HIGH-THROUGHPUT SCREENING FOR NOVEL ANTI-CANCER ... · high-throughput screening (HTS) approach....

HIGH-THROUGHPUT SCREENING FOR NOVEL

ANTI-CANCER RADIOSENSITIZERS FOR

HEAD AND NECK CANCER

by

Emma Ito

A thesis submitted in conformity with the requirements for the degree of

Doctor of Philosophy

Graduate Department of Medical Biophysics

University of Toronto

© Copyright by Emma Ito 2010

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High-Throughput Screening for Novel Anti-Cancer Radiosensitizers for Head and Neck

Cancer

Emma Ito

Doctor of Philosophy

Department of Medical Biophysics

University of Toronto

2010

ABSTRACT

Despite advances in therapeutic options for head and neck cancer (HNC), treatment-

associated toxicities and overall clinical outcomes have remained disappointing. Even with

radiation therapy (RT), which remains the primary curative modality for HNC, the most

effective regimens achieve local control rates of 4555%, with disease-free survival rates of

only 3040%. Thus, the development of novel strategies to enhance tumor cell killing, while

minimizing damage to the surrounding normal tissues, is critical for improving cure rates with

RT. Accordingly, we sought to identify novel radiosensitizing therapies for HNC, exploiting a

high-throughput screening (HTS) approach.

Initially, a cell-based phenotype-driven HTS of ~2,000 commercially available natural

products was conducted, utilizing the short-term MTS cell viability assay. Cetrimonium

bromide (CTAB) was identified as a novel anti-cancer agent, exhibiting in vitro and in vivo

efficacy against several HNC models, with minimal effects on normal fibroblasts. Two major

limitations of our findings, however, were that CTAB did not synergize with radiation, nor

was its precise cellular target(s) elucidated.

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Consequently, an alternative strategy was proposed involving a target-driven RNAi-

based HTS. Since the colony formation assay (CFA) is the gold standard for measuring

cellular effects of radiation in vitro, an automated high-throughput colony-formation read-out

was developed as a more appropriate end-point for radiosensitivity. Although successful as a

tool for the discovery of potent anti-cancer cytotoxics, a technical drawback was its limited

dynamic range. Thus, the BrdU incorporation assay, which measures replicative DNA

synthesis and is a viable CFA alternative, was employed. From an RNAi-based screen of

~7000 human genes, uroporphyrinogen decarboxylase (UROD), a key regulator of heme

biosynthesis, was identified as a novel tumor-selective radiosensitizing target against HNC in

vitro and in vivo. Radiosensitization appeared to be mediated via tumor-selective enhancement

of oxidative stress from perturbation of iron homeostasis and increased ROS production.

UROD was significantly over-expressed in HNC patient biopsies, wherein lower pre-RT

UROD levels correlated with improved disease-free survival, suggesting that UROD

expression could also be a potential predictor for radiation response.

Thus, employing a HTS approach, this thesis identified two novel therapeutic strategies

with clinical potential in the management of HNC.

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ACKNOWLEDGEMENTS

First and foremost, I would like to express my sincere gratitude towards my PhD

supervisor, Dr. Fei-Fei Liu, for her mentorship and professional support throughout the last

four years. My academic achievements and growth as an independent researcher would not

have been made possible without her invaluable guidance and encouragement. She will

continue to be a role model in my personal life and career development. I would also like to

thank the members of my supervisory committee, Dr. Aaron Schimmer and Dr. Anne Koch,

for their guidance and integral role in the completion of my PhD degree. Further, I wish to

acknowledge the members of my examination committee for their time and commitment

towards my thesis defense: Dr. Ernest Lam (Chair), Dr. Laurie Ailles (Medical Biophysics

Examiner), Dr. Meredith Irwin (University of Toronto Examiner), and Dr. Martin Gleave

(External Examiner).

Many present and past members of the Liu lab have contributed immensely to this

thesis. In particular, I would like to thank Angela Hui, Inki Kim, Nehad Alajez, Willa Shi,

Winnie Yue, David Katz, Ken Yip, Joe Mocanu, and Carlo Bastianutto for their conceptual

and technical advice. Other colleagues who have provided guidance and support along the way

include Eduardo Moriyama, Ken Lau, Alessandro Datti, Thomas Sun, and Frederick

Vizeacoumar.

Finally and most importantly, I would like to extend a special thanks to my father

(Hiroshi Ito), mother (Sumiko Ito), brother (Ryoma Ito), and best friend (Ryan Lim) for their

love and continued encouragement. They have been with me on every step of this journey and

I will always be grateful for their steadfast support. I dedicate this thesis to them.

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TABLE OF CONTENTS

ABSTRACT… ............................................................................................................................... II

ACKNOWLEDGEMENTS ....................................................................................................... IV TABLE OF CONTENTS ............................................................................................................ V LIST OF TABLES ...................................................................................................................... IX LIST OF FIGURES ..................................................................................................................... X LIST OF ABBREVIATIONS .................................................................................................... XI

CHAPTER 1: INTRODUCTION ................................................................................................ 1

1.1 Radiation Therapy .................................................................................................................. 2

1.1.1 Background ................................................................................................................ 2

1.1.2 Radiation Biology ...................................................................................................... 2

1.1.3 Cellular Response to Radiation ................................................................................. 4

1.1.3.1 DNA Damage Surveillance ......................................................................... 5

1.1.3.2 DNA Damage Cell Cycle Checkpoints ....................................................... 7 1.1.3.3 DNA Repair ............................................................................................... 10 1.1.3.4 Radiation-Induced Cell Death .................................................................. 13

1.2 Modulation of Radiation Response ...................................................................................... 14

1.2.1 Background .............................................................................................................. 14

1.2.2 Chemical Radiosensitizers ....................................................................................... 15

1.2.2.1 Oxygen ...................................................................................................... 17 1.2.2.2 Halogenated Pyrimidines.......................................................................... 20

1.2.2.3 Modifiers of Microtubule Structure and Function .................................... 22 1.2.2.4 Modifiers of the Nature or Repair of DNA Damage ................................. 23 1.2.2.5 Targets of Cell Signaling Pathways .......................................................... 25

1.3 High-Throughput Screens .................................................................................................... 27

1.3.1 Background .............................................................................................................. 27

1.3.2 Phenotype-Based High-Throughput Screens ........................................................... 29

1.3.3 Target-Based High-Throughput Screens ................................................................. 30

1.3.4 RNA Interference Screens ........................................................................................ 31

1.3.5 Radiosensitizer Discovery Screens .......................................................................... 32

1.4 Head and Neck Cancer......................................................................................................... 33

1.4.1 Background .............................................................................................................. 33

1.4.2 Treatment ................................................................................................................. 33

1.4.2.1 Radiation Therapy .................................................................................... 34 1.4.2.2 Chemotherapy ........................................................................................... 35 1.4.2.3 Molecularly-Targeted Agents ................................................................... 36

1.5 Research Objectives ............................................................................................................. 37

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CHAPTER 2: POTENTIAL USE OF CETRIMONIUM BROMIDE AS AN

APOPTOSIS-PROMOTING ANTICANCER AGENT FOR HEAD AND

NECK CANCER ................................................................................................. 40

2.1 Chapter Abstract .................................................................................................................. 41

2.2 Introduction .......................................................................................................................... 41

2.3 Materials and Methods ......................................................................................................... 43

2.3.1 Cell Lines ................................................................................................................. 43

2.3.2 Small Molecules ....................................................................................................... 43

2.3.3 Small-Molecule High-Throughput Screening .......................................................... 44

2.3.4 Cell Viability Assay .................................................................................................. 45

2.3.5 Colony Formation Assay.......................................................................................... 45

2.3.6 Fluorescence Microscopy ........................................................................................ 45

2.3.7 Caspase Activity Assay ............................................................................................ 46

2.3.8 Cell Cycle Analysis .................................................................................................. 46

2.3.9 Transmission Electron Microscopy ......................................................................... 46

2.3.10 Mitochondrial Depolarization, Calcium Content, and Propidium Iodide Uptake .. 47

2.3.11 ATP Synthase Activity Assay .................................................................................... 47

2.3.12 ATP Luminescence Assay ........................................................................................ 47

2.3.13 Plasma and Mitochondrial Membrane Potential Assays......................................... 48

2.3.14 In Vivo Tumor Model ............................................................................................... 48

2.3.15 Tumor Formation Assay .......................................................................................... 49

2.3.16 Therapeutic Tumor Growth Assay ........................................................................... 49

2.3.17 Statistical Analyses .................................................................................................. 50

2.4 Results .................................................................................................................................. 50

2.4.1 High-Throughput Screening .................................................................................... 50

2.4.2 Validation of HTS Hits and Evaluation of Anti-Cancer Specificity ......................... 51

2.4.3 Evaluation of Combination Therapy ........................................................................ 52

2.4.4 Cetrimonium Bromide Induces Apoptosis ............................................................... 54

2.4.5 Cetrimonium Bromide Perturbs Mitochondrial Function ....................................... 58

2.4.6 Role of M in Cetrimonium Bromide-Mediated Cell Death ................................. 60

2.4.7 Elimination of Tumor Formation ............................................................................. 62

2.4.8 Growth Delay in Established Xenograft Tumors ..................................................... 62

2.4.9 In Vivo Safety and Toxicity ...................................................................................... 63

2.4.10 Evaluation of Cetrimonium Bromide Analogues ..................................................... 65

2.5 Discussion ............................................................................................................................ 67

2.6 Acknowledgments................................................................................................................ 72

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CHAPTER 3: INCREASED EFFICIENCY FOR PERFORMING COLONY

FORMATION ASSAYS IN 96-WELL PLATES - NOVEL

APPLICATIONS TO COMBINATION THERAPIES AND HIGH-

THROUGHPUT SCREENING ......................................................................... 73


3.2 Introduction .......................................................................................................................... 74


3.3.1 Cell Lines ................................................................................................................. 76

3.3.2 6-Well Colony Formation Assay .............................................................................. 77

3.3.3 96-Well Colony Formation Assay ............................................................................ 77

3.3.4 High-Throughput Screening .................................................................................... 78

3.4 Results and Discussion ........................................................................................................ 79

3.5 Acknowledgements .............................................................................................................. 88

CHAPTER 4: UROPORPHYRINOGEN DECARBOXYLASE - A NOVEL

RADIOSENSITIZING TARGET FOR HEAD AND NECK CANCER

IDENTIFIED FROM AN RNAI HIGH-THROUGHPUT SCREEN ............ 90


4.2 Introduction .......................................................................................................................... 91


4.3.1 Cell Lines ................................................................................................................. 93

4.3.2 Patient Samples ........................................................................................................ 93

4.3.3 Reagents ................................................................................................................... 94

4.3.4 BrdU-Based siRNA High-Throughput Screen ......................................................... 94

4.3.5 Transfections ............................................................................................................ 95

4.3.6 Flow Cytometric Assays ........................................................................................... 95

4.3.7 γ-H2AX Detection .................................................................................................... 95

4.3.8 Hypoxia Treatment................................................................................................... 96

4.3.9 Iron Histochemistry ................................................................................................. 96

4.3.10 Porphyrin Detection................................................................................................. 96

4.3.11 Quantitative Real-Time PCR ................................................................................... 96

4.3.12 Western Blot Analysis .............................................................................................. 97

4.3.13 Colony Formation Assay.......................................................................................... 97

4.3.14 Cell Viability Assay .................................................................................................. 98

4.3.15 In Vivo Tumor Model ............................................................................................... 98

4.3.16 Tumor Formation Assay .......................................................................................... 98

4.3.17 Therapeutic Tumor Growth Assay ........................................................................... 98

4.3.18 In Vivo Knockdown Validation ................................................................................ 99

4.3.19 Statistical Analyses .................................................................................................. 99

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4.4 Results ................................................................................................................................ 100

4.4.1 High-Throughput Screening for Novel Radiosensitizers ....................................... 100

4.4.2 UROD is a Potent Radiosensitizing Target for HNC ............................................ 101

4.4.3 siUROD-Mediated Radiosensitization Differs from Photodynamic Therapy ........ 104

4.4.4 UROD Down-Regulation Promotes Radiation-Induced Apoptosis ....................... 107

4.4.5 siUROD-Mediated Radiosensitization Increases Cellular Oxidative Stress ......... 109

4.4.6 UROD Knockdown Perturbs Cellular Iron Homeostasis ...................................... 112

4.4.7 siUROD Radiosensitizes HNC Models In Vivo ..................................................... 115

4.4.8 UROD Knockdown Modulates Radiosensitivity of Several Cancer Models ......... 118

4.4.9 Clinical Implications of UROD in HNC ................................................................ 119

4.5 Discussion .......................................................................................................................... 121

4.6 Acknowledgments.............................................................................................................. 125

CHAPTER 5: DISCUSSION ................................................................................................... 126

5.1 Research Summary ............................................................................................................ 127

5.2 Future Directions ............................................................................................................... 128

5.2.1 Empirical to Target-Driven Cancer Drug Discovery ............................................ 128

5.2.2 RNAi in Drug Discovery and Therapeutics ........................................................... 130

5.2.3 Clinical Trials for Molecularly-Targeted Therapies ............................................. 133

5.2.4 Developing UROD as a Therapeutic Radiosensitizing Target .............................. 134

5.3 Conclusions ........................................................................................................................ 136

REFERENCES .......................................................................................................................... 137

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LIST OF TABLES

Table 2.1 HTS of the Spectrum Collection small molecule library for novel HNC cytotoxics .. 51

Table 3.1 Comparison of 96-well and 6-well clonogenic assays................................................. 84 Table 3.2 Confirmed hits in the LOPAC1280 library ................................................................. 88

Table 4.1 Primer sequences for mRNA expression analyses ...................................................... 97

Table 4.2 Top-scoring associated network functions ................................................................ 101 Table 4.3 Top scoring molecular and cellular functions ............................................................ 101

Table 5.1 Comparison of therapeutic modalities ....................................................................... 133

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LIST OF FIGURES

Figure 1.1 Direct and indirect effects of ionizing radiation on DNA ............................................ 4

Figure 1.2 DNA damage recognition pathway .............................................................................. 6 Figure 1.3 Cell cycle checkpoint pathways ................................................................................... 9 Figure 1.4 Double-strand break DNA repair pathways ............................................................... 12 Figure 1.5 Therapeutic ratio of radiosensitizing agents ............................................................... 16 Figure 1.6 High-throughput screening approaches ...................................................................... 28

Figure 2.1 Characterization of CTAB as a potential anti-cancer agent for HNC ........................ 53 Figure 2.2 Cetrimonium bromide induces apoptosis in human HNC cells ................................. 55

Figure 2.3 Evaluation of cetrimonium bromide-mediated apoptosis........................................... 57 Figure 2.4 Cetrimonium bromide induces mitochondrial dysfunction ........................................ 59

Figure 2.5 Role of M in cetrimonium bromide-mediated apoptosis ....................................... 61 Figure 2.6 In vivo efficacy of cetrimonium bromide ................................................................... 64 Figure 2.7 Anti-cancer efficacy of cetrimonium bromide analogues .......................................... 66

Figure 3.1 Schematic representation of the 96-well colony formation assay .............................. 81

Figure 3.2 Reproducibility of a 96-well CFA compared to a traditional 6-well CFA ................. 83 Figure 3.3 Dose response curves created using the 96-well CFA ............................................... 87

Figure 4.1 Identification of UROD as a novel radiosensitizing target ...................................... 103

Figure 4.2 Radiosensitizing effect of UROD knockdown is independent of porphyrin

accumulation ............................................................................................................. 106 Figure 4.3 UROD down-regulation promotes radiation-induced cytotoxicity .......................... 108

Figure 4.4 siUROD-mediated radiosensitization enhances cellular oxidative stress ................ 111 Figure 4.5 UROD knockdown induces intracellular iron accumulation.................................... 114 Figure 4.6 In Vivo efficacy of UROD knockdown plus irradiation in HNC models ................. 117

Figure 4.7 Clinical relevance of UROD in human cancers ....................................................... 120

xi

LIST OF ABBREVIATIONS

M Mitochondrial membrane potential

P Plasma membrane potential

-H2AX Gamma-H2AX

-ray Gamma ray

2D Two-dimensional

3D Three-dimensional

5-FU 5-fluorouracil

53BP1 P53 binding protein 1 60

Co Cobalt-60

AKT V-akt murine thymoma viral oncogene

ALA -aminolevulinic acid hydrochloride

ANOVA Analysis of variance

ASO Anti-sense oligonucleotide

ATM Ataxia telangiectasia mutated

ATP Adenosine-5'-triphosphate

ATPase ATP synthase

ATR Ataxia telangiectasia and Rad3-related

ATRIP ATR interacting protein

BAX BCL2-associated X protein

Bcl-2 B-cell leukemia/lymphoma 2

Br Bromo

BRCA1 Breast cancer 1

BrdU 5-bromo-2-deoxyuridine

Ca2+

Calcium

CCCP Carbonyl cyanide m-chlorophenylhydrazone

CDC25A Cell division cycle 25 homolog A

CDC25C Cell division cycle 25 homolog C

CDK Cyclin-dependent kinase

CFA Colony formation assay

CHK1 Checkpoint kinase 1

CHK2 Checkpoint kinase 2

CI Combination index

Cl Chloro

C-Map Connectivity Map

CM-H2DCFDA 5-(and 6-)chloromethyl-2,7-dichlorodihydrofluorescein diacetate

CML Chronic myelogenous leukemia

CPOX Coproporphyrinogen oxidase

CT Computed-tomography

CTAB Cetrimonium bromide

dATP Deoxyadenosine triphosphate

DDR DNA-damage response

DE Dihydroethidium

DFO Deferoxamine mesylate salt

DFS Disease-free survival

DiBAC4(3) Bis-(1,3-dibutylbarbituric acid)trimethine oxonol

xii

DiIC1(5) 1,1,3,3,3,3-hexamethylindodicarbocyanine

DLC Delocalized lipophilic cation

DMSO Dimethyl sulfoxide

DNA-PKCS DNA-dependent protein kinase catalytic subunit

dNTP Deoxyribonucleotide triphosphate

DSB Double-strand break

dsDNA Double-stranded DNA

dUrd Deoxyuridine

LIG4 DNA ligase IV

EC Effective concentration

EGFR Epidermal growth factor receptor

EM Electromagnetic

ER Endoplasmic reticulum

FdUMP 5-fluoro-2-deoxyuridine monophosphate

FdUrd Fluoro-deoxyuridine

Fe2+

Ferrous iron

Fe3+

Ferric iron

FFPE Formalin-fixed paraffin-embedded

FTI Farnesyltransferase inhibitor

FTMT Mitochondrial ferritin

GPX1 Glutathione peroxidase

Gy Gray unit

h Hours

H2O2 Hydrogen peroxide

HBO Hyperbaric oxygen

HER2 Human epidermal growth factor receptor 2

HIF-1 Hypoxia-inducible transcription factor-1

HNC Head and neck cancer

HP Halogenated pyrimidine

HPV Human papillomavirus

HR Homologous recombination

HRE Hypoxia response element

HTS High-throughput screen

I Iodo

IGFR Insulin-like growth factor receptor

IMRT Intensity-modulated radiation therapy

IP Intraperitoneal

IR Ionizing radiation

IV Intravenous

JC-1 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine

iodide

kDa Kilodalton

kV Kilovolt

LIG4 DNA ligase IV complex

mA Milliamp

MAP Mitogen-activated protein

MDC1 Mediator of DNA-damage checkpoint 1

xiii

MGMT O6-methylguanine DNA-methyltransferase

MOMP Mitochondrial outer membrane permeabilization

MRE11 Meiotic recombination 11

MRN MRE11–RAD50–NBS1

mRNA Messenger RNA

mTOR Mechanistic target of rapamycin

MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium, inner salt

NBS1 Nijmegen breakage syndrome 1 (nibrin)

NCI-DTP National Cancer Institute Developmental Therapeutics Program

NHEJ Non-homologous end joining

NIG Nigericin

NOE Normal oral epithelial

NOP Normal oropharyngeal

NPC Nasopharyngeal cancer

NSCLC Non-small cell lung cancer

OER Oxygen enhancement ratio

OLIG Oligomycin

OXPHOS Oxidative phosphorylation

PARP-1 Poly(ADP-ribose) polymerase-1

PBS Phosphate-buffered saline

PCT Porphyria cutanea tarda

PDT Photodynamic therapy

PFA Paraformaldehyde

PI Propidium iodide

PI3K Phosphoinositide 3 kinase

PIDD P53-induced protein with a death domain

PLK1 Polo-like kinase 1

PPIX Protoporphyrin IX

PPOX Protoporphyrinogen oxidase

PTP Permeability transition pore

PUMA p53-upregulated modulator of apoptosis

qRT-PCR Quantitative real-time PCR

RAD50 RAD50 homolog

Ras Rat sarcoma

RER Radiation enhancement ratio

RNAi RNA interference

RPA Replication protein A

RT Radiation therapy

s Seconds

SAPK Stress-activated protein kinase

SCID Severe combined immunodeficient

SD Standard deviaion

SEM Standard error of the mean

shRNA Small hairpin RNA

siCTRL Scrambled control siRNA

siRNA Small interfering RNA

siUROD UROD siRNA

xiv

SLRI Samuel Lunenfeld Research Institute

SOD Superoxide dismutase

SRB Sulforhodamine B

SSB Single-strand break

ssDNA Single-stranded DNA

TLD Tumor-plus-leg diameter

TOP1 DNA topoisomerase 1

TOPBP1 Topoisomerase II binding protein 1

TP Thymidine phosphorylase

TP53 Tumor protein 53

TS Thymidylate synthase

UROD Uroporphyrinogen decarboxylase

UV Ultraviolet

VEGF Vascular endothelial growth factor

VEGFR Vascular endothelial growth factor receptor

WEE1 WEE1 homolog

XLF Cernunnos

XRCC4 X-ray repair complementing defective repair in Chinese hamster cells 4

Z-VAD.FMK Benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone

1

CHAPTER 1: INTRODUCTION

2

1.1 Radiation Therapy

1.1.1 Background

Since the discovery of x-rays by German physicist, Wilhelm Conrad Roentgen in 1895,

x-ray technology has continued to evolve and revolutionize modern medicine. Since that time,

their clinical usefulness as a means of cancer treatment has developed into a recognized

medical specialty. Today, radiation therapy (RT) is a mainstay in the standard anti-cancer

therapeutic armamentarium, providing critical curative, adjuvant, and palliative roles in cancer

patient care. In the clinical setting, RT can be delivered as single or multiple treatments of

high-energy radiation to targeted areas of the patient’s body, with the ultimate aim of attaining

the highest probability of cure with the least morbidity. Thus, the dose of radiation that can be

delivered to a tumor is often limited by tolerance of the surrounding normal tissues and the

consequent risk of complications [1]. Over the past decade however, rapid advances in

radiation treatment planning and delivery have markedly improved patient outcomes,

particularly in reducing treatment-associated morbidities [2].

1.1.2 Radiation Biology

Ionizing radiation (IR) is radiation that has sufficient energy to remove electrons from

atoms [1]. Clinical radiotherapy typically utilizes IR to treat cancer patients, wherein waves or

packets of energy in the form of photons are delivered to a pre-defined tumor volume. Sources

of photons generally include x-rays (linear accelerators) and -rays (radioactive decay of 60

Co),

both of which are forms of electromagnetic (EM) radiation. X-ray and -ray photons have

essentially the same properties, but differ in origin; x-rays are emitted by electrons outside the

atomic nuclei (electronic shell), while -rays are released from unstable nuclei [1].

At the molecular level, when x-rays or -rays are absorbed by biological tissues, they

can directly ionize a critical site causing localized damage (direct effect) or interact with other

3

molecules to produce reactive free radicals (molecules with unpaired electrons), which can

subsequently damage key biological molecules (indirect effect) (Figure 1.1). Indirect effects

account for ~80% of the damage inferred by a given exposure of IR [3]. Since cells are

predominantly composed of water (~80%), the majority of the energy deposited is initially

absorbed by water (radiolysis), leading to the rapid generation (10-1410

-4 s) and propagation

of reactive radical species, with hydroxyl radicals (●OH) being the most lethal. Although IR is

capable of damaging a variety of intracellular molecules, DNA is considered to be the critical

target of both direct and indirect processes, resulting in DNA single- (SSB) or double-strand

breaks (DSB), DNA base damage, and/or DNADNA or DNAprotein cross-links [4, 5].

