeated measures ANOVA. **p-value<0.01, ****p-value<0.0001. Note: Data in graph (A) were generated from experiment performed by Stephanie Decollogne and Sylvia Chung; Data in graph (B) were generated from experiment performed in conjunction with Stephanie Decollogne. The preparation of cell suspension was performed by Han Shen, Sylvia Chung; tumour cell injection and pump implantation were performed by Stephanie Decollogne, Han Shen and Sylvia Chung; DCA gavage was performed by Han Shen; tumour volume measurements were conducted by Stephanie Decollogne and Han Shen.

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Consistent with the reduced tumour growth, immunohistochemistry (IHC) staining of the excised tumours showed a significant reduction in the global proliferation marker

(Ki-67 index) in 3 mg/kg/day PENAO (Figure 5.4 A, C) and 150 mg/kg/day DCA

(Figure 5.4 B, D) treated groups in contrast to the vehicle control groups.

Figure 5.4 Analysis of the proliferation index (Ki-67 index) in subcutaneous U87 tumour samples.

Tumour cell proliferation index was determined by counting Ki-67 positive cells using Image-Pro Plus software in ten 40x magnification fields. The index of proliferation (Ki- 67 index) was high in the vehicle-treated control samples but significantly lower in the tumour samples treated with PENAO (A, C) and DCA (B, D). The data points and error bars are expressed as mean ± SD for ten measurements. Statistical analysis for significance between the vehicle and drug treated tumour sections was performed using Mann-Whitney test. **p-value<0.01, ***p-values<0.001. Note: Experiment was performed in conjunction with Sylvia Chung, Fei Shang and Kerrie McDonald. Samples were collected by Han Shen, Sylvia Chung; IHC staining was performed by Fei Shang; data collection and analysis were performed by Kerrie McDonald and Han Shen.

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5.3.3 PENAO-DCA combination was well-tolerated by BALB/c nude mice at the

maximum effective doses of each individual drugs

Since the ultimate goal of this project is to evaluate the efficacy of PENAO-DCA combination which has never been tested on BALB/c nude mice, the MTD was tested on tumour-free animals treated with the combination with their maximum effective doses as single agents from section 5.3.2. Experimentally, combinations of PENAO with DCA at various doses (see Table 5.1) were administered for 28 days. The mice were monitored at least twice a week for their body weights and clinical signs of adverse reactions. 24 hr after the last dose, the mice were sacrificed and the major organs (liver, spleen and kidneys) were collected for weight and macroscopic examination. In addition, the harvested organs were also examined histologically. In all treatment groups with PENAO-DCA combinations, no significant indication of toxicity was observed when compared to the vehicle control group. No loss of body weight

(Figure 5.5) or abnormality in the macroscopic/histological examinations was present.

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Table 5.1 Combinations of PENAO with DCA for testing maximum tolerated dose (MTD).

Number of mice

DCA (mg/kg/day) 0 50 100 150

PENAO (mg/kg/day) 0 4 - - - 1 - 4 4 4 3 - 4 4 4

Figure 5.5 Body weight curves of tumour-free BALB/c nude mice administered with PENAO-DCA combinations over 28 days.

Female BALB/c nude mice were administered with PENAO-DCA combination or vehicle control. No weight loss was observed over 28 days. Note: Experiment was performed in conjunction with Stephanie Decollogne. The preparation of tumour cell suspension was performed by Han Shen; tumour cell injection was performed by Staphanie Decollogne and Han Shen; osmotic pump implantation was performed by Stephanie Decollogne and Han Shen; Animals were monitored by Han Shen.

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5.3.4 Effect of the combination therapy on orthotopic U87 model

To assess the effectiveness of PENAO in combination with DCA in an orthotopic setting, U87 cells were again used in preference to the patient-derived GBM cell line,

RN1. Although the U87 model lacks the diffuse infiltration feature, it was still chosen as our drug-testing model for the following reasons: (i) this model has highly efficient tumourigenicity; (ii) it offers reproducible growth rates and an accurate location of the tumour at the injection site; (iii) synergistic effect was observed when using PENAO-