DSBs in DNA are considered highly mutagenic and the most lethal type of radiation lesion;

cell lethality following IR has been shown to correlate with the level of residual DSBs [6]. It is

estimated that each gray unit (1 Gy) of radiation produces ~105 ionization events per cell,

leading to ~10003000 DNADNA or DNAprotein cross-links, 1000 damaged DNA bases,

5001000 SSBs, and 2550 DSBs [1].

4

Figure 1.1 Direct and indirect effects of ionizing radiation on DNA

Ionizing radiation induces direct DNA damage, and indirect damage via generation of reactive

free radicals (e.g. hydroxyl radical, ●OH) from secondary chemical reactions around the DNA,

often involving water radiolysis. These free radicals can in turn, react chemically with DNA to

induce damage. Indirect and direct damage can lead to DNA single- and double-strand breaks,

base damage, DNA-DNA or DNA-protein cross-links. This figure is adapted from [7].

1.1.3 Cellular Response to Radiation

Upon exposure to ionizing radiation, a complex cellular DNA-damage response (DDR)

cascade, involving genomic surveillance and repair mechanisms is triggered, in an effort to

maintain genetic integrity and stability. In the presence of sublethal chromosome aberrations,

the induction of cell cycle arrest prevents DNA replication and mitosis, providing time for

DNA repair. In cases where the damage is severe and irreparable, the cells irrevocably undergo

cell death. The following sections will focus on the intricate processes involved in cellular

response to radiation-induced DNA damage.

5

1.1.3.1 DNA Damage Surveillance

Irradiation-induced DSB lesions are first detected by the heterotrimeric MRN

(MRE11–RAD50–NBS1) complex, which is the primary DNA damage sensor (Figure 1.2).

The recruitment of MRN activates the key DDR signaling kinase ATM (ataxia telangiectasia

mutated), which associates with DSBs and phosphorylates the histone variant H2AX (-

H2AX) at nucleosomes flanking the DSB [8]. The activated ATM then triggers two pathways

(chromatin-response vs. DSB resection), culminating in local chromatin rearrangements and

DNA processing; events essential for initiating DSB repair, checkpoint and cell death

signaling.

In the chromatin-response pathway (CDK-independent), the MDC1 mediator protein

binds to γ-H2AX and recruits additional MRN and ATM proteins, as well as multiple

checkpoint/adaptor proteins (e.g. NBS1, 53BP1, and BRCA1) at sites of DNA breaks,

providing a molecular platform for the efficient amplification of the DNA damage signal [9,

10]. The locally accumulated active ATM then phosphorylates many targets, including the

effector kinase CHK2, to further spread the damage signal [11].

Double-strand break resection can also occur following the recruitment of MRN and

ATM at the DNA lesion. This process requires the activity of cyclin-dependent kinases

(CDK), and is restricted to the S and G2 phases of the cell cycle [12]. DSB resection creates

stretches of single-stranded DNA (ssDNA) that become coated and stabilized by the ssDNA-

binding protein, replication protein A (RPA); forming the critical structural intermediate for

DNA repair by homologous recombination (HR) and ATR (ataxia telangiectasia and Rad3-

related)-dependent signaling [12]. The ssDNARPA scaffold facilitates the recruitment of

ATR through its interacting partner ATRIP [13]. ATR is subsequently activated by TopBP1,

which is also recruited to the ssDNA [14]. The activated ATR is then able to target

6

downstream substrates, including the effector signaling kinase CHK1 via the Claspin mediator

protein [15]. Both the ATM- and ATR-dependent branches of the pathway, independently or

in concert, orchestrate the DNA repair, cell death, and checkpoint responses in the damaged

cell.

Figure 1.2 DNA damage recognition pathway

DSBs are initially detected by the MRN sensor complex, which activates the transducer kinase

ATM. ATM phosphorylates histone H2AX in the DSB-flanking chromatin, which serves as a

docking platform for the MDC1 mediator protein. MDC1 recruits more MRN and ATM, as

well as other DDR proteins (e.g. 53BP1 and BRCA1), spreading the damage response

machinery along the chromosome. DSB resection can also occur following MRNATM

recruitment at the break site, wherein ssDNA is formed and stabilized by RPA, which in turn

facilitates recruitment of the ATR-ATRIP complex. Both the ATM- and ATR-dependent

pathways, independently or jointly, orchestrate the DNA repair, cell death, and checkpoint

responses in the damaged cell via downstream effector kinases, CHK1 and CHK2. This figure

is adapted from [11].

7

1.1.3.2 DNA Damage Cell Cycle Checkpoints

Following DSB-recognition, accurate genome duplication is controlled by several cell-

cycle checkpoints to prevent cells from initiating DNA replication (G1S checkpoint),

progressing with replication (S checkpoint), or entering mitosis (G2M checkpoint) [16]. As

described above, radiation-activated ATM/ATR proteins phosphorylate the CHK2/CHK1

kinases, which in turn target downstream effectors that affect cell cycle progression (Figure

1.3).

At the G1-phase checkpoint, the dominant response to radiation-induced DNA damage

is the stabilization and activation of P53, a tumor suppressor protein. Initial checkpoint signals

originating from ATM and ATR are transmitted to P53 both directly, and indirectly via CHK2

and CHK1. Phosphorylation of P53 prevents the onset of S-phase via transcriptional up-

regulation of P21, an inhibitor of the CDK4cyclin D and CDK2cyclin E complexes

necessary for S-phase initiation [17]. ATM can also directly phosphorylate and inhibit MDM2,

an ubiquitin ligase of P53, preventing proteasome-mediated P53 degradation [18]. CHK2-

induced P53 phosphorylation further blocks P53MDM2 interactions, promoting nuclear P53

accumulation [19]. The role of ATR-activated CHK1 kinase in P53 phosphorylation and

subsequent stabilization has been demonstrated, but is less well-defined [20].

The S-phase checkpoint is of particular importance since this is when duplication of the

genome takes place. At least two parallel pathways involved in attenuating S-phase in

response to DNA damage and replication disruption have been identified:

ATMCHK2CDC25A and ATMNBS1/BRCA1/SMC1; with the former and latter

pathways communicating to the cell cycle and DNA replication machinery, respectively [21,

22]. Upon exposure to IR, ATM-activated CHK2 phosphorylates and promotes ubiquitin-

dependent degradation of the CDC25A phosphatase, a major regulator of CDK2 activation,

8

which is essential for the assembly of the replication initiation complex during S-phase

initiation and progression (via CDK2cyclin E/A complexes) [22]. ATM-mediated

phosphorylation of NBS1, BRCA1, and SMC1 can also arrest cells in S-phase following IR-

induced damage, wherein NBS1 and BRCA1 are required for optimal ATM-directed

phosphorylation and activation of SMC1, a downstream effector in the ATM/NBS1/BRCA1-

dependent S-phase checkpoint pathway [21]. The precise roles of these proteins and the

mechanism of this pathway remain to be elucidated. Although the involvement of ATR in the

S-phase checkpoint is also relatively undefined, ATR has been shown to initiate a slow IR-

induced S-phase checkpoint response via CHK1 phosphorylation, which in turn

phosphorylates CDC25A, targeting it for degradation [23].

The G2M phase checkpoint primarily serves to allow time for DNA repair prior to

mitosis entry, minimizing the extent of DNA damage passed on to daughter cells. The

ATRCHK1CDC25C/WEE1 pathway involves radiation-activated CHK1 phosphorylation

of the CDC25C phosphatase, which normally dephosphorylates CDK1 and activates the

CDK1cyclin B complex, a major mitosis-promoting factor [24]. CHK1-mediated CDC25C

phosphorylation results in the cytoplasmic sequestration of CDC25C via inhibitory binding by

14-3-3-, providing an effective G2M block upon recognition of DNA damage [24]. P53

reinforces the G2-checkpoint through its transcriptional up-regulation of 14-3-3- [25]. CHK1

also phosphorylates and activates the WEE1 kinase, which maintains the CDK1cyclin B

complex in an inactive form, delaying mitotic entry [26]. Similar to ATR, ATM can also

contribute to the G2M checkpoint through the ATMCHK2CDC25C pathway [27].

9

Figure 1.3 Cell cycle checkpoint pathways

In response to ionizing radiation-induced DNA damage, ATM and/or ATR trigger the

activation of a cell cycle checkpoint. These pathways are characterized by cascades of protein

phosphorylation events (P) that alter the activity, stability, or localization of the modified

protein. A simplified overview of the G1-, S-, and G2-phase checkpoint pathways is

illustrated. This figure is adapted from [16].

10

1.1.3.3 DNA Repair

The two major mechanisms involved in DSB repair are homologous recombination and

non-homologous end joining (NHEJ) (Figure 1.4). HR is a highly precise repair process

occurring primarily during the SG2 phase of the cell cycle, which relies on the presence of

extensive regions of DNA sequence homology on the undamaged sister chromatid or

homologous chromosome to use as a template [28]. In contrast, NHEJ is an error-prone

process predominant in the G1 phase that does not require the presence of a homologous

template; it is the major pathway for repairing non-replication-associated breaks [29].

During HR, a DNA lesion is recognized by the MRN complex, which is recruited to

the DSB to process the DNA ends via resection, generating 3 ssDNA tails [30]. These 3

overhangs are coated by RPA, to prevent secondary structure formation. RPA is subsequently

replaced by the RAD51 recombinase via mediator proteins, including RAD52 [31]. The

resulting RAD51 nucleoprotein filament undergoes an ATP-driven invasion of a homologous

double-stranded template to create a joint molecule intermediate that entails heteroduplex

DNA (D-loop) [32]; this process is mediated by the RAD54 helicase, which promotes invasion

and the dissociation of RAD51 off the dsDNA resulting from the strand-transfer reaction [33].

After strand invasion, a DNA polymerase extends the invading 3 strand, forming a Holliday

junction. Capture of the second resected ssDNA tail into the joint molecule is mediated via

RAD52, which facilitates annealing of the displaced strand with the other end of the DSB,

producing double Holliday junctions [31]. Ligation and resolution of the joint homologous

recombination partners via nicking endonucleases yields two intact DNA duplexes, ultimately

restoring the homologous template and genetic information that was disrupted by the DSB

[28].

11

NHEJ-directed repair is initiated by the binding of the Ku70-Ku80 heterodimeric

complex to both ends of a DSB. The DNA-Ku scaffold subsequently recruits the DNA-

dependent protein kinase catalytic subunit (DNA-PKCS) to the DSB, activating its kinase

activity and multiple roles [29]. DNA-PKCS is involved in the formation of the synaptic

complex, consisting of two DNA ends, two Ku70Ku80 and two DNA-PKCS molecules,

which brings both DNA ends together. Once the two DNA ends have been captured and

tethered, non-compatible DNA ends are processed to form ligatable termini before final repair

of the DSB can occur. Several processing enzymes have been identified, including Artemis,

polynucleotide kinase, and DNA polymerases of the Pol X family [29]; the exact roles and

mechanisms of these end-processors have not been fully elucidated. Finally, recruitment of the

XRCC4DNA ligase IV complex and XLF (LIG4XRCC4 binging protein) by DNA-PKCS,

allows for the final ligation step of the processed DNA ends [34].

12

Figure 1.4 Double-strand break DNA repair pathways

(A) During homologous recombination, DNA ends are first processed by the MRN complex to

create 3 single-strand overhangs, which are bound by RPA. RAD52 mediates RAD51-

recruitment to the ssDNA to form a nucleoprotein filament, which searches for homologous

DNA, leading to strand invasion, strand exchange, and joint molecule formation. Template-

guided DNA synthesis, ligation, and resolution of the two double helices joined by strand

exchange complete the repair of the DSB. (B) Non-homologous end-joining brings the ends of

the DSB together by initial recruitment of the Ku70Ku80 complex and DNA-PKCS. After

synaptic complex formation, non-compatible DNA ends are processed to form ligatable

termini, followed by the repair of the break by the XRCC4DNA ligase IV complex. This

figure is adapted from [28].

13

1.1.3.4 Radiation-Induced Cell Death

Following exposure to IR, cells can undergo apoptosis, mitotic catastrophe, and/or

terminal cell arrest (senescence-like phenotype). The extent to which one mode of cell death

predominates over another is unclear, but may be influenced by cell type, radiation dose, and

the cell’s microenvironment (e.g. relative oxygenation) [1]. Depending on the severity of

damage, the tumor suppressor protein P53 can trigger cell cycle arrest (as described above), or

initiate apoptosis via transcriptional activation of pro-apoptotic proteins, including those of the

Bcl-2 family (e.g. BAX, PUMA) [35, 36]. PIDD (P53-induced protein with a death domain),

another P53 pro-apoptotic target, also plays a critical role in DNA damage-induced apoptosis,

leading to caspase-2 activation and subsequent mitochondrial cytochrome c release [37].

In cells irradiated with lethal doses, whereby the amount of DNA damage is beyond

repair, IR can also induce terminal growth arrest leading to a senescent-like morphology (e.g.

senescence-associated -galactosidase activity). Terminally-arrested cells are metabolically

active, but incapable of division; they eventually die, days to weeks following IR, via necrosis

[38]. It is suggested that the terminal-arrest pathway begins with the transactivation of the

CDK2 inhibitor p21, which is involved in the initial induction of senescent-associated G1-

arrest. Expression of p21 subsequently declines, while stable expression of the CDK4 inhibitor

p16INK4A

is induced, thereby maintaining this arrest [38].

Although IR-induced DNA lesions are lethal if left unrepaired, cell membrane damage

can also contribute to apoptosis. Radiation-induced cleavage of plasma membrane-localized

sphingomyelin by sphingomyelinases results in the rapid formation of ceramide, a lipid second

messenger that is a potent inducer of apoptosis. Subsequent activation of the stress-activated

protein kinase (SAPK) signaling cascade via ceramide will then initiate apoptosis [39].

14

For the majority of cells, mitotic catastrophe-induced necrosis accounts for most of the

cell kill following IR. Mitotic catastrophe is characterized by abnormal nuclear morphology

(e.g. multiple micronuclei or multi-nucleated giant cells) following premature entry into

mitosis by cells manifesting unrepaired DNA breaks and lethal chromosomal aberrations,

often resulting in the generation of non-clonogenic aneuploid and polyploid cell progeny [1]. It

is suggested that the abrogation of the G2M checkpoint is due to over-accumulation of cyclin

B and premature activation of the CDK1cyclin B complex [40]. Radiation-induced mitotic

catastrophe is the predominant mode of cell death in P53-deficient tumor cells, which are

defective in the G1S checkpoint, and can be selectively arrested by the G2-checkpoint upon

DNA damage.

1.2 Modulation of Radiation Response

1.2.1 Background

The greatest challenge for radiation therapy or any cancer therapy is to attain the

highest probability of cure with the least morbidity. In the context of RT, the inherent

radiosensitivity of cells or tissues can be influenced by a number of chemical manipulations,

including endogenous substances (e.g. oxygen), or xenobiotic agents (e.g. chemotherapeutic

radiosensitizers). Clinically, tumor-selective modification of radiosensitivity would allow for

lower radiation doses to be administered, ultimately enhancing tumor response without

increasing damage to surrounding normal tissues within a treatment field. Modification of

radiosensitivity by specific agents of known mechanisms of action can also provide insights

into the molecular basis underlying cellular responses and repair to radiation damage.

15

1.2.2 Chemical Radiosensitizers

Although both RT and chemotherapy have been employed as single-modality cancer

treatments for more than 40 years, the combined chemo-radiotherapy approach has been

adopted only more recently. Optimal combinations and scheduling remain in evolution, and

precise mechanisms underlying the radiation-potentiating effects of chemotherapeutic drugs

are still not fully understood. Many agents act through diverse processes and thus, there is no

universal mechanism that defines the interaction of drugs with radiation leading to

sensitization. Nonetheless, chemo-radiotherapy has become the standard of care for many

cancer patients based on improvements in locoregional disease control and survival.

A theoretical framework defining the possible mechanisms by which chemotherapy

and radiation may interact was first introduced by Steel and Peckham in 1979 [41]. Spatial co-

operation describes the concept that different therapeutic modalities affect distinct anatomical

sites of disease; radiation targets the local tumor, while chemotherapy acts against distant

metastases beyond the radiation field. This co-operative effect requires that the two treatments

not interact with each other and have non-overlapping toxicity profiles. Spatial co-operation is

highly theoretical and rarely observed in clinical situations. The more clinically applicable

interactive scenario is radiation sensitization, whereby chemotherapy co-operates with

radiation within the radiation field, leading to increased cell killing; either to the same degree

as (additive), or more than (supra-additive or synergistic) the expected sum of the respective

single-modality responses [41]. The clinical benefit of this radiosensitizing effect is defined by

its therapeutic ratio (Figure 1.5). With a chemo-radiotherapy approach, the radiation alone

dose-response curves for both the tumor and surrounding normal tissues will shift to the left.

Ideal radiation sensitizers should induce a stronger shift in the tumor response curve compared

to that of the normal tissue, increasing overall efficacy of treatment (radiation enhancement).

16

Alternatively, chemotherapy and RT may interact in an antagonistic manner, wherein the

combined cytotoxic effect is less than the expected sum (infra-additive or radioprotective).

This scenario is clinically advantageous in cases where agents cause selective protection of

normal tissues, allowing administration of higher radiation doses.

Categorizing chemotherapeutic radiosensitizers into well-defined types is challenging

as many agents confer multiple effects. Thus, applying broad categories, some of the most

commonly used classical radiosensitizers and emerging agents, as well as their mechanisms of

sensitization will be reviewed in the following sections.

Figure 1.5 Therapeutic ratio of radiosensitizing agents

Radiosensitizers with a favorable therapeutic ratio induce a greater change in the radiation

dose required for 50% cytotoxicity in cancer tissues (DC; C1 to C2), than that in normal

tissues (DN; N1 to N2). This is represented by a greater leftward shift in the tumor radiation

dose-response curve. This figure is adapted from [42].

17

1.2.2.1 Oxygen

One of the best-studied biological entities that modulate cellular response to radiation

is molecular oxygen (O2). As early as 1909, Gottwald Schwarz reported normal mammalian

cells irradiated under conditions of hypoxia (2% O2) or anoxia (0.02% O2) were less

sensitive to radiation than those irradiated under normoxia (~21% O2; 150 mm Hg) [43]. Since

then, it has become well-established that oxygen can enhance the effectiveness of radiation in

cell killing by a magnitude of two to three compared to irradiation conducted under limited O2

conditions, a principle known as the O2 enhancement effect [43]. The corresponding oxygen

enhancement ratio (OER) describes the ratio of hypoxic to aerated radiation doses required to

achieve equivalent levels of cell kill. Oxygen is thought to act as a direct radiosensitizer

through its ability to stabilize radiation-induced DNA damage into a form that is not readily

repaired. IR exposure generates free radical-mediated broken DNA ends, which can react with

available O2 to generate stable, toxic peroxy radicals, thus chemically modifying the DNA

(“oxygen fixation”). In the absence of O2, the initial DNA radical is reduced, restoring the

DNA to its original composition.

The presence of hypoxia in tumors is a well-established source of resistance to RT.

Hypoxia generally occurs in solid tumors mainly due to insufficient vascularization, which is

unable to adequately satisfy the high nutrient and oxygen demands of the proliferating tumor

cells. Thus, cells situated long distances from a functional blood vessel will become oxygen-

deprived as a result of limited O2 diffusion and perfusion. Hypoxia is also a potent stimulus of

gene expression. The best-characterized biological pathway related to hypoxia is regulated via

the hypoxia-inducible transcription factor-1 (HIF-1), which mediates adaptive response to

changes in tissue oxygenation. HIF-1 is over-expressed in human cancers as a result of

intratumoral hypoxia, as well as genetic alterations [44]. The heterodimeric HIF-1 consists of

18

and subunits, which dimerize under hypoxic conditions and bind to DNA at hypoxia

response elements (HREs) in promoter or enhancer regions of numerous transcriptional target

genes involved in cellular hypoxic responses, such as initiating anaerobic metabolism,

increasing angiogenesis, protecting cells against oxidative stress, and promoting invasiveness

and motility [45]. Accordingly, there is an overwhelming body of evidence supporting the

notion that HIF-1 may be a potential therapeutic radiosensitizing target [45]. Indeed, studies

have reported HIF-1 deficient murine hepatomas to demonstrate increased radioresponsiveness

compared to wild-type tumors [46]. Furthermore, direct inhibition of HIF-1 target genes, such

as vascular endothelial growth factor (VEGF), has been shown to also enhance

radiosensitization. VEGF is a pro-angiogenic/permeability factor, which acts to improve the

availability of oxygen from capillaries via increased vascular permeability, as well as induce

formation of new vessels. Aberrant VEGF/VEGF receptor (VEGFR) signaling in cancer has

been associated with tumor progression and the formation of metastasis. Fittingly, blockage of

the tumor VEGF signal transduction cascade reverses the radioresistant phenotype of

glioblastoma multiforme and melanoma microvasculature and xenograft tumors [47]. Loss of

HIF-1 in in vitro and in vivo models also dramatically reduces VEGF expression and the

capacity to release VEGF during hypoxia [48]. Thus, hypoxia can impact tumor

radioresponsiveness via the physio-chemical reaction of oxygen with radiation-induced

radicals causing damage “fixation”, but also through hypoxia-induced expression of genes that

allow tumor cells to survive under these adverse conditions.

Clinically, tumor hypoxia is associated with poor tumor prognosis and local tumor

relapse after RT; it has also been linked to a more aggressive tumor phenotype [49, 50].

Various methods to overcome hypoxic radioresistance have emerged over the years. One

approach is to increase tumor oxygenation during radiation through the use of hyperbaric

19

oxygen (HBO), red blood cell transfusions, and erythropoietin administration, resulting in a

physical increase in the O2 content of blood. These approaches however, have not gained

widespread use due to their difficulty to translate into clinical practice routinely, and/or

conflicting reports of their efficacy in clinical trials [51]. Another approach that has received

much attention is the development of electron-affinic radiosensitizers. Molecular oxygen-

mimetics, such as nitroimidazoles, partially recapitulate the effects of O2 in the radio-chemical

process and enhance IR-induced DNA strand breaks. Despite initial promise, clinical trials

with nitroimidazoles and its derivatives (e.g. misonidazole, etanidazole) have demonstrated

limited therapeutic benefit in hypoxic radiosensitization, in part due to dose-limiting toxicities,

such as severe peripheral neuropathy [52, 53]. As an alternative to increasing tumor

oxygenation, more recent strategies have attempted to exploit hypoxia for tumor-selective

killing. These so-called “hypoxic cytotoxins” are aimed at destroying, rather than sensitizing,

cells under hypoxic conditions in the absence of radiation. Tirapazamine, the prototypic

hypoxic cytotoxin, shows ~100-fold increased potency under anoxic vs. normoxic conditions

due to its electron-donating property. It is a pro-drug that is specifically reduced in hypoxic

cells, forming radical species that poison topoisomerase II, leading to lethal DNA DSBs [54].

Preclinical studies have demonstrated tirapazamine to potentiate the efficacy of RT on tumor

response [55]. Furthermore, randomized phase I and II clinical trials with tirapazamine in

combination with RT have demonstrated clinical benefits in patients with HNC, warranting

further investigations [56, 57]. A phase III trial of RT tirapazamine was recently launched

for HNC, but has been discontinued by Sanofi-Aventis due to presumed lack of therapeutic

efficacy (personal communication).

20

1.2.2.2 Halogenated Pyrimidines

Halogenated pyrimidines (HPs) structurally mimic thymidine, a normal base required

for DNA synthesis; the difference resides in a replacement of the 5 methyl group of thymine

with a halogen (iodine, bromine, chlorine, or fluorine) [58]. HPs have found practical use in

clinical radiotherapy based on the premise that tumor cells have a higher demand for DNA

replication and therefore, should incorporate more drug than the surrounding normal tissues.

Accordingly, HPs increase the effectiveness of radiation chiefly when administered before and

during RT [58].

The methyl group of thymine is approximately the same size as iodine, bromine, and

chlorine atoms; thus, as the cells undergo DNA synthesis, iodo (I)-, bromo (Br)-, and chloro

(Cl)-deoxyuridine (dUrd) compete with thymidine pools for incorporation into cellular DNA.

As the percentage of replaced thymidine bases increases, so does the extent of HP-mediated

radiosensitization; a thymidine replacement of 1015% correlates with a radiation

enhancement ratio of ~2.0 [58]. The halogen moieties act as electron “sinks” during radiation,

wherein the carbonhalogen bond breaks on electron attachment to liberate free halide and a

carbon-centered free radical. In the presence of oxygen, a peroxyl radical is formed, leading to

DNA strand breaks. Incorporation of BrdUrd and IdUrd into DNA has been associated with

increased induction, and decreased rate of repair of radiation-induced DNA damage [59].

In contrast to iodine, bromine, and chlorine atoms, fluorine atoms are significantly

smaller than the methyl group of thymine. Consequently, fluoro-deoxyuridine (FdUrd) blocks

cells at the G1S interface, inhibiting DNA synthesis. Recent approaches to use the

radiosensitizing nucleosides have focused on other fluorine analogues, especially 5-

fluorouracil (5-FU), gemcitabine, and capecitabine. Among these, 5-FU, administered via

intravenous (IV) infusion, remains the predominant agent in the clinic. After cellular uptake, 5-

21

FU, a uracil analog, is converted to FdUrd by thymidine phosphorylase (TP), which is often

upregulated in tumor vs. adjacent normal tissues; thus, providing tumor-selectivity and a

therapeutic window [60]. Phosphorylation of FdUrd by thymidine kinase generates 5-fluoro-

2-deoxyuridine monophosphate (FdUMP), which then inhibits thymidylate synthase (TS)

activity [61]. TS inactivation results in the depletion of the intracellular pool of thymidine 5-

monophosphate and thymidine 5-triphosphate, which inhibits DNA synthesis and interferes

with DNA repair. Alternatively, 5-FU can be metabolized to 5-fluorouridine triphosphate, a

substrate for RNA polymerase which is readily incorporated into RNA, leading to inhibition of

mRNA polyadenylation with decreased mRNA stability, and alteration of the RNA secondary

structure [61]. The underlying mechanisms of the interaction of IR with 5-FU are still not fully

understood. However, 5-FU-induced radiosensitization appears to be mediated primarily by its

DNA-directed effects, and is dependent on inappropriate S-phase progression in the presence

of drug (i.e. from dysregulated S-phase checkpoints), and a decreased ability to repair

radiation-induced DNA damage [62, 63]. Phase III clinical trials of 5-FU and RT have

reported clinical benefit in cancers of the esophagus, cervix, and rectum [64-66] .

Capecitabine, an oral pro-drug of 5-FU, was developed to decrease the burden of 5-FU

IV administration and increase intra-tumoral bioavailability. Capecitabine is preferentially

metabolized to active 5-FU in tumors via a cascade of three enzymes, the last enzyme being

thymidine phosphorylase. Interestingly, studies have reported local RT to selectively

upregulate TP activity in tumor tissues via induction of tumor necrosis factor [67]; thus, RT

may further increase the therapeutic index of capecitabine due to the lack of TP upregulation

in normal tissues and the anatomically-targeted nature of RT. Preclinical studies have

demonstrated significant radiosensitization of several human cancer xenograft models [67].

22

Clinical trials investigating the efficacy of capecitabine in combination with RT have also

shown therapeutic benefits with low toxicity profiles [68, 69].

Gemcitabine is a pyrimidine analog that has also demonstrated potent radiosensitizing

effects against various solid tumor models, including HNC, colon, pancreatic, breast, and non-

small cell lung (NSCLC) cancers [70], as well as in phase II clinical trials for pancreatic

cancer [71]. Within the cell, gemcitabine is rapidly phosphorylated to its active di-and

triphosphate metabolites. Gemcitabine triphosphate serves as both an inhibitor and substrate

for DNA synthesis [72]. Gemcitabine diphosphate irreversibly inhibits ribonucleotide

reductase, resulting in the rapid decrease in cellular deoxyribonucleotide triphosphate (dNTP)

levels in a cell-specific manner [73]; the selective depletion of deoxyadenosine triphosphate

(dATP) appears to be a common response to gemcitabine in solid tumor cell lines [74].

Reduced dNTP pools may also contribute to the inhibition of DNA synthesis, as well as

promote the incorporation of gemcitabine into DNA through decreasing the level of its

endogenous competitor [72]. Thus, gemcitabine may radiosensitize cells that progress

inappropriately through S-phase by depleting dATP pools, leading to the misincorporation and

misrepair of incorrect bases, collectively enhancing radiation-inflicted DNA damage.

1.2.2.3 Modifiers of Microtubule Structure and Function

Taxanes, such as paclitaxel and its semi-synthetic analog docetaxel, are mitotic

inhibitors. They form high-affinity bonds with microtubules, promoting tubulin polymerization

and stabilization; ultimately interfering with normal microtubule function. At high cytotoxic

doses, both drugs inhibit mitotic spindle formation and block the progression of cells in

mitosis, between prophase and metaphase [75]. The radiosensitization observed after treatment

with paclitaxel or docetaxel in vitro is most likely due to the taxane-induced G2M block in

the cell cycle, leading to synchronization (i.e. cell-cycle pooling) of tumors cells at a point of

23

maximum radiosensitivity [76]. Improved overall outcomes have also been reported in

NSCLC and HNC patients treated with paclitaxel/docetaxel and radiotherapy in phase II

clinical trials, yielding good local regional control and survival rates [77, 78].

1.2.2.4 Modifiers of the Nature or Repair of DNA Damage

Platinum analogs, specifically cisplatin and more recently oxaliplatin, are DNA-

damaging agents being used clinically in combination with RT for the treatment of various

solid tumors. Cisplatin is one of the most commonly used anti-cancer agents for concurrent

chemo-radiotherapy. Its cytotoxicity is primarily ascribed to its interaction with nucleophilic

N7-sites of purine bases in DNA to form both DNAprotein and DNADNA inter-strand and

intra-stand cross-links, thereby distorting the DNA structure, and blocking DNA replication

and transcription [79]. Cisplatin-mediated radiosensitization can occur by several mechanisms.

It has been proposed that radiation-induced free radicals enhance the formation of toxic

platinum intermediates, which increase cell killing [80]. Moreover, IR has been reported to

increase cellular uptake of platinum [81]. Radiation-induced DNA damage that would

typically be repaired can become fixed and lethal via cisplatin’s capacity to scavenge free

electrons formed by the radiationDNA interaction. The resulting inhibition of DNA repair

leads to increased cell-cycle arrest and apoptotic cell death after radiation [82]. Clinically,

concurrent cisplatin-based radiotherapy trials have reported improved overall outcomes for

patients with HNC and cervix cancer [83, 84]. Oxaliplatin, a third-generation cisplatin

analogue, was developed to address the intrinsic or acquired cisplatin resistance that often

arises in tumors. The drug reacts with DNA forming mainly platinated intra-strand cross-links

with two adjacent guanines or adjacent guanineadenine residues [85]. These adducts appear

to be more effective at inhibiting DNA synthesis and are more cytotoxic than those formed by

cisplatin. Oxaliplatin consistently shows activity in cisplatin-resistant cell systems, as well as

24

human tumors [86, 87]. Although less well-studied, oxaliplatin exhibits significant in vitro and

in vivo radiation enhancement [88], and has shown promise in clinical trials with RT for the

treatment of rectal cancer [89].

DNA alkylating agents, such as temozolomide, cause DNA damage by methylating

guanine on the O6 position, activating the p53-regulated DNA damage response pathway [90].

These alkylated lesions are processed by the ubiquitous DNA repair enzyme, O6-

methylguanine DNA-methyltransferase (MGMT). Following removal of the alkyl groups,

MGMT is irreversibly inactivated such that de novo synthesis of MGMT is required for

cellular function [91]. Thus, administering temozolomide on schedules that result in

cumulative and sustained inactivation of MGMT reduces the cell’s capacity for DNA repair

[92], potentiating IR-inflicted DNA damage. Accordingly, tumors with MGMT mutations are

also preferentially radiosensitized by temozolomide [93]. Temozolomide also inhibits

signaling of radiation-induced cell migration and invasion, and decreases tumor cell

repopulation [94]. Temozolomide, which is orally administered, readily crosses the blood-

brain barrier [95], and is therefore commonly used to treat gliomas. A recent phase III clinical

trial has demonstrated a significant survival benefit with minimal additional toxicity in

glioblastoma patients treated with temozolomide plus RT [96].

Molecularly-targeted inhibitors of DNA repair proteins have also emerged as potential

radiosensitizers. Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that facilitates

DNA base excision repair, and also regulates HR- and NHEJ-mediated DSB repair [97].

PARP-1 activation and subsequent poly(ADP-ribosyl)ation are immediate cellular responses to

radiation-induced DNA damage [98]. Moreover, PARP-1-mediated DNA repair has been

associated with resistance to radiation; thus, inhibition of PARP-1 may be therapeutically

beneficial [99]. Accordingly, preclinical evaluations of PARP-1 inhibitors, such as AG14361,

25

have demonstrated signification radiosensitizing effects in vitro and in vivo, resulting in

enhanced radiation-induced cytotoxicity due to persisting DNA lesions that would normally be

repaired [100]. Ataxia telangiectasia mutated is a serine/threonine protein kinase that also

plays a critical role in regulating cell cycle arrest and DNA repair. ATM inhibitors, such as

wortmannin and caffeine, have garnered attention, demonstrating pre-clinical sensitization of

tumor cells in vitro; their clinical use as radiosensitizers however, are limited by potentially

lethal systemic toxicities [101]. More recently, KU-55933, a novel and specific inhibitor of

ATM was identified, exhibiting in vitro radiosensitization with an enhancement ratio of 2.6 at

2 Gy [102].

1.2.2.5 Targets of Cell Signaling Pathways

Molecularly-targeted therapies that inhibit radioresistance-associated signal

transduction pathways are also being investigated. The Ras protein family is well-studied in

the context of RT as they control key signaling pathways that regulate cell growth and

transformation. The Ras proto-oncogene is overexpressed in approximately 30% of human

tumors, and has been implicated in radioresistance by promoting aberrant survival signals

[103]. The activation of Ras is dependent on the post-translational addition of an isopreynl

group by the farnesyltransferase enzyme. Accordingly, farnesyltransferase inhibitors (FTI)

(e.g. FTI-277, L-744,832) have been used with RT in preclinical studies, successfully

reversing the radioresistant phenotype of human tumor xenografts and cells expressing the

mutant ras oncogene [104, 105]. FTI-induced radiosensitization has been proposed to be

mediated via downregulated signaling through the downstream PI3K/AKT and MAP kinase

pathways, and reduction of tumor hypoxia [104]. Furthermore, a phase I clinical trial of FTI L-

778,123 with concurrent RT for locally advanced pancreatic cancer patients reported no

increase in radiation-induced normal tissue damage, indicating the potential to increase the

26

therapeutic index. Radiosensitization of a patient-derived pancreatic cell line was also

observed [106].

The epidermal growth factor receptor (EGFR) family members are the most mature of

the molecular targets; upon activation, EGFR mediates various cellular responses important

for cell growth, differentiation, and survival. These receptors are overexpressed in a diverse

array of epithelial tumors [107], which often correlates with radioresistance and adverse

clinical outcomes [108, 109]. Currently, two therapeutic strategies have been developed to

inhibit EGFR activity. One approach targets the extracellular ligand-binding domain of EGFR

with monoclonal antibodies (e.g. cetuximab). The second targets the intracellular domain of

EGFR with tyrosine kinase inhibitors (e.g. erlotinib, gefitinib) that compete with the adenosine

triphosphate (ATP)-binding site [109]. Preclinical studies with the EGFR inhibitors

consistently show synergistic enhancement of radiosensitivity both in vitro and in vivo [110].

Due to the pleiotropic effects of EGFR signaling, the precise mechanism of radiosensitization

has not been fully elucidated; however, experimental evidence favors inhibition of cell

proliferation (preventing repopulation) and induction of apoptosis as major mediators of the

radiosensitizing properties of EGFR blockade [111]. The clinical application of EGFR-

mediated radiosensitization has been supported by phase III trials, wherein disease-free and

overall survival advantages were observed in HNC patients treated with RT and concurrent

cetuximab vs. RT alone [112, 113].

Another target attracting attention in the context of RT includes anti-angiogenesis

inhibitors. The process of angiogenesis is mediated by multiple pro-angiogenic and anti-

angiogenic factors, with VEGF playing a central role. Angiogenesis is essential for tumor

growth and progression. Accordingly, VEGF is over-expressed in many cancers, and its

expression can be induced by radiation, promoting tumor radioresistance [114]. Two strategies

27

for blocking VEGF signaling exist, those that target the VEGF ligand via neutralizing

antibodies (e.g. bevacizumab), and those that inhibit the receptor (e.g. PTK787 and SU5416

tyrosine kinase inhibitors). The importance of VEGF signaling in tumor radioresistance is

supported by preclinical observations demonstrating supra-additive tumor growth delays and

cytotoxicity when VEGF/VEGFR antagonists are combined with RT in radioresistant

xenograft models [47, 114] . The precise mechanism of how targeting angiogenesis promotes

tumor radiosensitization remains unclear. The initial concern that anti-angiogenic disruption of

the tumor blood supply may encourage tumor hypoxia, and in turn radioresistance, has been

discounted by recent evidence. Instead, VEGF/VEGFR antagonists are thought to induce

transient normalization of the tumor vasculature, leading to enhanced tumor oxygenation and

radiosensitization [115]. Phase I/II clinical trials to assess the efficacy and safety of the

addition of bevacizumab to concurrent RT have reported encouraging response rates with

acceptable toxicity profiles in rectal cancer [116, 117].

1.3 High-Throughput Screens

1.3.1 Background

High-throughput screening is an approach to anti-cancer drug discovery that has gained

widespread popularity over the past few decades. Initially developed for the pharmaceutical

sector, HTS has recently been adapted by academic institutions for the discovery of novel

therapeutics and biological pathways. HTS entails multiple-well microplates (96-/384-well)

and robotic processing to assay large numbers of potential effectors of biological activity

against targets, with the goal of accelerating drug discovery via large-scale screening of

chemical and genomic libraries often composed of thousands of molecules. As the number of

compounds available for screening has increased, the throughput of assay technology has also

28

kept pace; with the transition from 96-well to 384-well and nano (1,536)-well plate formats,

thereby accelerating screening times and decreasing overall costs [118]. In general, anti-cancer

drug discovery can be broadly divided into two distinct approaches, phenotype- and target-

based screening (Figure 1.6) [118]. Commonly utilized end-points include genetic or protein

markers (e.g. reporter-gene assays, antibody-based cellular immunoassays), functional assays

(e.g. cell division, proliferation, viability, apoptosis), or high-content automated microscope-

based imaging systems (cellular morphology, subcellular localization of protein markers).

Figure 1.6 High-throughput screening approaches

29

Phenotype-based discovery or the forward chemical-biology approach starts with the screening

of chemical libraries for compounds that induce a particular phenotype followed by the

identification of the responsible target. Target-based discovery or the reverse chemical-biology

approach employs chemical library screens to identify ligands for a specific molecular target

of interest. This figure is adapted from [118].

1.3.2 Phenotype-Based High-Throughput Screens

Forward chemical genetics, or phenotype-based screens, assay the response of an

experimental system to chemical perturbations via phenotypic read-outs, followed by

identification of the responsible target. The forward approach often yields novel targets and

insights into unexplored pathways upon identification of the drug target. Cell-based screening

for anti-proliferative effects of plant- and marine-derived natural product small molecule

libraries have recently uncovered new compounds with anti-tumor activity, including extracts

from the Helleborus cyclophyllus flower [119], and a class of DNA-intercalating plakinidines

isolated from the marine sponge, Crella spinulata [120]. The drug discovery process has also

been facilitated by the National Cancer Institute Developmental Therapeutics Program (NCI-

DTP), which has implemented an in vitro anti-cancer screening platform consisting of 60

different human tumor cell lines representing nine common forms of cancer: leukemia, colon,

lung, central nervous system, renal, melanoma, ovarian, breast, and prostate. To date, the NCI-

DTP has screened 3 million synthetic and natural compounds against the cancer panel

utilizing short-term colorimetric cell viability assays. Paclitaxel was discovered from a NCI-

sponsored large-scale plant-screening program, wherein ~35,000 plant species were screened,

identifying the bark extract from the Pacific yew tree, Taxus brevifolia to exert broad anti-

tumor efficacy [121]. It was not until years later that paclitaxel was shown to target

microtubules, uncovering a novel cellular mechanism as a viable anti-cancer therapeutic target

[122].

30

1.3.3 Target-Based High-Throughput Screens

Despite the large number and chemical diversity of the novel anti-cancer cytotoxics

being discovered via phenotype-driven screening, their clinical applications have been limited.

The ultimate goal of drug discovery is to improve the efficacy and selectivity of cancer

treatment by exploiting differences between cancer and normal cells. However, with the

forward-driven HTS approach, mechanism of action is not a primary determinant in selecting

agents for further development; thus, many discovered drugs have a low therapeutic index. As

we further unravel the underlying mechanisms of tumor initiation and progression, the

approach to drug discovery has transitioned from empirical compound-oriented preclinical

screening to target-focused drug screening.

Reverse chemical genetics, or target-based screens, target a specific cellular protein or

gene of interest with chemical perturbations, and elucidate the phenotypic consequences

thereafter. Reverse screens have been successful in identifying molecularly-targeted agents

currently in clinical use. For example, an initial lead compound targeting the Bcr-Abl onco-

protein, 2-phenylaminopyrimidine, was identified via random screening of large chemical

libraries for inhibition of its tyrosine kinase activity in vitro [123]. Further optimizations of the

lead compound led to imatinib mesilate, a 2-phenylaminopyrimidine derivative, which is now

FDA-approved as front-line treatment for chronic myelogenous leukemia (CML) [123]. Since

chemical screens can be costly and time-consuming, alternative approaches to facilitate target-

driven lead compound discovery include in silico virtual screening, which can readily filter

undesirable compounds based on criteria, such as target-binding affinity and chemico-physical

properties. To address the high incidence of imatinib resistance that often arises in patients

with advanced CML, a recent study has employed in silico screening of 200,000 commercially

31

available compounds, identifying two novel Bcr-Abl-targeted tyrosine kinase inhibitors for

further drug design and optimization [124].

1.3.4 RNA Interference Screens

The recent sequencing of the human genome and development of new genomic

technologies have brought promise of a myriad of new targets for the development of

molecularly-targeted therapies. RNA interference (RNAi) is an endogenous cellular process

that controls gene expression at the post-transcriptional level; messenger RNAs (mRNAs) are

targeted for degradation by complementary double-stranded interfering RNA, leading to

selective gene silencing. Since its discovery in the nematode Caenorhabditis elegans, RNAi

has been exploited as a standard tool for studying gene function via synthetic small interfering

RNAs (siRNAs) and small hairpin RNAs (shRNAs) [125]. Initially used to knock-down the

function of individual genes of interest, this technology has been harnessed on a global scale

with the production of RNAi libraries covering the entire human coding transcriptome,

allowing for genome-wide loss-of-function screening. Phenotypic read-outs can range from

simple and commonly used cell viability assays, to complex high-content screens involving

sophisticated microscopic image analyses. Thus, an RNAi screen is essentially a forward

genetics screen using a reverse genetics technique. The first genome-wide (21,127 genes)

synthetic-lethal siRNA screen for chemosensitizers was performed by Whitehurst et al.,

wherein gene targets that reduced the viability of human NSCLC cells in the presence of

sublethal concentrations of paclitaxel were identified, including components of the mitotic

spindle apparatus and proteasome machinery [126]. Thus, new classes of therapeutic targets

were revealed for combinatorial chemotherapy. High-throughput functional-genomic platforms

have also been exploited for the screening of key mediators of proliferation and survival in B-

32

cell lymphoma, utilizing a retroviral-based shRNA library targeting 2,500 human genes,

wherein genes regulating cell cycle, splicing, and NF-B signaling were identified [127].

1.3.5 Radiosensitizer Discovery Screens

As described above, the use of HTS has been fruitful in identifying drug candidates or

molecular targets for anti-cancer therapies. However, technical obstacles have impeded the

development of HTS for radiosensitizers, mainly with respect to read-outs. The colony

formation assay (CFA) is the accepted gold standard for measuring cellular radiation

susceptibility amongst radiation biologists. The long-term kinetics, difficulty in large-scale

automation, and limited robustness (i.e. colony-forming capacity) of the assay however, has

restricted its appeal and amenability to high-throughput platforms. Although CFA is the ideal

assay, radiosensitizers have been discovered using alternative read-outs. A recent forward

screen of 870 commercially available compounds utilizing the MTS cell viability as the end-

point, identified 4-bromo-3-nitropropiophenone (NS-123) as a tumor-selective radiosensitizer

of human glioma cells in vitro and in vivo by a mechanism involving inhibition of DNA repair

[128]. Several reverse screens employing known molecular targets of the DNA repair pathway

as read-outs via in vitro kinase activity assays, have identified KU-55933 (ATM inhibitor), and

NU7441 (DNA-PKCS inhibitor) as potential radiosensitizing agents from libraries of small-

molecule compounds [102, 129]. Novel radiation susceptibility targets have also been

discovered from small shRNA library screens (200 genes) using the sulforhodamine B (SRB)-

based cell growth assay as a read-out, identifying genes involved in cell cycle progression (e.g.

ZDHHC8) as potential molecular targets for radiosensitization [130].

33

1.4 Head and Neck Cancer

1.4.1 Background

Head and neck cancer is the eighth most common cancer worldwide, with an estimated

annual global incidence of approximately 650,000 cases and ~90,000 deaths attributed to this

disease per year [131]. In 2009, it was estimated that there would be 4,550 newly diagnosed

cases, with 1,660 deaths as a result of HNC in Canada [132]. HNC comprises a diverse group

of tumor types arising from the upper aerodigestive tract, including the lip, nasal and oral

cavities, sinuses, pharynx, larynx, and other sites in this anatomical region [133]. The vast

majority of HNC diagnoses (90%) are of squamous epithelial cell origin (oral cavity,

pharynx, larynx), and are thus termed head and neck squamous cell carcinomas (HNSCC)

[133]. Nasopharyngeal carcinoma (NPC) is a less common distinct HNC in that 90% of cases

harbor latent Epstein-Barr virus [134]. HNC is strongly associated with certain environmental

and lifestyle risk factors, including smoking or chewing tobacco, alcohol consumption, UV

light and occupational exposures (e.g. wood dust, paint fumes, asbestos), and certain strains of

viruses, such as the sexually-transmitted human papillomavirus (e.g. HPV16) [133, 135, 136].

1.4.2 Treatment

HNC treatment is complex and depends on the anatomical location of the tumor and

involvement of adjacent organs. The classification system is divided into three clinical stages:

early, loco-regionally advanced, and metastatic or recurrent, where treatment approaches can

vary depending on the disease stage [137]. At the time of presentation, ~3040% of HNC

patients typically have localized disease, 50% have associated regional disease, and ~10%

harbor distant metastases. For all sites and stages in the head and neck region, 5-year survival

rates average ~50% [137]. While treatment of HNC is complex due to the anatomical and

molecular heterogeneity of HNC, some general principles apply, wherein surgery,

34

radiotherapy, chemotherapy, and combinations of these are well accepted. Early-stage disease

is generally treated with single-modality treatments of either surgery or radiation alone. Late-

stage disease is best treated by a combination of surgery and radiation, radiation with or

without chemotherapy, or all three modalities [137]. A major challenge in treating HNC is

obtaining a high cure rate while preserving vital structures and function; tumors often reside in

close proximity to critical organs (e.g. brain, spinal cord, optic nerve), which when damaged

lead to long-term compromises in patients’ quality of life, not to mention fatality.