DCA combination in our in vitro model of U87 cells (chapter 4). Specifically, mice were injected with 5×104 U87 cells (day 0). The tested drugs or control vehicle treatment were administered from day 3 to day 28. No toxicity was noted in the treatment arms. PENAO alone (3 mg/kg/day) did not prolong the median survival time but a significant survival benefit was observed in mice treated with DCA alone (150 mg/kg/day) compared to the control-treated arm (p<0.05) (Figure 5.6 A). Unfortunately, no synergistic survival benefit was noted in the PENAO-DCA combination arm compared to each drug alone (Figure 5.6 A). To identify the reason why PENAO alone or in combination with DCA failed to demonstrate any survival advantage, arsenic levels in the mouse brain were measured. Arsenic is a surrogate indicator for PENAO levels as there are only traces of the metalloid in untreated animals. Both of the plasma and the brain were collected when the animals were sacrificed and the level of arsenic was measured by mass spectrometry. The analysis of PENAO distribution in the plasma and brain of the mice revealed that the level of arsenic compound in the tumour was significantly higher than in the adjacent healthy brain tissue (Figure 5.6 B) and in the plasma (Figure 5.6 C). Specifically, the level of PENAO in the tumour reached 1.5-2

µM, whereas the remaining part of the right hemisphere and the left healthy hemisphere only retained approximately 1 µM and 0.5 µM respectively (Figure 5.6 B). This finding

138 indicated PENAO successfully crossed the blood-brain-barrier and accumulated in the tumour. Intriguingly, the plasma level of PENAO in the PENAO-DCA combination treated group was slightly higher than that in the group treated with PENAO alone

(Figure 5.6 C), suggesting that DCA might prevent the clearance of PENAO from the systemic circulation.

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Figure 5.6 Effect of PENAO-DCA combination treatment on orthotopic U87 model and biodistribution of PENAO in the brain and plasma.

Kaplan-Meier survival analysis of orthotopic U87 model treated with vehicle control, PENAO (3 mg/kg/day), DCA (150 mg/kg/day) or the combination of PENAO (3 mg/kg/day) and DCA (150 mg/kg/day). Brain (B) and plasma (C) levels of PENAO were measured after 28 days of continuous administration. PENAO was delivered via a subcutaneous micro-osmotic pump and DCA was administered by intragastric gavage. The data points and error bars are expressed as mean ± SD for 3-5 measurements. Kaplan-Meier survival curves were compared using the Log-rank (Mantel-Cox) test.*p- value<0.05. The arsenic levels determination in the brain and plasma were analysed by Rabeya Akter from the Inductively Coupled Plasma ICP Elemental Analysis Laboratory, UNSW Australia.

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5.4 Discussion

Traditionally, pre-clinical cancer biology largely relies on the use of human cancer cell lines in vitro and xenograft tumour models derived from these cell lines. However, the process of establishing conventional GBM cell lines results in irreversible loss of important biological properties of human tumours. As a result, the xenograft tumour models do not maintain the genomic and phenotypic characteristics present in the original tumour (Martens et al., 2008; Sausville and Burger, 2006; Taillandier et al.,

2003). More importantly, the heterogeneity of GBM is compromised (Bonavia et al.,

2011; Lee et al., 2006; Verhaak et al., 2010). It has been suggested that in vitro and in vivo pre-clinical models using patient-derived GBM cells recapitulate the biology of the disease more precisely (Lee et al., 2006; Xie et al., 2008). In this chapter, I first characterised and compared two orthotopic GBM models bearing either immortalised

GBM cell line U87 or patient-derived GBM cell line RN1. Supporting the literature, significant histological and biological discrepancies were observed. RN1 displayed a highly invasive nature and recapitulated the biology of the disease more precisely than

U87 in the intracranial cavity. Moreover, the tumourigenic efficiency of U87 (median survival time 30 days) was much higher than that of RN1 (median survival time 74 days). This finding is consistent with previous studies reporting that tumour graft latency, measured as the time between implantation and the development of a progressively growing xenograft tumour can range from two to twelve months or even longer [336, 340]. The inefficient tumour initiation of implanted tumours suggest that these patient-derived tumour cells struggle to engraft for reasons that might include a dependence on hematopoietic cells and/or micro-environmental cues not present in mouse stroma or not compatible with human cells [341]. The relative long term growth

141 rate also compromises the practicality of patient-derived xenograft model to be used as a fast tool of drug screening, particularly in a time-limited PhD project.