1.4.2.1 Radiation Therapy

Radiation therapy remains a mainstay of curative therapy for HNC, and is typically

provided in a single fraction (~2 Gy per day) on a schedule of 5 days a week for 7 weeks (total

dose of ~70 Gy) [138]. Recent advances have focused primarily on altered fractionated

(hyperfractionation or accelerated fractionation) RT regimens, and the implementation of

intensity-modulated RT (IMRT).

In accelerated fractionation, the total treatment time is decreased, which in turn,

reduces the repopulation of tumor cells between sessions, allowing for improved locoregional

control. In hyperfractionated schedules, two to three lower-dose fractions (1.11.2 Gy) are

delivered daily, with a higher total dose administered over the same duration as conventional

RT, reducing the potential risk of late toxicities. A recent meta-analysis revealed altered

fractionation RT regimens to demonstrate a small, but significant absolute survival benefit of

3.4% at 5 years, with the benefit being greater for hyperfractionated vs. accelerated RT [138].

There was also improved locoregional control for the altered fractionation schedules vs.

conventional RT (6.4% at 5 years). These modest benefits however, came at the expense of

increased toxicity, mostly in the form of mucositis [138].

35

IMRT is a form of high-precision RT that delivers radiation in a more targeted and

conformal manner via computed-tomography (CT)-guided 3D tumor images; thereby

minimizing radiation exposure to surrounding normal tissues. Recent data suggest that IMRT

is as effective as conventional 2D RT with regard to locoregional control, but is superior in

reducing treatment-related toxicity [139, 140]. However, even with the most effective RT

regimens, the prognosis of patients with HNC remains poor, with 5-year overall survival rates

of 3040% [138].

1.4.2.2 Chemotherapy

Chemotherapy is also an important modality in the management of HNC. The most

commonly utilized cytotoxic agents include cisplatin, carboplatin, 5-FU, paclitaxel, docetaxel,

or mitomycin C, either as single-agents or in multi-agent chemotherapy platforms.

Chemotherapy is often administered concomitantly with RT (concurrent chemo-radiotherapy)

or before RT in the form of induction chemotherapy. A recent meta-analysis of 93 randomized

trials demonstrated that chemotherapy in general improves survival in non-metastatic HNSCC

treated with surgery and/or RT with an overall benefit of 4.5% at 5 years, from 31.1% to

35.6% [141]. Within this study, a greater benefit (6.5% vs. 2.4% at 5 years) was observed in

trials that delivered concomitant chemotherapy with RT, as compared to induction

chemotherapy. Similar conclusions were drawn from a meta-analysis comparing RT to radio-

chemotherapy in NPC, whereby an absolute overall survival benefit of 6% at 5 years was

observed in the chemoradiation arm; most of the benefit was again observed with concomitant

vs. induction chemotherapy [142]. Meta-analyses examining various chemoradiotherapy

regimens have revealed optimal types of drugs to be combined concomitantly with RT, which

include either platinum-based agents alone (cisplatin or carboplatin), or 5-FUplatinum

combinations.

36

Although the addition of chemotherapy to RT has improved outcomes, an increase in

both acute and late toxicities has also been reported. The sensitizing effects of most cytotoxic

agents are not tumor-specific and often affect adjacent normal tissues within the radiation

field. Concurrent chemoradiotherapy trials have consistently reported an increased incidence

of acute grade 3 and 4 toxicity, with mucositis and dermatitis being the most prominent [143].

Cisplatin for example, is a potent radiosensitizer, and the drug most commonly utilized for

chemoradiotherapy in HNC. Currently, the most widely used standard regimen is 100 mg/m2

of cisplatin administered every 3 weeks throughout the course of RT (~70 Gy in 2 Gy daily

fractions) [141]. This regimen, however, causes severe side-effects, both acute (nausea,

vomiting, severe mucositis), and late (nephro-, oto- and neuro-toxicity) toxicities [144].

1.4.2.3 Molecularly-Targeted Agents

Advances in our understanding of the molecular genetics underpinning HNC

development and growth have resulted in the emergence of novel molecularly-targeted

therapies, with the aim of improving efficacy and/or quality of life, while minimizing damage

to surrounding normal tissues. Since molecularly-targeted agents exploit tumor-specific

aberrations, they are thought to provide a theoretical advantage over conventional

chemotherapeutic cytotoxics, in which a major drawback is the exacerbation of normal tissue

toxicity. Evidence is accumulating that a variety of targets involved in different cellular

processes may provide novel opportunities for therapeutic targeting.

Epidermal growth factor receptor is one of the most attractive and widely-investigated

targets in HNC. High levels of EGFR tumor expression have been associated with poor

prognosis in HNC patients, including decreased response to RT, and increased loco-regional

recurrence following definitive RT [109]. Treatments targeting the function of EGFR appear to

show promise in improving clinical outcome for HNC. As mentioned previously, two

37

therapeutic strategies have been developed to inhibit EGFR activity: monoclonal antibodies

against the extracellular ligand-binding domain (e.g. cetuximab), or inhibitors targeting the

intracellular tyrosine kinase domain of EGFR (e.g. erlotinib, gefitinib) [109]. Currently,

cetuximab is the only FDA-approved EGFR-targeted therapy for the treatment of metastatic

HNSCC (as a single agent), and locally advanced HNSCC (in combination with RT). Recent

phase III multi-center trials have demonstrated RT with concurrent cetuximab to significantly

improve overall survival rates when compared to RT alone in patients with loco-regionally

advanced HNSCC [112, 113]. Moreover, the combinatorial regimen was well-tolerated, with

similar rates of toxicity compared with RT alone; in particular, cetuximab did not appear to

exacerbate radiation-induced mucositis or other toxicities. Despite these promising results

however, the 5-year overall survival rate for the combined cetuximab-plus-RT group was still

only 45.6%, underscoring a continued need for further improvements.

A number of other potential agents employing novel targeting approaches are currently

being explored for HNC, including those that target angiogenesis (VEGF inhibitor), protein

turnover (proteasome inhibitor), signal transduction pathways (mTOR and IGFR inhibitors),

and cell cycle regulators (aurora kinase inhibitors) [145]. These molecularly-targeted agents

are not yet FDA-approved for use in HNC, as their safety and activity in HNC are still under

active investigation in pre-clinical and clinical studies.

1.5 Research Objectives

Ionizing radiation therapy plays critical curative, adjuvant, and palliative roles in cancer

patient management; curability however, could be limited by tolerance of normal surrounding

tissues. Thus, the development of therapeutic strategies to enhance the therapeutic ratio is of

utmost importance. Unfortunately, many of the currently utilized radiosensitizers are neither

selective nor tumor-specific. This is particularly a concern in the management of HNC,

38

whereby RT is the primary curative modality, yet tumors often reside in close proximity to

critical organs, which when damaged, lead to long-term compromises in patients’ quality of

life.

Despite the advances in therapeutic options over the recent few decades, treatment

toxicities and overall clinical outcomes have remained disappointing; for all sites and stages in

the head and neck region, 5-year overall survival rates still average ~50% [137]. Even the most

effective RT regimens achieve local control rates of 4555%, with disease-free survival rates

of only 3040% for patients with locally advanced HNSCC [138]. Furthermore, meta-analyses

have documented concurrent RT with chemotherapy to offer an absolute survival advantage of

only 6.5% at 5 years [141]. These modest results underscore the urgent need to develop novel

therapeutic approaches in the treatment of HNC. Accordingly, the overarching objective of this

thesis is to identify novel radiosensitizing therapies for HNC utilizing a high-throughput

screening approach to expedite the discovery process. More specifically, two strategies will be

undertaken: a phenotype-based screen utilizing small molecules (Chapter 2), and a target-

driven screen employing RNAi technology (Chapter 4).

We have previously developed a cell-based phenotype-driven HTS for the large-scale

identification of novel HNC cytotoxics, wherein cell viability served as the read-out [146,

147]; two existing anti-microbials (benzethonium chloride and alexidine dihydrochloride)

were identified from the LOPAC1280 and Prestwick chemical libraries. Thus, employing the

same forward HTS, the Spectrum Collection natural product small molecule library will be

screened for novel anti-cancer radiosensitizing compounds (Chapter 2). The recent sequencing

of the human genome and development of new genomic technologies also provides

opportunities for the discovery of novel molecularly-targeted therapies, wherein genome-wide

loss-of-function screens have moved into the research forefront. Thus, we will also harness

39

high-throughput RNAi technology for the large-scale identification of novel radiosensitizing

targets for HNC (Chapter 4). Since the colony formation assay (CFA) is the gold standard for

measuring cellular effects of radiation in vitro, an automated high-throughput CFA will be

developed to be integrated into the RNAi screen, serving as a more appropriate read-out for

radiosensitivity (Chapter 3). The discovery of novel radiosensitizing compounds or molecular

targets from both screens will be followed by the characterization of in vitro and in vivo

efficacy, elucidation of mechanisms of radiosensitization, and assessment of their clinical

implications in the management of HNC.

40

CHAPTER 2: POTENTIAL USE OF CETRIMONIUM BROMIDE AS AN

APOPTOSIS-PROMOTING ANTICANCER AGENT FOR HEAD AND

NECK CANCER

The data presented in this chapter have been published in:

Ito E, Yip KW, Katz D, Fonseca SB, Hedley DW, Chow S, Xu GW, Wood TE,

Bastianutto C, Schimmer AD, Kelley SO, Liu FF. Molecular Pharmacology

2009; 76: 969-983.

Reprinted with permission from the American Society for Pharmacology and

Experimental Therapeutics. All rights reserved.

Copyright © 2009 The American Society for Pharmacology and Experimental

Therapeutics

41

2.1 Chapter Abstract

A potential therapeutic agent for human head and neck cancer, cetrimonium bromide

(CTAB), was identified through a cell-based phenotype-driven high-throughput screen of

2,000 biologically active or clinically used compounds, followed by in vitro and in vivo

characterization of its anti-tumor efficacy. The preliminary and secondary screens were

performed on FaDu (hypopharyngeal squamous cancer) and GM05757 (primary normal

fibroblasts), respectively. Potential hit compounds were further evaluated for their anti-cancer

specificity and efficacy in combination with standard therapeutics on a panel of normal and

cancer cell lines. Mechanism of action, in vivo anti-tumor efficacy, and potential lead

compound optimizations were also investigated. In vitro, CTAB interacted additively with -

radiation and cisplatin, two standard HNC therapeutic agents. CTAB exhibited anti-cancer

cytotoxicity against several HNC cell lines, with minimal effects on normal fibroblasts; a

selectivity that exploits cancer-specific metabolic aberrations. The central mode of cytotoxicity

was mitochondria-mediated apoptosis via inhibition of H+-ATP synthase activity and

mitochondrial membrane potential depolarization, which in turn was associated with reduced

intracellular ATP levels, caspase activation, elevated subG1 cell population, and chromatin

condensation. In vivo, CTAB ablated tumor-forming capacity of FaDu cells, and delayed

growth of established tumors. Thus, using a HTS approach, CTAB was identified as a

potential apoptogenic quaternary ammonium compound possessing in vitro and in vivo

efficacy against HNC models.

2.2 Introduction

Head and neck cancer, which comprises a diverse group of cancers affecting the nasal

cavity, sinuses, oral cavity, pharynx, larynx, and other sites in this anatomical region, has an

42

estimated annual global incidence of 533,100 cases [148]. It is the eighth most common cancer

worldwide with the majority being head and neck squamous cell carcinomas [148, 149].

Nasopharyngeal cancer is a distinct HNC in that 7581% of NPC patients globally harbor the

Epstein-Barr virus [134]. HNC is a challenging disease due to its heterogeneity and

complexity of treatments. Patients with locally advanced disease achieve an overall survival

rate of only 50%, despite combined radiation therapy and chemotherapy treatments, which is

unfortunately associated with significant morbidities and toxicities [150], underscoring a

critical need to develop novel therapeutic strategies to improve clinical outcome.

We have previously developed a rapid, cell-based phenotype-driven high-throughput

screen for the large-scale identification of novel HNC cytotoxics [146, 147]. Two existing

anti-microbials (benzethonium chloride and alexidine dihydrochloride) were thus identified

from the LOPAC1280 and Prestwick chemical libraries. In the current study, the Spectrum

Collection small molecule library was screened, identifying cetrimonium bromide as an

effective compound against multiple HNC cell lines, with minimal toxicity on normal

fibroblasts; a selectivity that appears to exploit cancer-specific metabolic aberrations.

CTAB belongs to a group of quaternary ammonium compounds, which also includes

benzethonium chloride and dequalinium chloride, both of which have demonstrated anti-

cancer properties in vitro and in vivo by targeting tumor mitochondria [146, 151, 152].

Quaternary ammonium derivatives have also been reported to show enhanced anti-tumor

activity compared to their parent compounds [153], suggesting that molecules possessing

quaternary ammonium moieties may be highly effective anti-cancer agents.

CTAB is a known component of the broad-spectrum antiseptic cetrimide, which is a

mixture of different quaternary ammonium salts that has been clinically used as a tumoricidal

irrigant in colorectal cancer surgery [154], and scolicidal adjunct to hydatid cyst operations

43

[155]. However, the role of CTAB in cetrimide-mediated anti-microbial and tumoricidal

activities has not been investigated extensively; previous studies have in fact described that

pure CTAB- and cetrimide-induced anti-microbial effects occur via different mechanisms

[156]. To our knowledge, the tumoricidal potential of pure CTAB has not yet been reported,

particularly in the context of HNC. This study therefore evaluated the cancer-specific

properties of pure CTAB, and assessed its mode of action in HNC models.

2.3 Materials and Methods

2.3.1 Cell Lines

FaDu (human hypopharyngeal squamous cell cancer), A549 (non-small cell lung

adenocarcinoma), MCF7 (breast adenocarcinoma), and MRC5 (normal lung fibroblasts) were

obtained from the American Type Culture Collection (Manassas, VA). GM05757 (human

primary normal) fibroblasts were obtained from the Coriell Institute for Medical Research

(Camden, NJ). All cell lines were cultured according to the manufacturer’s specifications.

C666-1 (undifferentiated nasopharyngeal cancer) cells [157] were maintained in RPMI 1640

supplemented with 10% fetal bovine serum (Wisent Inc., Quebec, Canada) and antibiotics

(100 mg/L penicillin and 100 mg/L streptomycin) as previously described [158]. UTSCC-8A

and -42A (human laryngeal squamous cell cancer) cells were maintained in Dulbecco's

modified Eagle's medium supplemented with 10% FBS and antibiotics (100 mg/L penicillin

and 100 mg/L streptomycin); UTSCC cells were a gift from R. Grénman (Turku, Finland). All

experiments were conducted when cells were in an exponential growth phase.

2.3.2 Small Molecules

The Spectrum Collection (2,000 compounds; MicroSource Discovery Systems,

Gaylordsville, CT) was provided by the Samuel Lunenfeld Research Institute High-

44

Throughput Screening Robotics Facility (Toronto, Ontario, Canada). The compounds were

initially dissolved using the BioMek FX (Beckman Coulter Inc., Fullerton, CA) in DMSO at a

concentration of 10 mM, then diluted in sterile H2O to 0.1 mM.

Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was obtained from Sigma-

Aldrich (St. Louis, MO) and dissolved in DMSO (Sigma-Aldrich). The pan-caspase inhibitor,

benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone (Z-VAD.FMK) was

purchased from BioVision (Mountain View, CA). Oligomycin (Calbiochem, San Diego, CA)

and nigericin (Sigma-Aldrich) were dissolved in ethanol with subsequent dilutions prepared in

H2O. Cisplatin (Mayne Pharma-Hospira, Lake Forest, IL), ouabain (Sigma-Aldrich),

cetrimonium bromide (Sigma-Aldrich), and all analogues (Alfa Aesar, Ward Hill, MA):

cetyltrimethylammonium chloride (analogue 1), dodecyltrimethylammonium bromide

(analogue 2), hexyltrimethylammonium bromide (analogue 3), tetramethylammonium bromide

(analogue 4), and butyltriethylammonium bromide (analogue 5) were dissolved and diluted in

H2O to the appropriate concentrations. In all cases, the vehicle (untreated) control was H2O.

2.3.3 Small-Molecule High-Throughput Screening

The BioMek FX and Samuel Lunenfeld Research Institute High-Throughput Screening

Robotics platform were used for cell seeding, treatment, and viability assessment as previously

described [146]. Briefly, FaDu or GM05757 cells were cultured to 85% confluency,

trypsinized, and re-suspended in growth media (2.5104 cells/mL). Cells were seeded

(5103/well in 96-well plates) in 200 µL of growth medium and incubated for 24 hours at

37°C with 5% CO2 and 95% humidity. Small molecules were then added to a final

concentration of 5 μM. Cells treated with 0.1% DMSO and 166.6 μM cisplatin were used as

negative and positive controls, respectively. After 48 hours, 100 μL of growth medium was

removed from each well. The CellTiter 96 AQueous One Solution Cell Proliferation Assay (3-

45

(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,

inner salt; MTS; Promega Corp., Madison, WI) was used to detect cell viability according to

the manufacturer’s specifications. A 1-hour MTS incubation time was utilized; and 490 nm

absorbance was measured on a SpectraMax Plus384

microplate reader (Molecular Devices

Corp., Sunnyvale, CA).

2.3.4 Cell Viability Assay

Cells were seeded (5103/well in 96-well plates) in 100

µL of growth medium and

incubated for 24 hours at 37°C. The chemicals were then added, to a total volume

of 5 µL.

After 48 hours, the MTS assay was performed with DMSO (0.1%)- and cisplatin (166.6 µM)-

treated cells serving as negative and positive controls, respectively.

2.3.5 Colony Formation Assay

Cells were seeded (10210

4/well in 6-well plates) in 3 mL of growth medium and

incubated overnight at 37°C. CTAB or vehicle alone (sterile H2O) was then added at the

specified concentrations to a total volume of 50 µL. After 48 hours, fresh growth medium was

added, and the plates were incubated at 37°C. Thirteen days after seeding, colonies were fixed

in 70% ethanol, stained with 10% methylene blue, and colonies of 50 cells were counted.

Where indicated, cells were irradiated 24 hours after small-molecule treatment, delivered at

room temperature using a 137

Cs unit (Gammacell 40 Extractor, MDS Nordion, Ottawa,

Ontario, Canada) at a dose rate of 1.1 Gy/min.

2.3.6 Fluorescence Microscopy

Cells were seeded (3105/T-25 flask), incubated for 24 hours, and treated with CTAB

(5 μM; EC75) or vehicle alone at 37°C. After 48 hours, detached and adherent cells were

pooled, pelleted at 200g, and stained with 10 µM Hoechst 33342 (Invitrogen Corp.,

Carlsbad, CA)-4% formalin-PBS

solution. Representative fields were visualized and

46

photographed with a Zeiss Axioskop HBO 40 microscope (Zeiss, Thornwood, NY) under UV

illumination.

2.3.7 Caspase Activity Assay

Cells were seeded (4105/well in 6-well plates), incubated for 24 hours, and treated

with CTAB or vehicle alone. Detached and adherent cells were then collected and stained

using the CaspGLOW In Situ Caspase Staining Kits (BioVision, Mountain View, CA) for

caspase-2, caspase-3,

caspase-8, and caspase-9 activity according to the manufacturer’s

specifications. Analysis was performed using flow cytometry (FACSCalibur, CellQuest

software, Becton Dickinson, San Jose, CA).

2.3.8 Cell Cycle Analysis

Cells were seeded (3105/T-25 flask), incubated for 24 hours,

and treated with CTAB

or vehicle alone. Detached and adherent cells were then pooled, pelleted at 200g, re-

suspended in 1.5 mL of hypotonic fluorochrome solution (50 µg/mL propidium iodide, 0.1%

sodium citrate, 0.1% Triton X-100; Sigma-Aldrich), and left in the dark at 4°C overnight. Flow

cytometric analysis was then performed, and cell cycle distribution was determined using

FlowJo software (Tree Star, Inc., San Carlos, CA). Apoptotic cells were defined as cells with

DNA content less than G0/G1 (hypodiploid).

2.3.9 Transmission Electron Microscopy

Cells were treated with CTAB or vehicle alone and then processed at the University of

Toronto, Faculty of Medicine Microscopy Imaging Laboratory (Toronto, Ontario, Canada).

Briefly, harvested cells were fixed with Karnosky style fixative (4% paraformaldehyde and

2.5% glutaraldehyde in 0.1 M Sorensen’s phosphate buffer, pH 7.2) followed with 1% osmium

tetroxide. Cells were then dehydrated with ethanol, washed with propylene oxide, treated with

epoxy resin, polymerized at 60°C for 48 hours, sectioned on a Reichert Ultracut E microtome

47

to 80 nm thickness, collected on 300 mesh copper grids, and counterstained with uranyl

acetate and lead citrate. Analysis was performed on a Hitachi H7000 transmission electron

microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 75 kV.

2.3.10 Mitochondrial Depolarization, Calcium Content, and Propidium Iodide Uptake

DiIC1(5) (1,1,3,3,3,3-hexamethylindodicarbocyanine; Invitrogen) was used to

estimate mitochondrial membrane potential (M); cell permeant indo-1 AM (Invitrogen) was

used to determine changes in cytosolic calcium, and propidium iodide (Invitrogen) uptake was

used to determine cell death as previously described [159]. Briefly, cells were seeded

(0.3106/T-25 flask), incubated for 24 hours, and then treated with CTAB or vehicle alone.

Detached and adherent cells were collected, pelleted at 200g, and re-suspended in medium at

a concentration of 106/mL. DiIC1(5) (40 nM final concentration) and indo-1 AM (2 µM final

concentration) were added to the cell suspensions and incubated at 37°C for 25 minutes,

followed by the addition of propidium iodide (1 µg/mL). Cells were analyzed with a Coulter

Epics Elite flow cytometer (Beckman Coulter; DiIC1(5) excitation 633 nm, 675 20 nm

bandpass; indo-1 AM excitation 360 nm, emission ratio 405/525 nm).

2.3.11 ATP Synthase Activity Assay

Cells were cultured to confluence in a 15-cm Petri dish and pelleted at 200g. Fresh

mitochondrial ATPase was isolated (130 μg/reaction), treated with test compounds or vehicle

alone, and measured for specific activity using the MitoProfile ATP Synthase

Activity/Quantity Rapid Microplate Assay Kit (MitoSciences, Eugene, OR) according to the

manufacturer’s specifications.

2.3.12 ATP Luminescence Assay

Cells were seeded (5103/well in 96-well plates) in 200

µL of growth medium,

incubated for 24 hours at 37°C, then treated with CTAB or vehicle alone. Cellular ATP levels

48

were determined using the luciferin-luciferase based ATP Luminescence Assay Kit

(Calbiochem) as instructed by the manufacturer.

2.3.13 Plasma and Mitochondrial Membrane Potential Assays

Cells were seeded (5105/well in 6-well plates) in 3 mL of growth medium and

incubated for 24 hours at 37°C. Mitochondrial membrane potentials were estimated using the

MitoProbe JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide)

Assay Kit (Invitrogen) according to the manufacturer’s specifications. DiBAC4(3) (bis-(1,3-

dibutylbarbituric acid)trimethine oxonol; Invitrogen) was used to estimate relative plasma

membrane potentials (P). Briefly, detached and adherent cells were collected, pelleted at

200g, and re-suspended in medium containing 30 nM of DiBAC4(3). Cells were incubated at

37°C for 30 minutes and washed with PBS. Cells were analyzed with a BD LSR II flow

cytometer (BD Biosciences, San Jose, CA; DiBAC4(3) excitation/emission: 488/516 nm; JC-1

excitation 488 nm, emission ratio 595/526 nm). Data were processed with FACSDiva software

(BD Biosciences).

2.3.14 In Vivo Tumor Model

All animal experiments utilized 6 to 8 week-old severe combined immunodeficient

(SCID) BALB/c female mice in accordance with the guidelines of the Animal Care

Committee, Ontario Cancer Institute, University Health Network (Toronto, Ontario, Canada).

The mice were euthanized by CO2 once tumor-plus-leg diameters (TLD) reached 14 mm. TLD

is a well-established tool for assessing in vivo therapeutic efficacy and was employed due to

the use of intra-muscular tumor models, which are not amenable to 2D-measurements of tumor

size.

49

2.3.15 Tumor Formation Assay

Cells were seeded (2106/T-75 flask), incubated for 24 hours, and treated as indicated.

After 48 hours, cells were harvested and implanted into the left gastrocnemius muscle of SCID

mice (2.5105 cells in 100 µL growth medium per mouse), then monitored for tumor

formation by measuring TLDs thrice weekly.

2.3.16 Therapeutic Tumor Growth Assay

The intra-muscular injection of tumor cells into the hind limbs of SCID mice is a well-

established method to generate xenograft models to evaluate in vivo efficacy and potential

toxicities of new therapeutic treatments for HNC, whilst allowing the delivery of local tumor

RT [146, 158, 160]. Briefly, cells were injected into the left gastrocnemius muscle of SCID

mice (2.5105 cells in 100 µL). Once the TLDs reached an average of 7.5 mm (range 7.258.0

mm), mice were randomly assigned to one of the following groups: vehicle, CTAB, RT-plus-

vehicle, or RT-plus-CTAB. Mice were administered one intraperitoneal (IP) injection (100 µL

bolus) daily of either vehicle (PBS) or CTAB (5 mg/kg dissolved in PBS) for five consecutive

days. This dosing regimen was selected based on the CTAB in vivo toxicity profiles (Acute

Toxicity Determination) provided by the National Cancer Institute/National Institutes of

Health Developmental Therapeutics Program In Vivo Screening Database

(http://dtp.nci.nih.gov). A well-tolerated treatment schedule with no evidence of toxicity or

lethality in mice was thus selected. Local tumor RT (4 Gy) was delivered on days 2 and 5,

immediately prior to the IP injections. Briefly, mice were immobilized in a Lucite box and the

tumor-bearing leg was exposed to 100 kV (10 mA) at a dose rate of 10 Gy/min, as previously

described [158]. TLDs and body weights were recorded thrice weekly. This drug-plus-RT

regimen has been established in our lab as a standard protocol that is generally well tolerated

http://dtp.nci.nih.gov/

50

in mice, thereby allowing for direct comparisons of therapeutic efficacy between different

experimental intervention strategies in vivo.

2.3.17 Statistical Analyses

All experiments were performed at least three independent times, with the data

presented as the mean SEM. The Z factor was utilized to evaluate the high-throughput

screening power [161]. The statistical differences between treatment groups were determined

using the Student’s t test and one-way ANOVA.

2.4 Results

2.4.1 High-Throughput Screening

The preliminary screen of the Spectrum Collection small molecule library (Z factor of

0.73) was conducted on FaDu cells, which represent a clinically relevant model for the study

of HNC [162, 163]; the counter-screen was performed on GM05757 fibroblasts due to their

ease of manipulation and similar growth kinetics (~20 h doubling-time). Potential hits were

defined as compounds that: (a) decreased FaDu cell viability by 50%, but 10% in GM05757

fibroblasts or (b) induced 3-fold reduction in FaDu viability compared to GM05757.

Eighteen compounds were thus identified to demonstrate preferential toxicity against FaDu

cells (Table 2.1), ranging in function from anti-microbial, apoptosis-promoting, anti-

metabolite, to DNA alkylation. The validity of the screen was corroborated by the

identification of existing chemotherapeutic agents such as novantrone, dactinomycin, and

mechlorethamine, as well as the two recently described anti-cancer agents [146, 147].

Amongst the 18 hits, only one compound, cetrimonium bromide (Figure 2.1A), was identified

with heretofore-unreported tumoricidal properties against HNC; hence, selected for further

evaluation.

51

Table 2.1 HTS of the Spectrum Collection small molecule library for novel HNC

cytotoxics

Eighteen compounds were identified with preferential toxicity against FaDu cells. Percent

inhibition of FaDu cell viability induced by each compound is shown. Validity of the screen

was corroborated by the identification of existing chemotherapeutic agents such as novantrone,

dactinomycin, and mechlorethamine.

Compound Molecular Formula Inhibition (%)

Mitoxantrone Hydrochloride C22H30Cl2N4O6 100

Mitomycin C C15H18N4O5 96

Mechlorethamine C5H11Cl2N 91

Antimycin A C28H40N2O9 91

Deguelin C23H22O6 90

Camptothecin C20H16N2O4 89

Beta-Dihydrorotenone C23H24O6 88

10-Hydroxycamptothecin C20H16N2O5 87

Actinomycin D C62H86N12O16 87

Dihydrorotenone C23H24O6 85

Aklavin Hydrochloride C30H36ClNO10 82

Pyrromycin C30H35NO11 80

Teniposide C32H32O13S 76

Floxuridine C9H11FN2O5 72

Cetrimonium Bromide C19H42BrN 71

Alexidine Dihydrochloride C26H57ClN10 60

Benzethonium Chloride C27H42ClNO2 57

Aminopterin C19H20N8O5 51

2.4.2 Validation of HTS Hits and Evaluation of Anti-Cancer Specificity

A dose-response evaluation of CTAB on six cancer and two normal cell lines was

performed to confirm the initial high-throughput screening results, and to further assess its

anti-cancer potential. HNC is a highly heterogeneous disease; hence, we selected cell line

models representing that spectrum, ranging from nasopharyngeal, laryngeal, to

hypopharyngeal subsites. The effective concentration required to reduce cell viability by 50%

after 48 hours of treatment (EC50) was ~2 μM in FaDu, ~3.8 μM in C666-1, ~3.5 μM in

52

UTSCC-8A, and ~4.2 μM in UTSCC-42A cells (Figure 2.1B). In contrast, the EC50 values

were much higher in normal cells; ~11 μM in MRC5 and ~18 μM in GM05757 fibroblasts.

Furthermore, the other human cancer models demonstrated differential sensitivity with higher

EC50 values of ~17 μM for A549 lung and ~12 μM for MCF7 breast cancer cells. Subsequent

studies focused primarily on FaDu cells, the most CTAB-sensitive cancer cell line.

2.4.3 Evaluation of Combination Therapy

To evaluate the effect of combining CTAB with traditional HNC therapeutics, FaDu

cells were exposed to increasing concentrations of CTAB combined with -radiation or

cisplatin. The clonogenic survival curves demonstrated that CTAB interacted additively with

radiation in a dose-dependent manner (Figure 2.1C), similar to the effect observed with

cisplatin (data not shown).

53

Figure 2.1 Characterization of CTAB as a potential anti-cancer agent for HNC

54

(A) Chemical structure of CTAB. (B) Cell viability dose-response curves for CTAB in six

cancer (FaDu, C666-1, UTSCC-8A, UTSCC-42A, A549, and MCF7) and two normal

(GM05757 and MRC5) cell lines. MTS viability assays were performed 48 h after drug

treatment. Line, 50% cell viability (EC50). (C) Effect of combining CTAB with -radiation on

the clonogenic survival of FaDu cells. Cells (102–10

4 per well) were seeded and incubated

with increasing concentrations of CTAB for 48 h; where indicated, cells were irradiated 24 h

after small-molecule treatment. Ten days later, colonies were counted. Each datum represents

the mean SEM from at least three independent experiments.

2.4.4 Cetrimonium Bromide Induces Apoptosis

In an effort to elucidate the mode of cell death induced by CTAB in HNC, apoptosis

and cell cycle analyses were conducted. Hoechst 33342 staining of FaDu cells treated with

CTAB revealed nuclear condensation and blebbing, consistent with apoptotic nuclear

morphology, which was not observed in CTAB-treated GM05757 fibroblasts (Figure 2.2A).

Flow cytometric DNA content analyses also revealed a dramatic increase in the population of

FaDu and C666-1 cells with subG1 DNA content, but not for the GM05757 fibroblasts (Figure

2.2B). On the other hand, cell cycle arrest was not detected in either HNC cell line (data not

shown). CTAB-induced caspase activation was also evaluated in cells treated for 6, 12, 24, 48,

72, 96, or 120 hours (Figure 2.2C, not all data shown). Activation of the caspase cascade, a

hallmark of apoptosis, was observed as early as 12 hours in CTAB-treated FaDu and C666-1

cells, and continued to increase in a time-dependent manner; in contrast to minimal activation

in the GM05757 fibroblasts. The use of a pan-caspase inhibitor, Z-VAD.FMK, revealed

CTAB-induced cytotoxicity to be highly dependent on caspase activation (Figure 2.2D).

55

Figure 2.2 Cetrimonium bromide induces apoptosis in human HNC cells

56

(A) Hoechst 33342 staining of CTAB-treated (48 h) FaDu cells revealed condensed chromatin

with nuclear blebbing, morphologic indicators of apoptosis, which were absent in GM05757

fibroblasts. Bar, 10 μm. (B) Flow cytometric DNA content analyses of CTAB-treated FaDu

and C666-1 cells revealed a dramatic increase in the population of cells with subG1 DNA

content, but not in GM05757 fibroblasts. (C) Fluorescent caspase inhibitor peptide-based

assays demonstrated significant CTAB-induced caspase activation in FaDu and C666-1 cells,

which increased in a time-dependent manner. Minimal increases in caspase activation were

observed in CTAB-treated GM05757 fibroblasts over 1272 h. **, P0.05 and *, P0.01,

statistically significant fold differences compared to vehicle control. (D) Inhibition of caspase

activation significantly suppressed CTAB-induced apoptosis. FaDu cells were incubated with

or without Z-VAD.FMK (25 μM; 1 h) prior to CTAB treatment for 24 h. Apoptotic fractions

were assessed by flow cytometry. *, P0.01, statistically significant difference compared to

CTAB alone. Each column represents the mean SEM from three independent experiments.

In all cases, cells were treated with 5 μM of CTAB (EC75); vehicle represents sterile H2O.

Transmission electron microscopy was utilized to better define the subcellular

morphological characteristics of apoptosis, such as chromatin condensation and membrane

blebbing. Progressive morphologic abnormalities in the mitochondria were observed after 24,

48, or 96 hours of CTAB treatment in FaDu cells (Figure 2.3A), but not in GM05757

fibroblasts (Figure 2.3B); the rough endoplasmic reticulum (ER) remained relatively intact.

To further investigate the mechanism of apoptosis in CTAB-mediated cell death,

cytosolic calcium increase, which may result from damage of the ER or Ca2+

plasma

membrane channels, as well as mitochondrial membrane potential depolarization, which has

been hypothesized to be a marker of apoptotic cells [164], were evaluated. The proportion of

FaDu cells with depolarized mitochondria increased with longer treatment times (1.5% at 2 h,

4.4% at 4 h, 11.4% at 6 h, and 24.2% at 12 h, vs. 1.2% at 12 h with vehicle alone; Figure 2.3C,

box A). Furthermore, increased cytosolic Ca2+

levels could be observed in cells with

depolarized mitochondria. Loss of membrane integrity and cell death, indicated by propidium

iodide (PI) uptake, also increased with incubation time (0.4% at 2 h, 2.5% at 4 h, 6.8% at 6 h,

and 11.7% at 12 h, vs. 0.3% at 12 h with vehicle alone; Figure 2.3C, box C). The presence of a

57

cell population with decreased M that excluded PI (Figure 2.3C, box D) confirmed that the

collapse of M was a primary cellular event leading to cell death.

Figure 2.3 Evaluation of cetrimonium bromide-mediated apoptosis

58

(A) Transmission electron microscopy was used to visualize the subcellular morphological

characteristics of CTAB-induced cytotoxicity in FaDu cells. Chromatin condensation (top;

black arrow) and membrane blebbing (top; white arrow), as well as mitochondrial autophagy

(middle; arrow) were observed. Rough endoplasmic reticulum (bottom; arrow) appeared to be

unaffected. Bar, 1 μm. (B) Mitochondria (arrow) of CTAB-treated GM05757 fibroblasts

remained intact up to 96 h. Bar, 1 μm. (C) FaDu cells treated for 2, 4, 6, or 12 h with CTAB

were simultaneously stained with DiIC1(5) (M), indo-1 AM (cytosolic Ca2+

), and propidium

iodide (PI; membrane integrity/cell viability). Gates for quantification are shown. Box A,

mitochondrial membrane potential (M) depolarized cells; Box B, M polarized cells; Box

C, M depolarized and dead cells; Box D, M depolarized and viable cells; Box E, M

polarized and viable cells. Each experiment was performed three independent times. In all

cases, cells were treated with 5 μM of CTAB (EC75); vehicle represents sterile H2O.

2.4.5 Cetrimonium Bromide Perturbs Mitochondrial Function

CTAB has previously been reported to compromise bioenergetic homeostasis by

inhibiting H+-ATP synthase [165]. To determine if CTAB induced apoptosis in HNC cells via

inhibition of ATP synthase (ATPase), freshly isolated mitochondria were solubilized, treated

with CTAB, and monitored for enzymatic activity. CTAB reproducibly decreased ATPase

activity in a dose-dependent manner; achieving ~90% inhibition at 50 μM (Figure 2.4A). The

extent of inhibition by CTAB was comparable to that of oligomycin, a potent mitochondrial

H+-ATP synthase inhibitor. Ouabain, a selective Na

+/K

+-ATPase inhibitor had minimal effect

on ATPase activity, validating the specificity of H+-ATPase inhibition by CTAB.

The inhibition of mitochondrial H+-ATPase should lead to a progressive reduction in

intracellular ATP levels; this was indeed observed after 12 hours of CTAB exposure, which

caused a modest (~10%), but statistically significant decrease in ATP content in FaDu cells

(Figure 2.4B). However, by 24 hours, total intracellular ATP level fell to ~12%, and continued

to decline in a time-dependent manner. In contrast, ATP levels in GM05757 fibroblasts were

minimally perturbed.

59

Figure 2.4 Cetrimonium bromide induces mitochondrial dysfunction

(A) Effect of CTAB (2.550 μM) on mitochondrial H+-ATP synthase activity in FaDu cells.

Percent inhibition was calculated by dividing the specific enzyme activity (normalized to

protein quantity) of CTAB- vs. vehicle-treated ATPase. *, P0.01, statistically significant

difference compared to untreated cells (ATPase inhibition set as 0%). (B) GM05757 and FaDu

cells were treated with CTAB for 696 h and assessed for changes in intracellular ATP levels.

In all cases, cells were treated with 5 μM of CTAB (EC75). **, P0.05 and *, P0.01,

statistically significant differences compared to untreated cells (ATP content set as 100%).

Each column represents the mean SEM from at least three independent experiments.

60

2.4.6 Role of M in Cetrimonium Bromide-Mediated Cell Death

Previous findings have suggested that the composition and function of mitochondria in

cancer and normal cells differ, including a higher M [166]. Hence, the relative intrinsic

mitochondrial transmembrane potentials of GM05757, MRC5, A549, and FaDu cells were

measured, demonstrating that FaDu cells had the highest M compared to the low values for

both types of fibroblasts, with an intermediate value for A549 cells (Figure 2.5A). This relative

difference in M reflects the respective differential sensitivity to CTAB (Figure 2.1B).

To further investigate the determinative role of M in CTAB-mediated cell death,

FaDu cells were pre-incubated with CCCP, a protonophore that dissipates the proton gradient;

a low concentration of CCCP was used to effectively uncouple M, without perturbing P

(Figure 2.5B). Mild M uncoupling prior to CTAB treatment significantly suppressed

CTAB-induced apoptosis by 50% (Figure 2.5C). In comparison, oligomycin, another potent

ATPase inhibitor (Figure 2.4A), did not respond to changes in M (Figure 2.5D) and

demonstrated no selective cytotoxicity amongst the different cancer cell lines tested (Figure

2.5E); an observation that was expected since oligomycins are neutral macrolide antibiotics

that could induce cell death independent of M.

To examine the involvement of the electrochemical pH-gradient in CTAB-mediated

apoptosis, FaDu cells were pre-treated with nigericin, a K+/H

+ exchange ionophore that

dissipates the pH gradient across the mitochondrial membrane. Perturbing the pH gradient

prior to CTAB treatment did not protect against cytotoxicity (Figure 2.5F). In fact, a modest

dose-dependent increase in apoptosis was observed, corresponding to the compensatory

increase in M that is anticipated with a pH gradient loss [167], which in turn could enhance

CTAB accumulation within the mitochondria.

61

Figure 2.5 Role of M in cetrimonium bromide-mediated apoptosis

62

(A) Mitochondrial transmembrane potentials of GM05757, MRC5, A549, and FaDu cells. In

cells with high M, the JC-1 dye forms red fluorescent J-aggregates. JC-1 remains in the

green fluorescent monomeric form in cells with low M. The ratio of red to green

fluorescence serves as a read-out for M. *, P0.01, statistically significant difference

compared to FaDu cells. (B) FaDu cells treated with or without CCCP (5 μM) were stained

with DiBAC4(3) and DiIC1(5) to measure relative changes in P and M, respectively. **,

P0.05, statistically significant fold difference compared to untreated cells. (C) Effect of M

on CTAB-mediated cytotoxicity. FaDu cells were incubated in medium with or without CCCP

(5 μM; 1 h) prior to CTAB treatment (5 μM) for 24 h. SubG1 apoptotic fractions were assessed

by flow cytometry. **, P0.05, statistically significant difference compared to CTAB alone.

(D) Effect of M on oligomycin-mediated cytotoxicity. FaDu cells were incubated in

medium CCCP (5 μM; 1 h) prior to oligomycin (OLIG) treatment (30 μM; EC75) for 48 h.

SubG1 apoptotic fractions were assessed by flow cytometry. (E) Cell viability dose-response

curves for oligomycin in four cancer (FaDu, C666-1, A549, and MCF7) cell lines. MTS assays

were performed 48 h after drug treatment. Line, 50% cell viability (EC50). (F) FaDu cells pre-

incubated in medium nigericin (5 or 10 nM; 1 h) prior to CTAB treatment (5 μM; 24 h) were

assessed for apoptosis via flow cytometry. Each datum represents the mean SEM from at

least two independent experiments.

2.4.7 Elimination of Tumor Formation

To evaluate the effect of CTAB on tumorigenesis in vivo, FaDu cells treated with

CTAB (EC75) were injected into the left gastrocnemius muscle of SCID mice (2.5105

cells/mouse); establishing a three-dimensional system that simulates the complex tumor micro-

environment. Mice implanted with CTAB-treated FaDu cells did not develop tumors even

after 100 days (Figure 2.6A). In contrast, mice with vehicle-treated cells (implanted with

6.25104 cells, representing the proportion of viable cells post-treatment with EC75),

developed tumors as early as 15 days, clearly demonstrating that CTAB effectively eliminated

the tumor-forming potential of FaDu cells in SCID mice.

2.4.8 Growth Delay in Established Xenograft Tumors

The therapeutic efficacy of CTAB in treating established FaDu xenograft tumors in

SCID mice was also evaluated. Once the TLDs reached an average of 7.5 mm, the mice were

treated with CTAB (daily 5 mg/kg IP for 5 days). The dosing regimen was not optimized for

63

absorption, distribution, metabolism, or excretion, but a delay in tumor growth (i.e. therapeutic

benefit) was nonetheless observed. CTAB induced a modest reduction in tumor development

compared to the vehicle-treatment arm; delaying the mean time to reach a TLD of 14 mm by

~3.7 days (P0.05; Figure 2.6B). When combined with local tumor RT, CTAB appeared to

have a modest additive effect by extending the mean time to reach 14 mm by ~7.2 days

(P0.05; Figure 2.6B). These data strongly suggest that improving the pharmacokinetics and

bioavailability of CTAB would render this compound highly effective; as the in vivo tumor-

forming capacity of FaDu cells was completely ablated when every tumor cell was exposed to

the drug (Figure 2.6A).

2.4.9 In Vivo Safety and Toxicity

To assess the in vivo safety and toxicity of our CTAB dosing regimen (~0.05% IP), the

body weights of tumor-bearing mice were monitored. The four treatment groups exhibited no

significant difference in overall body weight (Figure 2.6C), indicating that this treatment was

well tolerated, as no evidence of toxicity or lethality was observed.

64

Figure 2.6 In vivo efficacy of cetrimonium bromide

65

(A) FaDu cells treated with vehicle (H2O) or CTAB (5 μM) for 48 h were injected into the left

gastrocnemius muscle of SCID mice. CTAB-treated cells did not form tumors even after 100

days. (B) FaDu xenograft tumors were established in SCID mice; once the tumor-plus-leg

diameters (TLD) reached 7.5 mm, the mice were randomly allocated to one of the following

groups: vehicle, CTAB, local radiation therapy (RT)-plus-vehicle, or RT-plus-CTAB. The

mice were administered one IP injection (5 mg/kg) daily of either vehicle (H2O) or drug for

five consecutive days. Local tumor RT (4 Gy) was delivered on days 2 and 5 before the IP

injections. The mice were euthanized once TLDs reached 14 mm. Solid line, mean time to

reach a TLD of 14 mm. *, P0.05, statistically significant difference between CTAB vs.

vehicle or RT-plus-CTAB vs. RT-plus-vehicle. (C) Total body weight was also recorded for

each group, demonstrating no significant difference. Each datum represents the mean SEM

from three independent experiments (3 mice/treatment group/experiment).

2.4.10 Evaluation of Cetrimonium Bromide Analogues

To explore the structure-function relationship of CTAB with a focus on understanding

the importance of its chain length, five commercially available analogues were evaluated on

FaDu and GM05757 cells (Figure 2.7A). Substitution of Br− with Cl

− did not significantly

diminish the inhibitory actions of the compound (analogue 1). Complete removal of the alkyl

chain however, abolished any anti-cancer activity (analogue 4). Derivatives with carbon chains

Cn12 demonstrated a complete loss of inhibition (analogues 35); as did the sterically bulky

quaternary ammonium group of analogue 5. Only analogues 1 (cetyltrimethylammonium

chloride) and 2 (dodecyltrimethylammonium bromide) retained cytotoxicity and bioactivity

with EC50 values similar to those measured for CTAB: 2.5 μM vs. 14 μM for analogue 1, and 4

μM vs. 30 μM for analogue 2, in FaDu and GM05757 cells, respectively (Figures 2.7B and C).

66

Figure 2.7 Anti-cancer efficacy of cetrimonium bromide analogues

67

(A) Chemical structures of CTAB, cetyltrimethylammonium chloride (analogue 1),

dodecyltrimethylammonium bromide (analogue 2), hexyltrimethylammonium bromide

(analogue 3), tetramethylammonium bromide (analogue 4), and butyltriethylammonium

bromide (analogue 5). (B) Cell viability dose-response curves for CTAB and analogues 15 in

FaDu cells. (C) Dose-response curves for CTAB and analogues in GM05757 fibroblasts. Line,

50% cell viability (EC50). Only analogues 1 and 2 retained selective anti-cancer cytotoxicity

and bioactivity with EC50 values similar to CTAB. MTS viability assays were performed 48 h

after drug treatment. Each datum represents the mean SEM from three independent

experiments.

2.5 Discussion

In the current study, a phenotype-driven HTS of the Spectrum Collection small molecule

library was performed for the large-scale identification of novel HNC cytotoxics. Cetrimonium

bromide, an existing anti-microbial [168], was identified to have anti-cancer efficacy against

several human HNC cell lines with minimal toxicity towards normal cells. Our data document

CTAB to significantly compromise mitochondrial bioenergetic function, inducing cell death

primarily through the intrinsic caspase-dependent apoptotic pathway; non-apoptotic death such

as senescence and mitotic catastrophe were not observed (data not shown). FaDu cells, which

represent a highly aggressive HNC cell line, sustained sufficient damage upon CTAB

treatment to irreversibly inhibit the clonal growth of cultured carcinoma cells in vitro, and

ablate tumorigenicity in vivo. When combined with local RT, CTAB delayed tumor growth

whilst maintaining a favorable toxicity profile. CTAB is a known component of cetrimide,

which has been routinely used during hydatid cyst, and colorectal surgeries at concentrations

that are clinically well tolerated. In rare cases, cardiac ischemia, chemical peritonitis, and

methemoglobinemia have been reported with cetrimide concentrations ranging from ~15%

[168, 169]; 12% cetrimide solutions found in common household products have been

occasionally associated with erythema and skin blistering [170]. No clinical case reports of

toxicity have been described relating to administration of pure CTAB. Nonetheless, the CTAB

68

dosing utilized in our study was well tolerated in the treated mice, with good maintenance of

their body weights.