As described in chapter 4, PENAO in combination with DCA elicited proliferation arrest as well as apoptotic cell death synergistically in U87 cells in vitro by dual- targeting of the glucose metabolism. To further evaluate the efficacy of this combination, the two compounds were first tested individually on a heterotopic GBM model bearing U87 tumours. Both PENAO and DCA significantly delayed the tumour growth when acting alone compared to the vehicle control. These findings were also confirmed by IHC stain showing the proliferation index (Ki-67) was significantly lower in the drug-treated tumours compared to the control group. Unlike the dose-dependent tumour growth inhibition induced by PENAO, DCA hindered U87 tumour growth without any significant difference among the three tested doses (50, 75, 150 mg/kg/day).

It was previously reported that chronic administration of DCA at 30-50 mg/kg/day decreased the lactate levels by 60% and directly suppressed the activity of pyruvate dehydrogenase kinase (PDK) by 3-6 fold [342, 343]. Together with our reported in vivo efficacy of DCA, it may suggest that DCA produce the anti-tumour activity at a dose lower than 50 mg/kg/day, and the dose-independent growth inhibition was simply due to reaching the plateau of inhibition of PDK activity. It has been well documented that once DCA enters the circulation after intragastric administration, it initiates the stimulation of pyruvate dehydrogenase (PDH) activity by inhibiting PDK rapidly within minutes [344]. The half-life of DCA after the first administered dose is less than one hour, but it increases up to several hours with subsequent doses [345]. However, there is a plateau of this effect and the serum level of DCA does not continue to rise with chronic use. Specifically, the serum level of DCA after 5 years of continued oral

142 administration at 25 mg/kg/day is only slightly higher than the level after the first several doses [346]. Interestingly, the reduction on lactate level persists after the DCA serum level decreases, as DCA in part ‘locks’ PDK in a sustained inactive state such that the inhibition of PDK cannot be immediately reversed [345]. Additionally, the anti- tumour activity of DCA in vivo also appeared to be a paradox, as its modest in vitro anti-tumour effect could only be observed at suprapharmacological concentrations (e.g. dosage of 50-100 mg/kg/mouse/day translates into approximately 6.25-12.5 mg/kg/day in humans, which only gives a serum DCA trough level of 0.5-1 mM [128, 319], which is far lower than our derived IC50 value and barely triggered any proliferation arrest of

U87 cells in vitro). The majority of the data support the idea that clinically relevant concentrations of DCA (less than 1 mM) are only able to inactivate PDK (the apparent

Ki values of DCA to inactivate PDK2 is approximately 0.2 mM) rather than directly inducing any cytostatic/cytotoxic effect in vitro. The huge disparity of DCA efficacy between in vitro and in vivo models was in part attributed to its anti-angiogenic activity, which could only be thoroughly presented on in vivo models [174]. It was also thought that the limited in vitro anti-tumour efficacy of DCA possibly resided in the complex cellular physiology due to the enormous excess of metabolites presented in the in vitro culture media [316].

All of my in vitro work in chapter 4 indicated the combination of PENAO and DCA would result in significant survival benefit. Unfortunately, no synergistic tumour control was observed in vivo. This is most likely because PENAO did not reach the levels needed in the brain to work synergistically with DCA and this was confirmed with our post-mortem testing of arsenic in the brain. The resulting PENAO level in the tumour was 2-4 fold higher than those in the healthy brain tissue and the plasma, which could

143 be due to its inherited capability of accumulating within highly proliferating cells similar to GSAO [208]. However, the final drug concentration in the tumour was only

1.5-2 µM, which was far below the IC50 value of PENAO for U87 growth inhibition in vitro (4.5 ± 0.7 µM). This may in part explain the failure of PENAO as a single agent to extend the median survival time of the U87 orthotopic model. Regarding the combination, the level of PENAO was also well below the lowest required concentration (approximately 0.625×IC50 (3 µM), Figure 4.2) to synergise with DCA in vitro. Therefore, similar to the scenario of PENAO alone, the combination also failed to elicit synergistic survival benefit. Additionally, a recent study found DCA administration altered the MDR phenotype of tumour cells by down-regulating the expression of multidrug resistance protein 1 (MRP1), thereby enhancing the anti-tumour efficacy of cisplatin in the mouse model of lymphoma [318]. Although MRP is known to blunt the efficacy of PENAO by exporting it from tumour cells [241], the level of

PENAO in the tumour treated with PENAO-DCA combination was not significant higher than its level in the PENAO single agent arm. Therefore, further investigation is still needed to unravel the relationship between DCA treatment and MDR phenotypes in

GBM cells.