CTAB is a quaternary ammonium compound, belonging to a group of small molecules,

known as delocalized lipophilic cations (DLCs). Due to their lipophilic nature and delocalized

positive charge, DLCs can penetrate the hydrophobic barriers of plasma and mitochondrial

membranes, and accumulate in the mitochondria in response to the negative transmembrane

potential, resulting in mitochondriotoxicity [171]. Accordingly, the determinative role of M

in CTAB-mediated cytotoxicity was demonstrated as mild M uncoupling prior to CTAB

treatment significantly suppressed the overall level of apoptosis in FaDu cells (Figure 2.5C);

while perturbation of the mitochondrial pH gradient and corresponding compensatory M

increase via nigericin enhanced CTAB-induced apoptosis (Figure 2.5F).

Dysregulation of mitochondrial functions and aberrant metabolic bioenergetics are

mechanisms cancer cells have developed to resist mitochondrial-mediated apoptosis, thereby

surviving in the toxic tumor micro-environment [172]. These features however, can be

exploited for the development of novel anti-cancer therapies targeting mitochondrial proteins

and membranes to promote cell death. Elevated intrinsic plasma and/or mitochondrial

membrane potentials have been reported for various cancer cells [167, 173, 174]; with higher

M attributed to the buildup of the mitochondrial proton gradient, resulting from reduced

oxidative phosphorylation (OXPHOS) [171, 175]. Such M differences of 60 mV can

therefore result in a 10-fold accumulation of DLCs in tumor vs. normal mitochondria [176].

The degree of glycolytic up-regulation also varies between different tumors, which

might in part explain the differential sensitivity to CTAB amongst the various cancer cell lines.

Head and neck cancers, which are often hypoxic, are commonly associated with high aerobic

glycolytic activity and increased aggressiveness [177, 178]; while the MCF7 breast and A549

69

lung cancer cells have relatively lower aerobic glucose consumption rates [179]. Accordingly,

we observed A549 cells to have lower intrinsic M than FaDu cells (Figure 2.5A);

correlating with their relative cytotoxicity profiles (Figure 2.1B). Taken together, the basis of

selectivity of CTAB against HNC cells appears to be rooted at the mitochondrial level, with

subtle differences in M being a key regulator. Thus, CTAB would be predicted to be more

effective against tumors that rely heavily on glycolysis, and are dependent on the Warburg

effect.

Once CTAB is concentrated into the tumor mitochondria, the H+-gradient across the

inner mitochondrial membrane may begin to dissipate, with the consequent M decline

sensed by the mitochondrial permeability transition pore (PTP) [180]. Opening of the PTP

causes mitochondrial outer membrane permeabilization (MOMP), a pivotal event in the

intrinsic apoptotic pathway, leading to the disruption of essential mitochondrial functions,

along with release of apoptogenic factors, such as cytochrome c [181]. We detected M

depolarization as early as 2 hours post-treatment (Figure 2.3C) with caspase-9 activation after

12 hours (Figure 2.2C), indicating that mitochondrial damage is an early event in CTAB-

induced cell death. Structural abnormalities observed at 24 hours (Figure 2.3A) may represent

dysfunctional mitochondria that are being eliminated via autophagy [182]. The high levels of

initiator caspase-9 activation (Figure 2.2C) suggest that mitochondria-mediated apoptosis may

be the primary mechanism by which CTAB exerts its cytotoxic effect.

Increased cytosolic Ca2+

levels were also detected in cells with depolarized

mitochondria, which may be associated with endoplasmic reticulum Ca2+

release during ER-

stress induced apoptosis. Increased cytosolic Ca2+

can trigger mitochondrial Ca2+

overload,

resulting in M collapse, with subsequent MOMP and cytochrome c release, thereby

activating the caspase cascade [183]. The activation of initiator caspases-2 and -8, which was

70

observed to a lesser extent in CTAB-treated HNC cells (Figure 2.2C), is also involved in the

ER-stress response [183, 184]. Collectively, this suggests that activation of both ER- and

mitochondria-mediated apoptotic pathways is responsible for CTAB-induced cytotoxicity.

We and others [165] have demonstrated that CTAB compromises mitochondrial

bioenergetic regulation via inhibition of ATP synthase, which consists of the membrane-

embedded F0 (H+-translocation) and peripheral catalytic F1 (ATP synthesis/hydrolysis)

subcomplexes [185]. The ATPase couples the electrochemical H+-gradient to ATP

synthesis/hydrolysis and is responsible for maintaining the M in response to changes in the

proton motive force [185, 186]. Thus, mitochondrial repolarization via ATP hydrolysis may

occur to counteract the CTAB-induced depolarization in cancer cells. The ability of CTAB to

directly bind and inhibit ATPase will prevent M repolarization, serving as another means of

committing cancer cells to death. It should be noted that the CTAB concentrations required to

significantly inhibit ATPase activity were higher than the cytotoxic doses in FaDu cells

(Figures 2.4A and 1B). Previous studies have observed that higher levels of CTAB are

necessary to inhibit the activity of purified ATPase vs. the enzyme in the presence of sub-

mitochondrial particles (membrane-bound ATPase) [165], which could potentially explain the

difference in concentrations. In comparison, the neutrally charged oligomycin, which was

unresponsive to subtle M changes (Figure 2.5D), and thus demonstrated no selective

cytotoxicity amongst different cancer cell lines (Figure 2.5E), was able to inhibit ATPase at

concentrations much lower than its cytotoxic doses (Figures 2.4A and 2.5E). Thus, CTAB-

induced cell death involves, at least in part, ATPase inhibition, although this might not be its

primary mode of action.

Since mitochondria-mediated cytotoxicity is complex, and can proceed simultaneously

via multiple mechanisms, additional mitochondriotoxic effects cannot be ruled out. The

71

preliminary biological action of cationic CTAB may be the M-driven accumulation in the

tumor mitochondria, initiating a multitude of secondary effects (M depolarization, lipid

peroxidation, ATPase inhibition, etc.) that collectively perturb mitochondrial function and

ultimately, induce apoptosis. The CTAB-induced onion-skin appearance of damaged

mitochondria (Figure 2.3A) is consistent with lipid peroxidation, which has also been reported

with other DLCs via membrane intercalation and reactive oxygen species, resulting in

membrane permeabilization [187, 188]. Thus, the possibility of CTAB promoting membrane

lipid peroxidation also warrants further evaluation.

Interestingly, CTAB has recently been implicated in the regulation of OXPHOS

expression [189], whereby it decreased the transcription of nuclear-encoded OXPHOS genes,

including atp5a1, atp5c1, and atp5o, all of which encode subunits of the ATPase F1 complex.

Furthermore, CTAB has been shown to specifically interact with negatively charged acidic

residues buried in the hydrophobic environments of the F1 moiety [165]. These findings point

towards a unique mechanism by which CTAB appears to be able to down-regulate the

transcription of certain ATPase subunits, as well as physically inhibit their enzymatic

activities.

The desirable anti-cancer activity of CTAB suggests that analogues based on structural

modification may result in more efficacious lead compounds. As such, commercially available

derivatives were exploited to examine the structure-function relationship of CTAB. Our results

indicate that the combination of both the positively charged quaternary nitrogen and non-polar

hydrophobic alkyl chain are indispensable for its cytotoxic effect. This inhibitory action also

appeared to be highly dependent on the length of the alkyl chain, as analogues with shorter

tails exhibited reduced cytotoxicity. Additional testing of cetrimonium analogues with longer

alkyl chains may provide useful starting points for further lead optimization. Taken together,

72

our results suggest that the positively charged polar head of CTAB provides the basis for its

anti-cancer specificity, while the non-polar hydrophobic tail may aid in its insertion into the

plasma membrane. The lipophilic nature, delocalized positive charge, and structural similarity

to sphingosine, a primary component of sphingolipids, may allow CTAB to readily penetrate

the hydrophobic barriers of the lipid bilayer and accumulate within the tumor cell.

In conclusion, we have identified CTAB as a clinically relevant, novel anti-cancer agent

for HNC. P53 is mutated in over ~50% of human cancers [190] and is correlated with poor

prognosis and enhanced resistance to commonly used chemotherapeutic agents [191].

Examination of CTAB-treated wild-type (p53+/+

) and mutant (p53-/-

) colon cancer HCT116

cells [192] demonstrated very similar sensitivity (data not shown), suggesting that CTAB-

mediated toxicity is independent of p53 status; thereby increasing the potential applicability of

CTAB to many different human cancers. Moreover, its favorable toxicity profile, ability to

induce apoptosis in cancer cells at much lower concentrations than its anti-microbial

application [168], and capacity to delay tumor growth in FaDu xenograft models comparable

to paclitaxel [193], a commonly used chemotherapeutic agent in the clinical management of

HNC patients [194], all suggest that optimizing the bio-availability and pharmacokinetics of

CTAB could provide an exciting opportunity for the development of a highly effective drug

candidate, capable of exploiting the metabolic aberrations of human head and neck cancers.

2.6 Acknowledgments

We thank Alessandro Datti, Thomas Sun, and Frederick Vizeacoumar from the Samuel

Lunenfeld Research Institute High-Throughput Screening Robotics Facility for their assistance

with the high-throughput screen.

73

CHAPTER 3: INCREASED EFFICIENCY FOR PERFORMING

COLONY FORMATION ASSAYS IN 96-WELL PLATES - NOVEL

APPLICATIONS TO COMBINATION THERAPIES AND HIGH-

THROUGHPUT SCREENING

The data presented in this chapter have been published in:

Katz D*, Ito E*, Lau KS, Mocanu, JD, Bastianutto, C, Schimmer, AD, Liu, FF.

Biotechniques 2008; 44: ix-xiv. *These authors contributed equally to this work.

Reprinted with permission from BioTechniques. All rights reserved.

Copyright © 2008 BioTechniques.

74


The colony formation assay is the gold standard for measuring the effects of cytotoxic

agents on cancer cells in vitro; however, in its traditional 6-well format, it is a time consuming

assay, particularly when evaluating combination therapies. In the interest of increased

efficiency, the 6-well CFA was converted to a 96-well format using an automated colony

counting algorithm. The 96-well CFA was validated using ionizing radiation therapy on the

FaDu (human hypopharyngeal squamous cell) and A549 (human lung) cancer cell lines. Its

ability to evaluate combination therapies was investigated by the generation of dose-response

curves for the combination of cisplatin and RT on FaDu and A549 cells. The 96-well CFA was

then transferred to a robotic platform for evaluating its potential as a high-throughput

screening read-out. The LOPAC1280 library was screened against FaDu cells, and 8 putative

hits were identified. Using the 96-well CFA to validate the 8 putative chemicals, 6/8 were

confirmed, resulting in a positive hit rate of 75%. These data indicate that the 96-well CFA can

be adopted as an efficient alternative assay to the 6-well CFA in evaluating single and

combination therapies in vitro, providing a possible read-out that could be utilized on a HTS

platform.

3.2 Introduction

The colony formation assay has been the gold standard for determining the effects of

ionizing radiation therapy on in vitro cellular systems since first described by Puck and

Marcus in 1956 [4]. Despite being the gold standard, the CFA can be time consuming when

counting the number of colonies manually under the microscope. For this reason, many

different assays have been used in lieu of the CFA in order to assess the effects of cytotoxic

agents on cancer cell growth in vitro. While many of these techniques are able to detect

75

specific cellular processes such as apoptosis [195], proliferation [196], or senescence [197],

the CFA is the only assay that monitors a cancer cell’s ability to produce a viable colony after

treatment. Unlike most other assays, the CFA is unbiased to the mode of cell death. It is able to

detect the cytotoxic effect of an agent, regardless of mechanism, as long as the agent affects

the cell’s reproductive ability to form progenies.

Most of the advancements made in cancer therapy in recent years have resulted from

the combination of previous individual modalities, such as radiation and chemotherapy.

Chemotherapy with such agents as cisplatin, 5-fluorouracil, doxorubicin, temozolomide, or

cetuximab, have been combined with radiotherapy for the treatment of head and neck cancer

[198], non-small cell lung carcinoma [199], glioblastoma [96], cervix [84], and bladder

cancers [200], to name a few. Initially, discovery of such combinations was conducted in the

laboratory using tissue culture as the primary testing platform. To assess novel potential

combinations, the majority of experiments were performed in a traditional 6-well tissue culture

plate CFA, with each plate representing a different combination of two potential treatments.

Commonly, such experiments test up to six different doses of radiation (010 Gy) and up to

seven different doses of drug [201]. Using the traditional CFA, this would result in utilizing 42

individual plates. Beyond the technical challenges of conducting an experiment with 42

individual plates, a significant amount of time would be required to manually count the

colonies on all such plates. This underscores the need for a more modern approach to this

assay. With recent improvements in fluorescent probes and high-content microscopy, it is now

possible to adapt the traditional method to a more efficient approach using a 96-well plate and

an automated colony counting algorithm. While this article describes this novel approach in

evaluating combination therapies including radiation and chemical treatment, it can be

76

extended to any combination of treatments including two different chemical treatments

administered concurrently.

Another area of advancement in cancer research is the use of high-throughput

screening for the identification of novel anti-cancer compounds. There are two basic

approaches to HTS. The forward chemical biology approach identifies a phenotype of interest,

after treatment with chemical compounds, and the mechanism is subsequently elucidated

[118]. The reverse chemical biology approach identifies a target, and “hits” are selected as

compounds that modulate that specified molecule [118]. Both forward and reverse approaches

have yielded clinically useful anti-cancer drugs. The forward chemical biology approach is

illustrated by the use of paclitaxel, which was shown to be effective against tumors long before

it was identified to target microtubules [121]. The reverse chemical biology approach has

been highly successful in the identification of the bcr-abl inhibitor, Imatinib (Novartis), used

for treatment of chronic myelogenous leukemia [123], and the src-abl kinase inhibitor,

Dasatinib (Bristol-Myers Squibb), used to treat imatinib-resistant chronic myelogenous

leukemia [202].

In the current study, we have adapted the 6-well CFA to a more efficient 96-well CFA

that will allow for rapid analysis of combination therapies, and opens the potential for high-

throughput drug screening using the CFA as the read-out.


3.3.1 Cell Lines

Human head and neck squamous cell carcinoma FaDu cells were cultured in MEM-

F15 medium containing 10% FBS, 1 mM pyruvate, 1.5 g/L sodium bicarbonate, 100 mg/L

77

penicillin and 100 mg/L streptomycin. Human lung adenocarcinoma A549 cells were cultured

in RPMI media containing 10% FBS, 100 mg/L penicillin and 100 mg/L streptomycin.

3.3.2 6-Well Colony Formation Assay

Cells were trypsinized and plated in 6-well dishes at different densities depending on

the potency of the treatments (from 50104 cells/well). Cells were allowed to attach overnight

and then exposed to RT (016 Gy) or chemical treatment at the corresponding dilution. Forty-

eight hours after chemical treatment, the media was replaced with fresh media, and the plates

were incubated at 37°C. Seven to eleven days later, the cells were fixed and stained with 10%

methylene blue in 70% ethanol. The number of colonies, defined as 50 cells/colony were

counted, and the surviving fraction was calculated as the ratio of the number of colonies in the

treated sample to the number of colonies in the untreated sample. Triplicate wells were set up

for each condition.

3.3.3 96-Well Colony Formation Assay

Cells were trypsinized and plated in 96-well plates at different densities depending on

the stringency of the treatments (from 502500 cells/well). The cells were allowed to attach

overnight and then exposed to RT (016 Gy) or chemical treatment at the corresponding

dilution. RT was administered 24 hours after addition of the chemical, and the media was

replaced 48 hours later. Six days after seeding, cells were fixed in 3.7% formaldehyde at room

temperature for 15 minutes followed by staining with 10 μM Hoescht 33342 (Invitrogen;

Carlsbad, CA, USA) and 10 μM Cell TrackerTM

Orange CMTMR (Invitrogen; Carlsbad, CA,

USA) in serum-free media, and incubated at 37°C for 30 minutes. After staining, the wells

were scanned (4 fields/well) at 4x objective using the IN Cell Analyzer 1000 (GE Healthcare;

Buckinghamshire, England). The excitation and emission filters used for Hoescht 33342 and

Cell TrackerTM

Orange were 360 nm/460 nm and 535 nm/620 nm, respectively. Colonies were

78

recognized by an algorithm setup on the Developer Toolbox software (GE Healthcare;

Buckinghamshire, England) using the overlay of the blue and red images. The algorithm’s

output indicated the location of the colony within the plate, and the number of blue nuclei

contained within the colony. The number of colonies per well was then filtered by a custom

computer program (http://www.uhnres.utoronto.ca/labs/liu/ACC/) to include all colonies with

6 cells/colony and 350 cells/colony. The surviving fraction was then calculated by dividing

the number of colonies by the number of cells seeded, multiplied by 2.217 (to account for the

proportion of the well that was scanned), then divided by the surviving fraction of untreated

cells. Three to eight replicates were set up per treatment.

3.3.4 High-Throughput Screening

FaDu cells were seeded in 96-well plates at a dilution of 250 cells per well in 100 μL of

media using a Biomek®

FX liquid handler (Beckman Coulter; Fullerton, CA, USA). After

allowing the cells to attach overnight, the LOPAC1280 (Sigma-Aldrich; St. Louis, MO, USA)

library of compounds was added to the cells at a final concentration of 0.5 μM. On each plate,

column 1 was the vehicle control and column 12 was 0.5 μM cisplatin, providing the positive

cytotoxic control. After 48 hours, the chemical containing media was removed and fresh media

was replaced in all wells. After 72 hours, the cells were dual stained using Hoescht 33342 and

Cell TrackerTM

Orange CMTMR; the number of colonies per well was then determined, as

described previously for the 96-well CFA. All reagents and media were added and removed

using the robotic platform available at the Samuel Lunenfeld Research Institute Robotics

Facility. The B-score was then used to normalize the data with respect to systematic variation

between plates using the HTS Corrector software [203]. Putative hits were then determined to

be any compounds with a B-score lower than 3 standard deviations from the median B-score.

The LOPAC1280 library was supplied by the Samuel Lunenfeld Research Institute and

79

screened in two independent experiments, the first using plates 1 through 8, and the second

using plates 9 through 16. The data were analyzed and putative hits were selected separately.

All putative hits were re-ordered from Sigma-Aldrich, and fresh batches were used for follow

up testing.

3.4 Results and Discussion

The primary objective of this study was to automate the conduct of the CFA, thereby

increasing efficiency in performing large-scale high-throughput experiments. This task was

addressed by using high-content microscopy, with differentially staining fluorescent dyes for

morphologic distinction of the cell’s nucleus vs. cytoplasm (Figure 3.1A). This allowed the

determination of not only the number of colonies, but also the size of each colony. This

additional level of assessment thereby allowed the filtering of data by removing any colonies

that resulted from a radiation-induced giant cell, or a small aggregate of cells that would not

score as a legitimate colony. The IN Cell Analyzer 1000 provided the necessary flexibility that

could automate both image acquisition and analysis. Upon acquiring the images and utilizing

the Developer Toolbox software, the images were analyzed using an algorithm that first

identified a colony based on adjacent signals from the cytoplasm stained with Cell TrackerTM

Orange CMTMR. Once the colony was defined, the number of cells within this colony was

determined by counting the number of Hoescht 33342 stained nuclei (Figure 3.1B). After

analysis of all the images on a single plate, a list of the well location of each colony, and its

corresponding number of nuclei, was produced (Figure 3.1C). A customized program then

converted this list into a matrix representing the number of colonies per well (Figure 3.1D).

In this process, a filter was also included to define the minimum and maximum number

of nuclei necessary in order to qualify as a colony. The minimum number of cells per colony is

80

determined by assuming a “least growth” scenario wherein cells do not start dividing until the

chemical has been removed, allowing for 72 hours of growth. Given the doubling time of the

cell line being investigated, one could then determine the number of divisions a single cell will

undergo, thereby providing an estimate for the minimum number of cells in a colony. In the

case of FaDu cells, with a doubling time of ~22 hours [204], approximately 3 doublings should

have occurred within 72 hours, thus a colony should constitute at least 8 cells. For practical

purposes, the threshold was set between 6 to 350 nuclei per colony. Once the number of

colonies per well has been determined, the surviving fraction was then calculated in the same

manner as the traditional CFA (Figure 3.1E).

81

Figure 3.1 Schematic representation of the 96-well colony formation assay

(A) Flourescence image obtained using the INCell Analyzer 1000 at 4x objective of FaDu cells

stained with Cell TrackerTM

Orange CMTMR and Hoescht 33342. (B) Colonies are defined

using the Developer Toolbox software as an adjacent set of cytoplasmic signals (blue outline)

containing stained nuclei (green outline). (C) The readout from Developer Toolbox is an excel

worksheet that lists all colonies with their corresponding number of nuclei within a specific

well. (D) A custom software program was created to convert the list output into a matrix

containing the number of colonies per well on the original plate, filtered to include only

colonies with 6 and 350 nuclei. (E) The matrix data are then plotted as a clonogenic

survival curve.

82

Historically, the CFA was first described as a method for assessing the effects of

radiation [4]. Since that time there have been many modifications to this assay with the use of

fluorescent dyes and smaller formats [205], however it has yet to be clearly illustrated whether

these modified protocols are able to recapitulate the traditional gold-standard assay. In order to

clearly validate the previously described protocol, the first set of 96-well experiments was

conducted using RT on the FaDu cells since they produced well-defined single colonies,

facilitating miniaturization of the CFA. At a dilution of 100 cells/well in the control

(untreated) sample, the colonies remained discrete and separable by the image analysis

software after 7 days’ growth. This was achieved in the untreated samples, and could be

extended out to 2500 cells/well in samples exposed to 16 Gy. Survival curves were then

generated using the traditional 6-well CFA format, and compared with that of the 96-well CFA

(Figure 3.2A). The two curves overlap almost completely, indicating that the 96-well CFA

could recapitulate the 6-well CFA. The survival curves generated with the 96-well format

however could only be extended to 8 Gy RT, since beyond this dose, too many radiation-

induced giant cells would confound the analysis. To determine if this method could be

applicable to other cell lines, the experiments were repeated using the A549 cell line (Figure

3.2B). Once again, the 96-well data set was able to reproduce the data from the 6-well assay,

indicating that the 96-well format could recapitulate the traditional assay in the context of

radiation treated colony forming cell lines.

83

Figure 3.2 Reproducibility of a 96-well CFA compared to a traditional 6-well CFA

(A) CFAs were performed on FaDu cells using the 96-well and the 6-well assays. (B) CFAs

were performed on A549 cells using the 96-well and the 6-well assays. Each datum represents

the mean SEM from 3 independent experiments for 6-well data and 2 independent

experiments for 96-well data.

The subsequent studies proceeded to evaluate combination treatments, a major objective

in improving cancer therapy. Hence, FaDu cells were treated with RT combined with

cisplatin, a common clinical regimen for treatment of head and neck squamous cell carcinoma

patients. Again, the 96-well assay appeared to be an excellent representation as the RT alone

curve replicated that of the 6-well assay results (Figure 3.3A). Similarly, the cisplatin data

agree with previously published reports whereby ~50% survival level was observed when

FaDu cells were treated with 0.5 μM cisplatin [206]. The corresponding experiments were

performed with the A549 cells, with similar results obtained (Figure 3.3B).

84

From the time of staining of the plates, the data were obtained within 24 hours using

automated counting. Comparison of estimated time required for these assays indicated time

savings at almost every step of the protocol with the 96-well CFA, but the most substantial

gain was observed at the colony counting step (Table 3.1). Given that the approximate reagent

and consumable costs of both the 6-well and the 96-well assays are similar; this demonstrates

the substantial gain in efficiency of generating and analyzing such volumes of data,

particularly for the determination of multiple permutations of combinatorial therapies. From an

analytical perspective, many different statistical methods could be used to test for additive,

synergistic, or sub-additive interactions of combined therapies. These methods include

isobologram analyses [207], mean inactivation dose [208], or the median effect principle

[209], all of which require the generation of data using a broad range of concentrations of

different treatments. Such experimental data are time consuming to generate using the

traditional 6-well format, which is easily overcome by the efficiency of the 96-well CFA,

enabling the collection of masses of data required for such analyses in a much shorter time

frame.

Table 3.1 Comparison of 96-well and 6-well clonogenic assays

Estimated time savings calculated when using the 96-well CFA vs. the 6-well CFA in a typical

combination therapy experiment with 8 compound doses and 4 RT doses (32 individual

conditions).

Parameters 96-well 6-well

Number of plates used 4 32

Cell seeding 10 min 90 min

Addition of compounds 30 min 90 min

Radiation 15 min 60 min

Change media 10 min 90 min

Colony counting 3 h 12 h

Data analysis Same

Time savings ~14 h

85

The final question we sought to address using the 96-well CFA was its utilization for

HTS of chemical libraries. Our lab has previously identified novel anti-cancer activities of

existing anti-microbial compounds, such as alexidine dihydrochloride [147] and benzethonium

chloride [146]. However the read-out for these screens was the tetrazolium-based MTS assay,

which is a measure of mitochondrial enzymatic activity. There are other read-outs available for

determining cytotoxicity [210], or other specific cellular events [211, 212], however these

assays do not measure reproductive potential. Hence, the 96-well CFA was adopted for the

HTS, anticipating the identification of novel and more potent anti-cancer agents. As an initial

proof of concept experiment, the protocol for performing the assay was transferred to a robotic

platform. The LOPAC1280 library was screened at 0.5 μM final concentration of all

compounds for their ability to inhibit clonogenic growth, as measured by the number of

colonies per well normalized to untreated samples. To correct for systematic variation, the data

were normalized using the B-score, a statistical analysis that accounts for variability across

rows, columns, and plates [213]. After normalization by the B-score, only compounds whose

score was 3 standard deviations from the median B-score value were selected as putative

“hits”. This resulted in the identification of eight such “hits”, many being known anti-cancer

agents such as mitoxantrone [214], idarubicin [215], vincristine [216], and vinblastine sulfate

[216]. All eight of these “hits” were secondarily screened using the 96-well CFA; if the

subsequent IC50 value was 0.5 μM, then they were considered as a confirmed “hit” (Figures

3.3C and D). If the compounds had an IC50 0.5 μM, then they were rejected as a false positive

result. Six of the eight “hits” (75%) were thus confirmed as positive hits (Table 3.2). This

experiment confirmed the proof of concept and illustrated that the 96-well CFA can be used as

a HTS read-out to discover anti-cancer drugs. In this case, the 6 confirmed anti-cancer drugs

were identified from a library of 1,280 compounds for a confirmed hit rate of ~0.5%. In

86

previous screenings of this library conducted by our lab, a confirmed hit rate of ~2% was

observed [146]. However, in these experiments the library was screened at a 10 times higher

concentration. This suggests that the CFA is a suitable read-out in terms of its ability to

identify anti-cancer compounds. One drawback of this read-out is related to the restricted

dynamic range in the 96-well CFA format. Subsequent to the miniaturization of the assay, the

variability observed in the screen as a percentage of the difference between the number of

colonies between the positive and negative controls becomes significant. This underscores the

possibility of a higher false positive hit rate of ~25%, thereby relying on a more rigorous

secondary screen to filter out the unconfirmed “hits”. The advantage of this novel read-out for

HTS is that it is an unbiased assay with respective to the mode of cell death. As long as a

compound inhibits a cancer cell’s ability to grow into a larger population it will be detected

using this assay.

87

Figure 3.3 Dose response curves created using the 96-well CFA

(A) Evaluation of the 96-well CFA on FaDu cells for the analysis of combination therapy of

RT (06 Gy) and cisplatin (01 μM). (B) Evaluation of the 96-well CFA on A549 cells for the

analysis of combination therapy of RT (06 Gy) and cisplatin (00.5 μM) (C) Survival curve

created using the 96-well CFA on FaDu cells treated with ouabain (00.4 μM), an example of

a confirmed hit from the HTS. (D) Survival curve created using the 96-well CFA on FaDu

cells treated with mitoxantrone (05 nM), another example of a confirmed hit from the HTS.

Each datum represents the mean SEM from 2 independent experiments.

88

Table 3.2 Confirmed hits in the LOPAC1280 library

Summary of confirmed hits in the LOPAC1280 library screened at 0.5 μM final concentration

on FaDu cells using the 96-well colony formation assay as the read-out.

Compound B-Score Value

Emetine Dihydrochloride -4.479

Idarubicin -4.781

Mitoxantrone -4.792

Ouabain -4.648

Vincristine Sulfate -5.367

Vinblastine Sulfate Salt -5.754

In conclusion, we have demonstrated a novel CFA conducted in a 96-well plate format,

using high content microscopy with dual fluorescent labeling, as being a highly efficient

method of performing CFA, which faithfully recapitulates the results from the traditional

approach. This newer approach could be transferred to a robotic platform, with potential in

identifying novel anti-cancer compounds using HTS. However, the most significant advantage

of this novel CFA methodology is facilitating the evaluation of multiple permutations of

combination therapies, such as between radiation and drug treatments, whereby such analyses

of these many interactions could be completed within a single day.

3.5 Acknowledgements

This work was supported by funds from the Canadian Institutes of Health Research, and

the Elia Chair in Head and Neck Cancer Research. David Katz is a recipient of a Canadian

Institutes of Health Research Fellowship in the Excellence of Radiation Research for the 21st

Century Program, which is funded by the Lawrence, Ila and William Gifford Scholarship

Fund. Emma Ito is a recipient of a Natural Sciences and Engineering Research Council of

Canada Scholarship. We thank Dr. Alessandro Datti, Thomas Sun, and Frederick Vizeacoumar

from the Samuel Lunenfeld Research Institute HTS Robotics Facility for their assistance with

89

the high-throughput screen, as well as Cyrus Handy of the High-Content Microscopy Facility

at the Samuel Lunenfeld Research Institute.

90

CHAPTER 4: UROPORPHYRINOGEN DECARBOXYLASE - A NOVEL

RADIOSENSITIZING TARGET FOR HEAD AND NECK CANCER

IDENTIFIED FROM AN RNAI HIGH-THROUGHPUT SCREEN

The data presented in this chapter have been submitted to Science Translational

Medicine on January 26, 2010 and is currently under revision.

Ito E, Yue S, Moriyama EH, Hui AB, Kim I, Shi W, Alajez NM, Bhogal N, Li

GH, Datti A, Wrana J, Schimmer AD, Wilson BC, Liu PP, Durocher D, Neel

BG, Sullivan BO, Cummings B, Bristow R, Liu FF.

91


Uroporphyrinogen decarboxylase (UROD), a key regulator of heme biosynthesis, was

identified from an RNA interference-based high-throughput screen as a novel tumor-selective

radiosensitizing target against head and neck cancer. UROD knockdown plus irradiation

induced caspase-mediated apoptosis and cell cycle arrest in vitro, while delaying tumor growth

in vivo. Radiosensitization appeared to be mediated via enhancement of tumor oxidative stress

from perturbation of iron homeostasis and increased reactive oxygen species production. We

found UROD to be significantly over-expressed in HNC patient biopsies, wherein lower pre-

radiation therapy UROD levels correlated with improved disease-free survival, suggesting that

UROD expression could be a potential predictor for radiation response. UROD down-

regulation radiosensitized several different human cancer models; it also sensitized standard

chemotherapeutic agents, including 5-FU and cisplatin. Thus, our study has uncovered UROD

as a novel potent sensitizer for both radiation and chemotherapy, with potentially broad

applicability for many human malignancies.

4.2 Introduction

Ionizing radiation therapy plays critical curative, adjuvant, and palliative roles in cancer

patient management; curability however, could be limited by tolerance of normal surrounding

tissues. Thus, the development of therapeutic strategies to enhance the therapeutic ratio is of

great importance. Unfortunately, many of the currently utilized radiosensitizers are neither

selective nor tumor-specific. This is particularly a concern in the management of head and

neck cancer, whereby RT is the primary curative modality, yet tumors often reside in close

proximity to critical organs (e.g. brain, spinal cord, optic nerve), which when damaged lead to

long-term compromises in patients’ quality of life, not to mention fatality.

92

HNC is the eighth most common malignancy worldwide, comprising a diverse group

of cancers affecting the sinuses, nasal and oral cavities, pharynx, larynx, and other sites in this

region [131, 133]. In addition to the anatomic and molecular heterogeneity of HNC, most

patients present with locally advanced disease, and/or suffer from other co-morbidities,

rendering HNC particularly challenging to treat. Despite the advances in therapeutic options

over the recent few decades, treatment toxicities and overall clinical outcomes have remained

disappointing [217]. Even the most effective RT regimens achieve local control rates of

4555%, with disease-free survival rates of only 3040% for patients with locally advanced

head and neck squamous-cell carcinomas [138]. Furthermore, meta-analyses have documented

concurrent RT with chemotherapy to offer an absolute survival advantage of only 6.5% at 5

years [141]. These modest results underscore the urgent need to develop novel therapeutic

approaches in the treatment of HNC.

Amongst the new therapies, molecularly-targeted agents have gained momentum [112,

113, 218], but an effective strategy to select appropriate patients does not yet exist, perhaps

due to the complexities of radiation response. Ionizing radiation induces a myriad of physico-

chemical changes at the cellular and molecular level, most of which have not yet been clearly

elucidated. In the current study, we describe an RNA interference-based high-throughput

screen for the large-scale identification of novel anti-cancer radiosensitizing molecular targets.

Uroporphyrinogen decarboxylase, a key regulator of heme biosynthesis, was identified as a

heretofore unreported potent modulator of tumor radioresponse. Here, we present in vitro and

in vivo characterizations of UROD-mediated radiosensitization, and its clinical implications in

the management of HNC.

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4.3.1 Cell Lines

FaDu, A549, SiHa, ME-180, T47D, MDA-MB-231, DU-145, and MRC5 cells were

obtained from the American Type Culture Collection (Manassas, VA). Normal human

oropharyngeal (NOP) and oral epithelial (NOE) cells were purchased from Celprogen (San

Pedro, CA). Untransformed fibroblasts from familial porphyria cutanea tarda (type II) patients

(GM01482, GM00977, GM00961, GM01041) and GM05757 (primary normal human skin)

fibroblasts were obtained from Coriell Institute (Camden, NJ). All cell lines were cultured

according to the manufacturer’s specifications. C666-1 undifferentiated nasopharyngeal cancer

cells [157] were maintained in RPMI 1640 supplemented with 10% fetal bovine serum

(Wisent, Quebec, Canada) and antibiotics (100 mg/L penicillin and 100 mg/L streptomycin).

UTSCC-8 and -42a laryngeal squamous cell cancer cells were a gift from R. Grénman (Turku,

Finland) and maintained as previously described [219]. All cells were maintained in 5% CO2,

21% O2, and 95% humidity at 37°C unless otherwise stated.

4.3.2 Patient Samples

Thirty-eight formalin-fixed paraffin-embedded (FFPE) tissue biopsies from locally

advanced HNSCC patients (Stage III or IV; oropharynx, hypopharynx, or larynx primary SCC

subsites), who participated in a randomized clinical study of two RT fractionation regimens

[220] were utilized with Institutional Research Ethics Board approval. FFPE samples were

macro-dissected for regions of invasive SCC (70% malignant epithelial cell content). Five

normal human larynx and tonsillar FFPE tissues were purchased from Asterand (Detroit, MI).

Total tumor RNA was extracted with RecoverAll Total Nucleic Acid Isolation Kit for FFPE

(Ambion, Austin, TX) as specified by the manufacturer.

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4.3.3 Reagents

Cisplatin, 5-fluorouracil, -aminolevulinic acid hydrochloride (ALA), and

deferoxamine mesylate salt (DFO) were obtained from Sigma-Aldrich (St. Louis, MO). All

compounds were dissolved and/or diluted in complete media.

4.3.4 BrdU-Based siRNA High-Throughput Screen

The Human siGENOME Druggable and Protein Kinase siRNA Libraries (Dharmacon,

Lafayette, CO) were provided by the Samuel Lunenfeld Research Institute (SLRI) HTS

Robotics Facility (Toronto, Canada). Automation of the 96-well siRNA transfection and

bromodeoxyuridine (BrdU) cell proliferation assay (Exalpha Biologicals, Shirley, MA) were

performed using the BioMek FX (Beckman Coulter, Fullerton, CA), SpectraMax Plus384

microplate reader (Molecular Devices, Sunnyvale, CA), and SLRI robotics platform.

Working stock solutions of siRNA were prepared in Opti-MEM I reduced-serum media

(Invitrogen, Carlsbad, CA). Reverse transfections (final concentration of 40 nM siRNA) were

performed with Lipofectamine 2000 (Invitrogen) as specified by the manufacturer. Columns 1

and 2 of each plate contained siRNA targeting DNA ligase IV (LIG4 siGENOME

SMARTpool; Dharmacon), serving as the positive radiosensitizing control, and scrambled

negative siRNA control (ON-TARGETplus Non-Targeting Pool; Dharmacon), respectively.

Twenty-four h post-transfection, 100 µL of complete media was added to each well, then cells

were irradiated using a 137

Cs unit (Gammacell 40 Extractor; MDS Nordion, Ottawa, Canada) at

a dose rate of 0.84 Gy/min. Cells were incubated for an additional 72 h, at which time, BrdU

(Exalpha Biologicals) was added to each well. After 24 h, cells were monitored for BrdU

incorporation on a SpectraMax Plus384

microplate reader according to the manufacturer’s

specifications.

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4.3.5 Transfections

siRNAs targeting UROD (Hs_UROD_2/8 HP GenomeWide siRNAs) and a scrambled

control (AllStars Negative Control siRNA) were purchased from Qiagen (Valencia, CA). A

plasmid vector containing the protein-coding sequence of UROD (Hs_UROD_IM_1 QIAgene

Expression Construct) and an empty vector control (pQE-TriSystem Vector) were also

purchased from Qiagen. All transfections were performed in complete media without

antibiotics using Lipofectamine 2000 and 40 nM of siRNA and/or 1 μg of plasmid DNA.

4.3.6 Flow Cytometric Assays

Flow cytometric analyses were performed on a FACSCalibur Flow Cytometer (BD

Biosciences, San Jose, CA), equipped with FlowJo software (Tree Star, Ashland, OR). Cell

cycle distributions, caspase activation, and mitochondrial membrane potentials were measured

as previously described [219]. Intracellular ROS levels were quantified using the non-specific

5-(and 6-)chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) dye, and

the superoxide-selective dihydroethidium (DE) dye as instructed by the manufacturer

(Invitrogen).

4.3.7 -H2AX Detection

Global cellular -H2AX protein levels were quantified by flow cytometry using the

H2AX Phosphorylation Assay Kit (Upstate Biotechnology, Lake Placid, NY) as specified by

the manufacturer. To image -H2AX nuclear foci, cells transfected on coverslips were fixed

with 2% paraformaldehyde (PFA)-0.2% Triton X-100, then probed with -H2AX mouse

monoclonal antibody (clone JBW301; Upstate Biotechnology), followed by donkey anti-

mouse Alexa 488 antibody (Invitrogen) and DAPI (4,6-diamidino-2-phenylindole; Invitrogen)

for nuclear staining. Cells were imaged with an Olympus IX81 inverted microscope equipped

with a 16-bit Photometrics Cascade 512B EM-CCD camera (Roper Scientific, Tucson, AZ).

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4.3.8 Hypoxia Treatment

Transfected cells were immediately exposed to a continuous flow of humidified 0.2%

O2 with 5% CO2 and balanced N2 (Praxair, Ontario, Canada) in an In Vivo2 400 Hypoxia

Chamber (Ruskinn Technology, Pencoed, UK). An OxyLite 4000 oxygen-sensing probe

(Oxford Optronix, Oxford, UK) was used to verify target O2 levels.

4.3.9 Iron Histochemistry

Intracellular Fe2+

and Fe3+

were detected according to Turnbull’s blue and Perl’s

Prussian blue staining protocols [221], respectively. Images were captured with a Nikon

ECLIPSE E600 microscope equipped with a Nikon DXM1200F digital camera (Nikon

Instruments, Melville, NY) for quantitative analysis using SimplePCI imaging software

(Hamamatsu, Sewickley, PA).

4.3.10 Porphyrin Detection

Transfected cells were treated with ALA (500 μM) for 4 h. Cells were lysed with

SOLVABLE (PerkinElmer, Waltham, MA), and intracellular porphyrin levels were measured

spectrofluorometrically using a SpectraMax Plus384

microplate reader (excitation 405 nm,

emission 635 nm). To visualize porphyrin accumulation, transfected cells ALA were stained

with MitoTracker Green FM (Invitrogen) and Hoechst 33342 (Invitrogen) as specified by the

manufacturer. Live cells were imaged on a Zeiss LSM510 confocal microscope (Carl Zeiss

MicroImaging).

4.3.11 Quantitative Real-Time PCR

Primers for PCR amplifications (Table 4.1) were designed using Primer3 software

(http://primer3.sourceforge.net). Total RNA from transfected cells was harvested using the

RNeasy Mini Kit (Qiagen). Total RNA (1 μg) was reverse-transcribed using SuperScript II

Reverse Transcriptase (Invitrogen) as specified by the manufacturer. qRT-PCR was performed

97

using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), and an ABI

PRISM 7900 Sequence Detection System (Applied Biosystems) with cycle parameters

previously described [158]. Relative mRNA levels were calculated using the 2Ct

method

[222].

Table 4.1 Primer sequences for mRNA expression analyses

Gene Forward Reverse

-ACTIN 5-CCCAGATCATGTTTGAGACCT-3 5-AGTCCATCACGATGCCAGT-3

UROD 5-AGGCCTGCTGTGAACTGACT-3 5-CCTGGGGTACAACAAGGATG-3

SOD1 5-AGGGCATCATCAATTTCGAG-3 5-ACATTGCCCAAGTCTCCAAC-3

SOD2 5-TTGGCCAAGGGAGATGTTAC-3 5-AGTCACGTTTGATGGCTTCC-3

GPX1 5-CTCTTCGAGAAGTGCGAGGT-3 5-TCGATGTCAATGGTCTGGAA-3

FTMT 5-ACGTGGCCTTGAACAACTTC-3 5-ATTCCAGCAACGACTGGTTC-3

4.3.12 Western Blot Analysis

Total protein extracts from transfected cells were harvested and prepared for

immunoblotting as previously described [158]. Membranes were probed with anti-UROD

polyclonal (clone L-19; 1:300 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-

GAPDH monoclonal (1:15000 dilution; Abcam, Cambridge, MA) antibodies, followed by

secondary antibodies conjugated to horseradish peroxidase (1:2000 dilution; Abcam). GAPDH

protein levels were used as loading controls. Western blots were quantified with the Adobe

Photoshop Pixel Quantification Plug-In (Richard Rosenman Advertising & Design, Toronto,

Canada).

4.3.13 Colony Formation Assay

Cells were irradiated (06 Gy) 48 h post-transfection and harvested immediately for

seeding (5005000 cells/well in 6-well plates). Twelve days later, colonies were fixed in 70%

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ethanol, stained with 10% methylene blue, and colonies of 50 cells were counted. Clonogenic

survival curve data were utilized to evaluate the interactive effects of combinatorial therapies

via the Chou-Talalay combination index method [209]. Radiosensitivity was also expressed in

terms of the mean inactivation dose (D-bar), which represents the area under the survival curve

[208]. Radiosensitization was expressed as an enhancement ratio, defined as the mean

inactivation doses of control to treatment.

4.3.14 Cell Viability Assay

The CellTiter 96 AQueous One Solution Cell Proliferation MTS Assay (Promega,

Madison, WI) was used to detect cell viability according to the manufacturer’s specifications.

4.3.15 In Vivo Tumor Model

All animal experiments utilized 68 week-old SCID BALB/c female mice in

accordance with the guidelines of the Animal Care Committee, Ontario Cancer Institute,

University Health Network (Toronto, Canada). TLDs and body weights were recorded thrice

weekly; mice were euthanized by CO2 once TLDs reached ~14 mm.

4.3.16 Tumor Formation Assay

Cells transfected with siCTRL or siUROD for 48 h were harvested and implanted into

the left gastrocnemius muscle of SCID mice (2.5105 viable cells in 100 µL growth medium

per mouse), followed immediately by administration of local tumor RT (4 Gy). Mice were

immobilized in a Lucite box and the tumor-bearing leg was exposed to 225 kV (13 mA) at a

dose rate of 3.37 Gy/min (X-RAD 225C Biological X-Ray Irradiator; Precision X-Ray, North

Branford, CT).

4.3.17 Therapeutic Tumor Growth Assay

Cells were implanted into the left gastrocnemius muscle of SCID mice (2.5105 viable

cells in 100 µL). Once the TLDs reached an average of ~8 mm, mice were injected

99

intraperitoneally (IP) with 600 pmol of siRNA complexed to in vivo-jetPEI (Polyplus-

Transfection, New York, NY), thrice a week for up to 2 weeks. siRNAs were mixed with in

vivo-jetPEI following the manufacturer’s specifications (nitrogen/phosphate ratio: 8). Local

tumor RT (4 Gy) was delivered on days 5 and 13 post IP-injections.

4.3.18 In Vivo Knockdown Validation

To assess the extent of UROD knockdown in vivo, mice were sacrificed 24 h after the

last treatment described in Section 4.3.17. Tumors were excised, immediately fixed in 10%

formalin for 48 h, 70% alcohol for an additional 48 h, paraffin embedded, and then sectioned

(5 μm). Immunohistochemical analysis was performed using microwave antigen retrieval with

anti-UROD polyclonal antibody (clone B02; 1:500 dilution; Abnova, Walnut, CA) and Level-

2 Ultra Streptavidin Detection System (Signet Laboratories, Dedham, MA). For

immunoblotting, tumors were excised and immediately snap-frozen in liquid nitrogen. 30 mg

of tumor tissue was lysed and homogenized as detailed elsewhere [223]; 30 μg of protein was

analyzed for UROD expression via immunoblotting as described above.

4.3.19 Statistical Analyses

All experiments were performed at least three independent times, with the data

presented as the mean SEM. Statistical differences between treatment groups were

determined using the Student’s t test and one-way ANOVA. The Ingenuity Pathways Analysis

software (Ingenuity Systems, Redwood City, CA) was used to identify functional biological

networks from the HTS data. The right-tailed Fisher Exact test was employed to calculate p-

values and scores (p-score = -log10 p-value), indicating the likelihood of genes being observed

together in a network due to random chance.

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4.4 Results

4.4.1 High-Throughput Screening for Novel Radiosensitizers

The preliminary screen of the Human siGENOME Druggable and Protein Kinase

siRNA Libraries identified 188 target sequences with potential radiosensitizing effects at 2 Gy

in FaDu cells (human hypopharyngeal squamous cell cancer), a clinically relevant model for

the study of HNC [162]; the “hit” threshold was defined as 4 standard deviations below the

mean after b-score normalization (Figure 4.1A). The validity of the screen was corroborated

by the identification of known radiosensitizing targets, such as ATM (ataxia-telangiectasia

mutated), ATR (ataxia-telangiectasia and Rad3-related), and aurora kinase A [224, 225]. To

confirm the initial HTS results, FaDu cells were transfected with the 188 siRNAs IR (Figure

4.1B), and those that decreased the surviving fraction by 30% in the absence of IR were

eliminated, leaving 67 potential hits. Targets which reduced the surviving fraction by 50% at

2 Gy relative to their un-irradiated counterparts were selected for further evaluation. Ingenuity

Pathways Analysis identified the top-scoring functional biological network common amongst

the radiosensitizing targets to involve cell death, cancer, and/or cellular compromise (Table

3.2); the top molecular and cellular functions included cell growth and proliferation (Table

3.3).