Unfortunately, to resolve the problem, it is not a matter of simply increasing the dose of

PENAO in the mouse model. The administration of PENAO at 3 mg/kg/day led to a local inflammatory response at the delivery site of the osmotic pump due to the high drug concentrate, while this dosage of PENAO was still far below its MTD (40 mg/kg/day) when administered as a single intraperitoneal injection daily over two weeks (data not shown). Additionally, dose escalation of PENAO was restricted by its maximum solubility when high concentrate of PENAO was loaded into the micro-pump,

144 which was proven as the most efficient route of delivery pharmacokinetically. In this regard, future studies will focus on enhancing the efficacy of PENAO by either inhibiting the drug efflux pumps of the MRP [241], or combining glutathione (GSH) depletion using buthionine sulphoximine (BSO) [241], to maximise its anti-tumour activity in the central nervous system without inducing apparent adverse effects.

Currently, the patient-derived xenograft model harbouring RN1 cells is being optimised to further test this combination. Due to relative long latency after tumour inoculation, the starting time of PENAO treatment, duration of PENAO delivery via osmotic pump and using in vivo imaging (MRI) to evaluate the tumour response to drug treatment all need to be optimised. Although long term growth rate of patient-derived xenograft would be of high cost of agistment and treatment as well as time consuming, as a better pre-clinical model of GBM that more accurately recapitulates the biology of human

GBM in situ, pre-clinical results from it would maximally mirror the response of patients to the tested treatment, thereby being translated to clinical trials more efficiently. This combination will be ultimately tested in the patient-derived GBM model in the near future.

In conclusion, PENAO and DCA significantly inhibited tumour growth as single drugs when applied on a heterotopic GBM xenograft model, but no synergism was observed when the orthotopic U87 model was used. The efficacy of PENAO alone and in combination with other therapeutic agents needs further investigation in a more translational orthotopic xenograft model bearing a more sensitive patient-derived GBM cell line.

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6 CHAPTER 6: Summary and Future Directions

The treatment of GBM has been a formidable challenge as the prognosis of these patients is dismal, despite aggressive surgery, radiotherapy and chemotherapy. The majority of GBM patients either do not have well response to chemotherapeutic regimens or quickly progress on standard therapy and develop resistance [347].

Therefore, it is necessary to fully understand the biology of malignant gliomas in order to significantly improve patient survival. In recent years, the understanding of the regulation of tumour metabolism has significantly improved. An increasing number of evidence show that tumour cells reprogram their metabolism to meet high energy demands, coordinate markedly elevated biosynthetic processes and energy production, which in turn promote rapid growth and division of tumour cells [118, 348-350]. In addition, elevated glycolysis [351, 352], enhanced glutaminolysis [242, 353], and exacerbated lipogenesis [354] have been demonstrated as prominent characteristics in

GBM. Thus, targeting metabolism has become a novel promising strategy for treating cancers. The objective of my thesis was to identify alternative therapeutic approaches for GBM and investigate the efficacy of targeting aberrant glucose metabolism of GBM in vitro and in vivo.

The enhanced aerobic glycolysis, or the Warburg effect, is a wide spread phenomenon that has been identified in over 90% of all tumour forms. Cells that exhibit the Warburg effect take on alternative routes of energy homeostasis to maintain their rapid

146 proliferation [141]. Nobel laureate Otto Warburg stated that cancer cells rely on glycolysis or substrate phosphorylation to generate ATP, and suppress their mitochondrial activities [290]. Recent studies using advanced technologies have confirmed the Warburg’s hypothesis on the aspect of ATP production yet revealed that mitochondrial activity is not suppressed in cancer cells. On the contrary, mitochondria play important roles in providing substrates to maintain the proliferation of cancer cells

[154].

The anti-cancer effect of inhibiting aerobic glycolysis (reversing the Warburg effect) has been described recently and an old compound dichloroacetate (DCA) that has been used for treatment of lactic acidosis over 40 years, has been tested as a repurposing drug and demonstrated moderate anti-cancer activity both in vitro [300, 301, 355] and in vivo

[317-319]. As a pyruvate dehydrogenase kinase (PDK) inhibitor, DCA results in increased pyruvate dehydrogenase (PDH) activity [19], which leads to the increased conversion of pyruvate to acetyl-CoA rather than lactic acid, and stimulates mitochondrial respiration by increasing the supply of acetyl-CoA. Consequently, cancer cells treated with DCA showed increased levels of ROS, depolarisation of the mitochondrial trans-membrane potential and increased apoptosis [17, 20]. As DCA can redirect substrates into mitochondrial respiration and ATP production, it could have a synergistic activity with anti-cancer drugs that impair mitochondrial activity. Therefore, my work focused on studying the pharmacological effect of PENAO, a novel arsenic- based small molecule with mitochondria-targeting activity, alone and in combination with DCA.

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The overarching aim of my thesis was to prove that the dual-targeting of glucose metabolism in GBM cells would be an efficacious therapeutic to delay tumour growth.

Specifically, I proposed that (i) by acting on the mitochondria in GBM cells, PENAO would uncouple the oxidative phosphorylation, increase oxidative stress via inducing superoxide production, and depolarise mitochondrial trans-membrane potential, which would in turn inhibit proliferation and invasion of GBM cells and trigger apoptotic cell death; and (ii) by combining the mitochondrial toxin PENAO with the glycolytic inhibitor DCA, the dual target therapy could block both oxidative phosphorylation and glycolysis simultaneously and induce proliferation arrest and mitochondria-mediated apoptotic cell death in GBM cells synergistically. To test these hypotheses, a series of in vitro and in vivo experiments were conducted to evaluate the effects of PENAO,

DCA and their combination.

PENAO was designed and synthesised as a metabolic inhibitor primarily targeting the adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane of rapidly proliferating tumour cells [233, 241]. It has been proved as an effective anticancer agent in several cancer models both in vitro and in vivo [241]. In my thesis, I first investigated the anti-tumour efficacy and the mechanism of action of PENAO in a panel of GBM cells in vitro. As we expected, PENAO demonstrated substantial anti- tumour activity in GBM cells including immortalised GBM cell lines and patient- derived GBM cell lines. Specifically, the IC50 values of PENAO in GBM cells ranged from 0.7-4.5 µM while its IC50 values in non-cancerous cells lied within 7-10 µM, indicating the selectivity of PENAO in targeting cancer cells. The anti-proliferative effect of PENAO was further confirmed by cell cycle analysis, suggesting PENAO treatment arrested GBM cells at G2/M phase, the most radiosensitive phase [275]. This 148 finding suggests that PENAO treatment may have the potential in sensitising GBM cells to ionising irradiation, the standard-of-care for GBM patients [22]. Furthermore, low micro-molar concentrations of PENAO impeded the invasiveness of GBM cells, another key propensity of this deadly disease limits the effectiveness of surgery as well as radiotherapy [356]. It has been reported that arsenic compound, such as arsenic trioxide, inhibits invasion/migration in cancer cells in vitro by activating the ROS-dependent matrix metalloproteinase-2 (MMP) pathway [278]. Although the MMP expression was not examined after PENAO treatment, my results confirmed that the invasion of GBM cells could be effectively hindered by PENAO treatment modulating ROS production.

There is accumulating evidence that current therapeutic modalities, including anti- angiogenic therapy [112, 279] and radiotherapy [280, 281], can enhance glioma invasiveness. Therefore, the invasiveness of glioma cells induced by anti-angiogenic or radiation therapy may be blocked by combining PENAO or other arsenic compounds.

Future work would be useful to unravel the underlying mechanism of anti-invasive effect of PENAO by investigating the MMP expression, alteration of extracellular matrix in 3D culture model and Rho signalling pathway.

In addition to the proliferation arrest and inhibition of invasiveness triggered by

PENAO, another important observation is apoptotic cell death was activated in GBM cells after PENAO treatment. Killing of tumour cells by most anticancer strategies currently used in clinical oncology, for example, chemotherapy, suicide gene therapy or immunotherapy, has been linked to activation of apoptotic pathways in cancer cells such as the intrinsic and/or extrinsic pathway [357]. PENAO treatment activated the apoptotic cell death, as per our data, through the intrinsic apoptotic pathway in mitochondria. Further mechanistic studies showed by acting on the mitochondria of 149

GBM cells, PENAO induced oxidative stress (elevation of mitochondrial superoxide level) and depolarised mitochondria, which led to mitochondria-mediated apoptosis. A recent study examining freshly isolated GBM specimen from 49 patients noticed the mitochondrial trans-membrane potential was increased (mitochondrial hyperpolarisation) compared to normal brain tissues [128]. This finding implies GBM cells are more vulnerable to mitochondrial depolarisation than normal astrocytes. Our data, through showing the selectivity of PENAO treatment, reaffirms the observation above. In saying that, the findings outlined should not be confined to in vitro testing only, but should be examined in an in vivo model to identify the mitochondrial trans-membrane potential change after arsenic compound treatment. In addition, accumulating evidence suggest that apoptosis is insufficient to account for the therapeutic effect of radiotherapy whereas mitotic catastrophe is the most important cell death triggered by irradiation.

From this aspect, future investigation would be useful to evaluate the combination of irradiation and PENAO, as by doing so both mitotic catastrophe and apoptotic cell death would be induced to eliminate GBM cells.

As a candidate for cancer treatment, PENAO and its previous generation GSAO are being tested in phase I dose escalation studies in patients with solid tumours refractory to standard therapy. The main aim of these trials is to work out the safest dose of

GSAO/PENAO to give to patients with advanced solid tumours. The primary outcomes of the trial is to define the recommended phase II dose of PENAO as a 21 day continuous intravenous infusion and the secondary outcomes is to identify the toxicity profile of GSAO/PENAO. To date, treatment with GSAO has not shown significant side effects in 31 patients (NCT01147029). The trial for PENAO is still at the stage of recruitment and patients with CNS malignancy have been included. 150

Moving from the cytostatic/cytotoxic assays, we assessed the efficacy of PENAO at the metabolic level. Treatment with PENAO led to an uncoupling of oxidative phosphorylation, which further confirmed its mitochondria-targeting property. This is also consistent with previous report that arsenic is recognised as an uncoupler of mitochondrial oxidative phosphorylation [259]. In addition to the uncoupling of oxidative phosphorylation, an increase in acid production (glycolysis) in GBM cells treated with PENAO was also observed, which indicated the impaired mitochondrial respiration in GBM cells could be compensated by the intact glycolytic pathway to balance and maintain their bioenergetic metabolism. This finding inspired me to combine PENAO with a glycolytic inhibitor to maximally disturb the homeostasis of bioenergetic metabolism in GBM cells. As a matter of fact, several studies have tested this strategy to elicit synergistic cell killing by dually blocking glucose metabolism of various cancer cells, such as breast cancer cells [306] and hepatocellular carcinoma

[307]. DCA, a glycolytic inhibitor, was selected to be combined with PENAO, because

I proposed that by reversing the glycolytic phenotype with DCA and directing more pyruvate into mitochondrial oxidative phosphorylation, simultaneous targeting of the mitochondria with PENAO would be more efficacious than PENAO alone. This hypothesis was validated by the results in chapter 4, demonstrating DCA enhanced

PENAO-induced cytotoxicity in GBM cells selectively while sparing non-cancerous cells. These results are also supported by another study, which reported that DCA induced a small but significant increase in caspase 3/7 activity without activating apoptosis [317]. Mechanistically, the synergistic effect was achieved as both agents acted by increasing mitochondrial superoxide production and depolarising mitochondrial trans-membrane potential, which in turn triggered apoptotic cell death.

More importantly, this dual-targeting combinatorial strategy led to severe metabolic

151 disturbance of the bioenergetic homeostasis in GBM cells (oxidative phosphorylation and glycolysis were simultaneously blocked by PENAO-DCA combination), which further confirmed the underlying mechanism of PENAO-DCA combination as a potent anti-metabolic therapeutic for GBM. Interestingly, a recent study reported DCA treatment led to an alteration in the multidrug resistance protein (MDR) phenotype of tumour cells by inhibiting the expression of multidrug resistant protein 1 (MRP1), thereby enhancing the effectiveness of cisplain in a Dalton's lymphoma mouse model

[318]. As MRP is known as a protein that blunts the effectiveness of PENAO in tested cancer cells, future work would require confirming the association of DCA with the

MRP expression in GBM cells, and to unravel more underlying mechanism of the interaction between PENAO and DCA.

The discovery process in the development of cancer therapeutics may start with either empiric screening or rational drug design. In either case, animal model systems play an essential role in the critical steps of drug development. Just as drug screening systems and rational drug design have benefited from recent advances in cell culture techniques and molecular biology, the role of animal model systems in novel drug development has also been irreplaceable and accelerating the progress of translational medical research from bench to bedside. In this regard, I examined the response of in vivo GBM models to a dual-targeting therapeutic strategy, PENAO-DCA combination, in chapter 5. Both agents were first tested individually using a heterotopic GBM model (subcutaneous tumour implantation) and promising results were observed. In specific, 14 days of continuous PENAO delivery via a subcutaneous osmotic pump induced significant delay of tumour growth dose-dependently with two tested doses (1 and 3 mg/kg/day) and 17 days of DCA administration via intragastric gavage also significantly impeded

152 tumour growth, independent of dose. The effect of DCA is consistent with a study performed by Duan et al. showing that there were no significant differences among the

DCA treatment groups although three tested doses (25, 75, 125 mg/kg), all significantly inhibited subcutaneous tumour growth [358]. These data indicate DCA might possibly produce the antitumor activity at a dose lower than 25 mg/kg, and the efficacy of DCA on tumour growth inhibition may not be benefitted from dose escalation.

To better mimic the in vivo environment of brain tumour growth and more accurately assess the response of GBM cells to the tested compounds, I further evaluated the effect of PENAO-DCA combination using an orthotopic GBM xenograft bearing U87 cells.

Although the orthotopic model harbouring U87 does not recapitulate the diffused growth and invasiveness of GBM in situ, we still chose it as a strong synergy between

PENAO and DCA was observed when they act on U87 cells in vitro. In contrast to the in vitro results, synergistic tumour growth control was not observed in vivo. This finding led us to analyse the biodistribution of PENAO in mouse brain, as most of the arsenic compounds, not like DCA, are not fully competent in crossing blood-brain- barrier (BBB) efficiently [359]. Further analysis of drug distribution by mass spectrometry revealed the concentrations of arsenic in the brain post-mortem. PENAO crossed the BBB and accumulated in the tumour. However, the synergism between

PENAO and DCA could not occur as the accumulated level of PENAO was too low.

Hence, it can be concluded that the anti-tumour effect of PENAO was restricted due to its insufficiency to reach and maintain at an effective concentration in the brain tumour of the orthotopic U87 model. In a recent study, the orthotopic U87 model was used to investigate the function of the blood-brain-barrier and its restriction of drug delivery to glioma cells [360]. Their findings demonstrate that the BBB is sufficiently intact in

153 areas of brain adjacent to the tumour core to significantly restrict erlotinib delivery.

Inhibition of P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) by the dual inhibitor elacridar dramatically increased erlotinib delivery to the tumour core, rim, and normal brain. These results provide conclusive evidence of the impact that active efflux at the BBB has on the delivery of molecularly targeted therapy to different tumour regions in glioma. The results of this study also support the possibility that the repeated failure of clinical trials of new drugs for gliomas may be in part due to a failure to achieve effective concentrations in the tumour cells that reside behind an intact BBB.

In light of this paper, concurrent administration of a modulator of drug transporters can be used as a strategy to enhance delivery of PENAO and other arsenic compounds to the brain.

Our previous pharmacokinetic study demonstrated PENAO administered as a single intraperitoneal injection (40 mg/kg) to BALB/c nude mice reached 334 μM and 3.6 μM as the peak concentrations in the blood and brain, respectively, and these concentrations were maintained for 10 min in both tissues (data not shown). The half-life of the compound was only 16 min and 24 min in the blood and brain, respectively, which meant chronic continuous delivery of PENAO was the only effective route to maintain the level of PENAO in the plasma and target organs. Currently, 3 mg/kg/day of PENAO is the highest dose that can be delivered via osmotic pumps in our mouse models. The dose escalation of PENAO is limited by its solubility when loaded in pumps and inflammation at the delivery site due to localised high concentration of PENAO.

Therefore, future experiment will be aimed at enhancing the efficacy of PENAO by either blocking the MRP that is known to blunt the effectiveness of PENAO [241] or depleting glutathione (GSH) using buthionine sulphoximine (BSO) [361] to maximise

154 the anti-tumour activity of PENAO. In addition, our in vitro data showed the PENAO-

DCA combination synergistically arrested GBM cells in the G2/M phase (the most radiosensitive phase) whereas decreased the proportion of cells in the S phase (the most radioresistant phase). Thus, combining PENAO-DCA dual therapy with ionising radiation may be proposed as a promising combinatorial therapeutic approach for GBM treatment. Another theoretical basis of combining irradiation with PENAO-DCA therapy is that ionising radiation has been thought to mediate transient permeability of the BBB [362, 363], which would favour the improvement of PENAO efficacy for tumours cell killing in the central nervous system.

Despite the millions of dollars spent on target validation and drug optimisation in pre- clinical models, most therapies still fail in phase III clinical trials. Our current model systems, or the way we interpret data from them, clearly do not have sufficient clinical predictive power. Cell lines provide an unlimited supply of material that is widely available, easy to propagate, thereby forming the basis for relatively high-throughput assays. This makes cell line studies on large numbers of drug combinations quite feasible. A great number of cell lines have now already been exhaustively characterised

[364, 365]. They represent the spectrum of mutations found in cancers, have similar patterns of chromosomal gains and losses, mRNA expression and epigenetic characteristics, and show no evidence of genetic alterations in major driver mutations after long-term in vitro culture. However, current opinion suggests that the failure of translating pre-clinical results to clinical settings is primarily because the cell lines and xenografts that are widely used are inadequate models that do not effectively/accurately mimic and predict the responses of human patients. This has become such a widespread belief that it approaches dogma in the field of drug discovery and optimisation and has 155 led to increasing studies devoted to the development of more sophisticated animal models such as orthotopic patient-derived xenografts in an attempt to obtain more accurate estimates of whether particular cancers will respond to given treatments. As a standard commonly used model, U87 has been criticised as a model due to its dissimilarity to human GBM when compared to patient-derived GBM cells. U87 tumour lacks the diffusely invasive feature of GBM, which significantly contributes to its resistance to chemoradiotherapy and high recurrence rate [330]. However, this model is the gold standard and experiments based on it can be performed and repeated efficiently. To further accurately evaluate the efficacy of this dual-targeting therapy, a better pre-clinical model of GBM harbouring patient-derived cells is urgently needed.

RN1, an in-house established patient-derived GBM xenograft model displays highly invasive nature of the disease and recapitulates the biology of GBM more precisely than

U87 model does, is being optimised in our laboratory at this stage. The invasiveness of

GBM is a key aspect of brain tumour pathology and the main cause of recurrence in patients with GBM after multimodality treatment [366]. The anti-invasive property of

PENAO is promising, and would certainly require validation in the proper in vivo model bearing the patient-derived GBM cells.

Although recent studies have suggested that patient-derived xenograft models might hold great promise for testing novel drug candidates as well as to identify rational combination therapies [367], it is worth noting that the range of mutations have been found narrowed in engrafted tumours compared with their parental cancers and that to capture tumour heterogeneity, several implants of each tumour may be necessary [368,

369]. Additionally, patient-derived xenograft models also own the general limitations of mouse model, such as the species incompatibility with respect to human tumour–mouse 156 microenvironment interactions [370]. The overall usefulness of patient-derived xenograft model may be further hampered by relatively low engraftment rates in comparison with current success rates in growing cells in vitro from primary tumour material [371]. Despite these shortcomings of patient-derived xenograft models, they remain a prerequisite tool with more advantages over conventional mouse model bearing established cell lines for target validation and toxicity studies of in vitro validated compounds.

In conclusion, I explored the dependence of GBM cells on its abnormal glucose metabolic pathways and demonstrated that the PENAO-DCA combination synergistically targeted GBM cells in vitro by perturbing both glycolytic and mitochondrial metabolism. Notably, recent findings showed a metabolic shift to glycolysis occurred in GBM cells during anti-angiogenic therapy with bevacizumab, which was thought to be associated with resistance to anti-angiogenic therapy and enhanced tumour cell invasion [112]. Most importantly, thus far there is no effective treatment for recurrent GBM patients who progress following the failure of bevacizumab-containing treatment. Reversal of the bevacizumab-induced shift in glucose metabolism using DCA has been shown to effectively inhibit the GBM growth in vivo [319]. Therefore, adjuvant therapy with drugs targeting both glycolytic and mitochondrial metabolism could be beneficial in the anti-angiogenic therapy for GBM.

Taken together, although the inhibitory action of PENAO-DCA combination needs further verification in vivo using different paradigms, the in vitro data of this thesis provide the proof of concept that dual-targeting of glucose metabolism is a novel therapy for GBM.

157

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