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Table 4.2 Top-scoring associated network functions

Sixty-seven radiosensitizing targets identified from the HTS were subjected to Ingenuity

Pathways Analysis. Each functional biological network was assigned a score according to the

number of focus genes present from the HTS dataset. Scores indicate the likelihood of focus

genes found together in a network due to random chance. Scores of 4 have 99.9% confidence

level of significance. UROD was identified in the third network (score 23).

Network Score

Cell death, cancer, cellular compromise 59

Amino acid metabolism, molecular transport, small molecule biochemistry 25

Genetic disorder, hematological disease, DNA replication, recombination, and repair 23

Lipid metabolism, small molecule biochemistry, behavior 20

Table 4.3 Top scoring molecular and cellular functions

Top molecular and cellular functions amongst the 67 radiosensitizing targets identified by

Ingenuity Pathways Analysis.

Function p-value

Cellular growth and proliferation 1.97×10-7

2.73×10-2

Cell death 1.03×10-5

3.09×10-2

Cell cycle 3.52×10-5

2.51×10-2

Cellular compromise 4.56×10-5

2.65×10-2

DNA replication, recombination, and repair 4.56×10-5

2.65×10-2

4.4.2 UROD is a Potent Radiosensitizing Target for HNC

Uroporphyrinogen decarboxylase, a key regulator of heme biosynthesis, was identified

from the HTS as a potent modulator of tumor response to IR, and was selected for further

evaluation due to its novelty in the context of human cancers, and being a well-characterized

enzyme; thereby increasing its potential “druggability”. Clonogenic survival curves confirmed

that UROD down-regulation significantly enhanced radiosensitivity of FaDu cells, a highly

aggressive radioresistant HNC cell line, in a dose-dependent manner (Figure 4.1C). Radiation

enhancement ratios (RER) of 2.0, 1.7, and 1.6 were observed at 2, 4, and 6 Gy, respectively; a

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RER 1 denotes synergistic radiosensitization [208]. The Chou-Talalay combination index

(CI) [209] further confirmed the synergistic interaction between siUROD with IR, wherein the

CI remained significantly below 1 for all tested combinations (Figure 4.1D). Corroboration of

siRNA-mediated UROD knockdown was determined via qRT-PCR and immunoblotting,

demonstrating significant suppression of both mRNA (~88% knockdown) and protein

expression by 48 h post-transfection (Figures 4.1E and F). To ensure this observation was not

due to off-target effects, a rescue plasmid expressing target mRNA refractory to siRNA via

silent mutations was utilized. Co-transfection of FaDu cells with siUROD and the rescue

plasmid completely neutralized any siUROD-mediated effects, with or without IR (Figure

4.1G), further confirming a siUROD-specific process.

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Figure 4.1 Identification of UROD as a novel radiosensitizing target

104

(A) Preliminary screen of the Human siGENOME Druggable (6080 genes) and Protein Kinase

(800 genes) siRNA Libraries at 2 Gy in transfected FaDu cells. (B) 67 target sequences with

potential radiosensitizing effects (50% reduction in surviving fraction at 2 Gy vs. 0 Gy) were

identified. Targets that decreased the surviving fraction by 30% in the absence of IR were not

considered (grey box). Known radiosensitizing targets (grey circles); UROD (black circle);

scrambled siRNA control (black triangle). (C) Clonogenic survival curves of FaDu cells

transfected with siCTRL or siUROD for 48 h, then irradiated (06 Gy). Colonies were counted

12 days post-IR. *p0.05 and **p0.01, siCTRL vs. siUROD for each IR dose. (D) As in (C),

but FaDu cells were transfected with a range of siRNA concentrations (060 nM), combined

with IR (06 Gy) for Chou-Talalay combination index analyses. (E) Relative UROD mRNA

levels in FaDu cells transfected with siCTRL or siUROD for 24, 48, and 120 h, as measured

by qRT-PCR. **p0.01, siCTRL vs. siUROD. (F) UROD protein expression was detected by

immunoblotting at 2472 h post-transfection. (G) FaDu cells were co-transfected with siRNA

(siCTRL or siUROD) and plasmid DNA (empty vector control, pVector or siRNA-resistant

rescue plasmid, pUROD) for 48 h, and then irradiated (4 Gy). Apoptotic fractions were

assessed by flow cytometry 72 h post-IR. **p0.01, siCTRL-pVector vs. siUROD-pVector or

siUROD-pUROD IR. Each datum represents the mean SEM from three independent

experiments.

4.4.3 siUROD-Mediated Radiosensitization Differs from Photodynamic Therapy

UROD is the fifth enzyme in the heme biosynthetic pathway (Figure 4.2A) that

catalyses the decarboxylation of uroporphyrinogen to coproporphyrinogen [226]. Since

porphyrinogens are unstable and readily oxidized to fluorescent porphyrin molecules, UROD

down-regulation was functionally validated by indirectly measuring uroporphyrinogen

accumulation via overall changes in oxidized porphyrin levels (uroporphyrin and other highly

carboxylated porphyrins). Spectrofluorometrically, porphyrin accumulation with siUROD

alone was negligible (~1.3 fold-increase vs. untreated control; Figure 4.2B); thus, FaDu cells

were pre-treated with -aminolevulinic acid (ALA) to artificially induce porphyrin synthesis.

ALA-plus-siUROD significantly increased intracellular porphyrin levels relative to ALA alone

or siCTRL-treated cells (~18.1 vs. ~9.9 and ~10.1 fold-increase). Similar observations were

made via fluorescent microscopy (Figure 4.2C), wherein cells treated with ALA-plus-siUROD

105

exhibited enhanced porphyrin accumulation, reflecting the disruption of heme biosynthesis by

siUROD.

Since the majority of currently utilized photosensitizers in photodynamic therapy

(PDT) are porphyrin based [227], it was of interest to compare the radiosensitizing effects of

siUROD to commonly used photosensitizers. ALA-based PDT is a well established anti-

cancer therapy that utilizes the heme precursor ALA, to induce accumulation of

protoporphyrin IX (PPIX) in neoplastic cells [228, 229]. When ALA-treated cells are exposed

to visible light, PPIX become excited and induce ROS formation, leading to oxidative stress-

mediated cell death. In this study, siUROD-plus-IR was dramatically more cytotoxic compared

to the negligible effects of ALA-plus-IR (Figure 4.2D), indicating that the effects of siUROD

were independent of intracellular porphyrin accumulation (Figures 4.2B and C), thus distinct

from PDT.

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Figure 4.2 Radiosensitizing effect of UROD knockdown is independent of porphyrin

accumulation

107

(A) Heme biosynthetic pathway. CPOX, coproporphyrinogen oxidase; PPOX,

protoporphyrinogen oxidase; Fe, iron. (B) Porphyrin synthesis in mock-, siCTRL-, or

siUROD-transfected FaDu cells was artificially induced with ALA (500 μM, 4 h) prior to

porphyrin extraction at 24 h post-transfection. Porphyrin levels were quantified

spectrofluorometrically and normalized to total cell number. Representative spectral scans

(575750 nm) are shown. **p0.01, siUROD vs. siCTRL or untreated ALA. (C)

Fluorescent microscopy images of transfected cells ALA (500 μM, 1 h). Mitochondria and

nuclei were stained with MitoTracker Green and Hoechst 33342, respectively. Intracellular

porphyrin excited with a wavelength of ~400 nm emits red fluorescence at a peak of ~635 nm.

Scale bar, 10 μm. (D) ALA-treated (2501000 μM, 4 h) and siCTRL- or siUROD-transfected

(48 h-transfection) FaDu cells were irradiated (4 Gy), then cell viability was assessed 96 h

later via MTS assay. **p0.01, siCTRL vs. siUROD IR; untreated vs. ALA IR. In all

cases, each datum represents the mean SEM from three independent experiments.

4.4.4 UROD Down-Regulation Promotes Radiation-Induced Apoptosis

The enhanced tumor radiosensitivity observed with UROD suppression (Figure 4.1C)

was mediated in part by G2-M cell cycle arrest (Figure 4.3A), along with induction of double-

strand DNA breaks, reflected by increased overall -H2AX expression and nuclear foci

formation in siUROD-plus-IR-treated FaDu cells vs. IR alone (Figures 4.3B and C). The

significantly prolonged G2-M arrest and concomitant increase in the subG1 population

suggested that the DNA damage induced by siUROD-plus-IR was more lethal than IR alone,

thereby significantly augmenting apoptosis (Figure 4.3A). The central role of apoptosis in

siUROD-plus-IR-mediated cytotoxicity was further evident by the induction of caspase

activation (Figure 4.3D) and depolarization of the mitochondrial membrane potential (M)

(Figure 4.3E), both classical hallmarks of apoptosis.

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Figure 4.3 UROD down-regulation promotes radiation-induced cytotoxicity

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(A) Flow cytometric DNA content analyses of siCTRL- or siUROD-transfected FaDu cells at

1272 h post-IR (4 Gy). Representative histograms with gates for cell cycle distributions are

shown. *p0.05 and **p0.01, siCTRL vs. siUROD IR at each time point. (B) Flow

cytometric analyses of cellular -H2AX expression levels in transfected FaDu cells at 0240

min post-IR (4 Gy). **p0.01, siCTRL vs. siUROD at each time point. (C) Representative

images of -H2AX nuclear foci formation in siCTRL- and siUROD-transfected FaDu cells 30

min post-IR. Scale bar, 10 μm. (D) Flow cytometric analyses of caspase 9, 8, and 3 activation

in siCTRL or siUROD-transfected FaDu cells at 1248 h post-IR (4 Gy). *p0.05 and

**p0.01, siCTRL vs. siUROD IR at each time point. (E) M depolarization was

quantified by flow cytometry 48 h post-IR in transfected FaDu cells. **p0.01, siCTRL vs.

siUROD IR. Each datum represents the mean SEM from three independent experiments.

4.4.5 siUROD-Mediated Radiosensitization Increases Cellular Oxidative Stress

Heme biosynthesis occurs within the cytoplasm and mitochondrion (Figure 4.2A); the

latter being a major source of intracellular free radicals [230]. Thus, to investigate whether

siUROD mediated its radiosensitizing effects via perturbation of ROS homeostasis,

intracellular levels of oxidants were measured. Mitochondrial superoxide anion radicals (O2●–

),

a primary ROS species, were more prevalent in siUROD-plus-IR vs. IR- or siUROD-treated

FaDu cells (Figure 4.4A). Similarly, global ROS production, as measured by CM-H2DCFDA

which detects many other ROS species (hydrogen peroxide, hydroxyl radical, peroxyl radical,

peroxynitrite anion), was highest in siUROD-plus-IR-treated cells, which increased in a time-

dependent manner (Figure 4.4B).

Many tumors display lower anti-oxidant enzyme levels compared to their normal

counterparts [231-233]. Hence, ROS production in normal oropharyngeal (NOP) and oral

epithelial (NOE) cells was assayed after exposure to siUROD IR (Figures 4.4C and D), after

attaining equivalent degrees of UROD knockdown as verified by qRT-PCR (data not shown).

At 72 h post-IR, both normal cell lines demonstrated significantly less ROS accumulation

compared to FaDu cells, particularly in combination with UROD down-regulation (DE: 1.4

and 1.3 vs. 1.8 fold-increase in NOP and NOE vs. FaDu, respectively; CM-H2DCFDA: 1.5 and

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2.0 vs. 2.7 fold-increase in NOP and NOE vs. FaDu, respectively). These differential ROS

levels translated into higher survival for the normal vs. FaDu cells after siUROD IR (Figure

4.4E; NOP or NOE vs. FaDu RERs, p0.01), exposing a therapeutic window to exploit the

differential anti-oxidant capacity between normal vs. tumor cells to achieve tumor-selective

siUROD radiosensitization.

Given that ROS production is regulated by oxygen tension, and hypoxia diminishes

radiosensitivity, we also examined the effects of O2 on siUROD radiosensitization.

Interestingly, siUROD alone retained activity under hypoxia comparable to that under

normoxic conditions, and displayed only a partial reduction in radiosensitization (Figure 4.4F).

To further understand the mechanisms of siUROD-plus-IR-mediated cytotoxicity, relative

expression levels of a panel of genes involved in oxidative stress responses were examined. As

expected, anti-oxidants involved in maintaining cellular redox homeostasis, including

superoxide dismutases (SOD1 and SOD2), glutathione peroxidase (GPX1), and mitochondrial

ferritin (FTMT) were all up-regulated in FaDu cells in response to siUROD-plus-IR (Figure

4.4G).

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Figure 4.4 siUROD-mediated radiosensitization enhances cellular oxidative stress

112

(A) Intracellular superoxide anions in siCTRL- or siUROD-transfected FaDu cells at 372 h

post-IR (4 Gy) were detected by flow cytometry with dihydroethidium (DE). *p0.05 and

**p0.01, siCTRL vs. siUROD IR at each time point. (B) Overall ROS levels in transfected

FaDu cells were measured with CM-H2DCFDA at 372 h post-IR (4 Gy). *p0.05 and

**p0.01, siCTRL vs. siUROD IR at each time point. (C) Superoxide radical levels in two

transfected normal head and neck epithelial cells (NOP and NOE) 72 h post-IR (4 Gy).

**p0.01, normals vs. FaDu at 72 h post-IR. (D) Overall ROS levels in transfected NOP and

NOE cells 72 h post-IR (4 Gy). *p0.05 and **p0.01, normals vs. FaDu at 72 h post-IR. (E)

Cell viability of siCTRL or siUROD-transfected FaDu, NOP, and NOE cells at 96 h post-IR (2

Gy) via MTS assay. **p0.01, siCTRL vs. siUROD IR. (F) FaDu cells were transfected with

siCTRL or siUROD and irradiated under normoxia (21% O2) or hypoxia (0.2% O2). Apoptotic

fractions were assessed by flow cytometry 72 h post-IR. *p0.05 and **p0.01, normoxic vs.

hypoxic treatments. (G) Relative mRNA expression of a panel of genes involved in cellular

oxidative stress responses in siCTRL- or siUROD-transfected FaDu cells 48 h post-IR.

Relative fold changes represent average ΔCt values normalized to those of -actin, then

compared to siCTRL-transfected cells. **p0.01, siCTRL vs. siUROD IR. Each datum

represents the mean SEM from three independent experiments.

4.4.6 UROD Knockdown Perturbs Cellular Iron Homeostasis

The induction of mitochondrial ferritin, a nuclear-encoded iron (Fe)-sequestering

protein, in FaDu cells transfected with siUROD IR prompted further investigations into the

role of Fe-homeostasis in siUROD-mediated effects. Mitochondria are intimately involved in

Fe-trafficking for heme biosynthesis and the formation of Fe-sulfur clusters [234]. These

organelles, also being the major source of ROS production, have developed efficient

mechanisms to segregate free Fe from ROS, thereby preventing the production of harmful

hydroxyl radicals (●OH) via Fenton-type reactions [235]. Accordingly, up-regulation of the

FTMT anti-oxidant in siUROD IR treated cells (Figure 4.4G) was associated with markedly

elevated levels of intracellular ferrous (Fe2+

) and ferric (Fe3+

) iron, visible as diffuse deep-

purple staining within the cells (Figure 4.5A). The relative changes in iron species (Figure

4.5B), with Fe2+

reduction vs. Fe3+

increase after IR are likely related to the Fenton reaction,

whereby IR can induce hydrogen peroxide (H2O2) formation, which consumes Fe2+

, and in the

113

process of generating ●OH, converts Fe

2+ to Fe

3+. To corroborate the central role of excess

cellular Fe in mediating siUROD radiosensitization, the Fe-chelator deferoxamine was

introduced prior to IR. Significant suppression (~50%) of siUROD-plus-IR-induced apoptosis

was observed (Figure 4.5C), underscoring the critical role of Fe in mediating this

radiosensitization process.

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Figure 4.5 UROD knockdown induces intracellular iron accumulation

115

(A) Ferrous (Fe2+

) and ferric (Fe3+

) iron staining of siCTRL or siUROD-transfected FaDu cells

at 48 h post-IR (4 Gy). Scale bar, 50 μm. (B) Quantification of intracellular Fe2+

and Fe3+

levels from (A). Deep-purple areas and total area of cultured cells were measured. The ratio

(% area) was calculated by dividing the sum of deep-purple areas by the sum of the total area

from sections. *p0.05 and **p0.01, siCTRL vs. siUROD IR. (C) FaDu cells transfected

with siCTRL or siUROD for 24 h were treated with deferoxamine (DFO; 5 μM), and then

irradiated (4 Gy) 24 h later. Apoptotic fractions were assessed by flow cytometry 72 h post-IR.

**p0.01, - DFO vs. + DFO treatments. Each datum represents the mean SEM from at least

two independent experiments.

4.4.7 siUROD Radiosensitizes HNC Models In Vivo

To evaluate the radiosensitizing efficacy of UROD knockdown in vivo, transfected

FaDu cells were injected into the left gastrocnemius muscle of SCID mice, followed by local

tumor RT. Mice implanted with siUROD or siCTRL-transfected cells started to form tumors at

~23 vs. ~9 days, respectively; delaying the time to reach a tumor-plus-leg diameter (TLD) of

14 mm by ~14 days (p0.001; Figure 4.6A). When combined with RT, siUROD appeared to

synergistically suppress tumor-forming capacity of FaDu cells, wherein tumors developed at

~37 vs. ~12 days in the siUROD-plus-RT vs. siCTRL-plus-RT groups, respectively; extending

the mean time to reach 14 mm by ~27 days (p0.001; Figure 4.6A).

The therapeutic efficacy of siUROD-plus-RT in treating established FaDu tumors was

also evaluated. Tumor-bearing mice were systemically treated with siRNA complexed to a

cationic polymer polyethylenimine with or without local tumor RT. Although no difference in

tumor growth was observed between the siUROD vs. siCTRL groups, siUROD-plus-RT

caused a significant reduction in tumor size compared to the siCTRL-plus-RT arm (p0.001;

Figure 4.6B). The extent of tumor growth delay was reflected by the in vivo level of UROD-

knockdown, verified by both immunoblotting and immunohistochemistry (Figures 4.6C and

D). Despite the fact that this treatment regimen was not optimized for absorption, distribution,

metabolism, or excretion, a therapeutic benefit was nonetheless observed. These data strongly

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suggest that improving the pharmacokinetics and bioavailability of siUROD would render this

therapeutic approach highly effective, based on the significant suppression of tumor-forming

capacity when FaDu cells were fully exposed to siRNA-mediated UROD knockdown (Figure

4.6A). This therapeutic regimen was well-tolerated based on the minimal differences in mice

body weights between the treatment groups (Figure 4.6E).

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Figure 4.6 In Vivo efficacy of UROD knockdown plus irradiation in HNC models

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(A) Mock, siCTRL, or siUROD-transfected FaDu cells were implanted into the left

gastrocnemius muscle of SCID mice, followed immediately by local RT (4 Gy). Each

treatment group comprised of 9 mice. ***p0.001, siUROD vs. mock or siCTRL RT. (B)

FaDu tumors were established in SCID mice; once TLDs reached ~8 mm, mice were randomly

assigned to siCTRL, siUROD, siCTRL-plus-RT, or siUROD-plus-RT. Mice were IP-injected

with 600 pmol of jetPEI-complexed siRNA thrice a week for up to 2 weeks (white arrows).

Local tumor RT (4 Gy) was delivered on days 5 and 13 post IP-injections (grey arrows). Each

treatment group comprised of 5 mice. ***p0.001, siUROD vs. siCTRL + RT. (C) UROD

knockdown was assessed in FaDu tumors 24 h after the last treatment as described in (B).

Excised tumors were subjected to immunoblotting for UROD expression. Western blots were

quantified and relative fold changes in UROD protein levels were determined by normalizing

to corresponding GAPDH loading controls, then compared to siCTRL-treated tumors. (D)

UROD knockdown in tumors (black arrows) was also verified by immunohistochemistry. (E)

Average mouse body weight for each treatment group from (B). Each datum represents the

mean SEM from at least two independent experiments.

4.4.8 UROD Knockdown Modulates Radiosensitivity of Several Cancer Models

To assess the applicability of siUROD-induced radiosensitization to other human

cancers, additional HNC, cervix, breast, lung, and prostate cancer cell lines were evaluated;

almost all the cell lines were radiosensitized by siUROD, albeit to different degrees (Figure

4.7A). Examination of the relationship between the extent of radiosensitization and basal

UROD mRNA levels revealed a general trend, wherein cells with lower basal UROD

expression were more readily radiosensitized than those with higher levels (data not shown),

possibly due to greater ease in achieving siRNA-mediated UROD knockdown. To further

corroborate the role of UROD in modulating tumor radiosensitivity, exogenous expression of

UROD was introduced into the most sensitive HNC cell line UTSCC-42a (Figure 4.7B), to

determine whether this phenotype could be reversed. Indeed, over-expression of UROD prior

to IR protected the UTSCC-42a cells against radiation-induced apoptosis (~53% reduction vs.

empty vector control; Figure 4.7C), substantiating the critical role of UROD in modulating

radiosensitivity.

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4.4.9 Clinical Implications of UROD in HNC

The clinical importance of our findings was determined from the analysis of pre-

treatment tumor biopsies from patients with Stage III or IV non-metastatic HNSCC, who were

all participants in a RT clinical trial [220]. Of note, UROD mRNA expression was

significantly higher (~11-fold) compared to that of normal laryngeal and tonsillar epithelial

tissues (p0.05; Figure 4.7D). Furthermore, patients with the lowest quartile level of UROD

expression experienced a superior disease-free survival (DFS) compared to those with the

highest UROD expression (p=0.06; Figure 4.7E); consistent with the notion that higher UROD

levels conferred radioresistance (Figure 4.7C), and supporting the strategy of reducing UROD

to increase radiocurability.

UROD deficiency is responsible for the clinical syndrome of porphyria cutanea tarda

(PCT), a rare non-fatal metabolic disorder, characterized by elevated cellular porphyrin and Fe

levels [226]. Thus, it was of interest to examine whether a naturally occurring state of UROD

deficiency could recapitulate our findings. Indeed, untransformed fibroblasts from familial

PCT patients demonstrated minimal cytotoxicity comparable to UROD-functional primary

normal human fibroblasts (Figure 4.7F), corroborating our previous data that siUROD-

mediated radiosensitization is tumor selective (Figure 4.4E).

The breadth of application of the siUROD-sensitization strategy was further broadened

when non-toxic doses of cisplatin or 5-fluorouracil were significantly sensitized in FaDu cells,

in a dose-dependent manner (Figure 4.7G). These two drugs are commonly utilized in HNC

management; hence, siUROD could play a significant role in enhancing the outcome for both

radiotherapy and chemotherapy in HNC patients, allowing lower treatment doses to be

administered without compromising cure.

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Figure 4.7 Clinical relevance of UROD in human cancers

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(A) Cell viability assessment of siCTRL or siUROD-transfected cancer cells at 96 h post-IR (2

Gy) via MTS assay. Human HNC (red), cervix (blue), breast (green), lung (black), and

prostate (orange) cancer cell lines. *p0.05 and **p0.01, siCTRL vs. siUROD IR. (B)

Relative UROD mRNA expression in UTSCC-42a cells transfected with UROD-expressing

plasmid (pUROD) or empty vector control (pVector) for 48 h, determined via qRT-PCR.

***p0.001, pVector vs. pUROD. (C) UTSCC-42a cells transfected with pUROD or pVector

for 48 h were irradiated (2 Gy). Apoptotic fractions were assessed by flow cytometry 72 h

post-IR. Representative histogram of cell cycle distribution is shown. ***p0.001, pUROD vs.

pVector + IR. (D) Total RNA was extracted from 38 HNSCC patient tumor biopsies and 5

normal laryngeal and tonsillar epithelial tissues, and assessed for relative levels of UROD

mRNA expression. Fold change was determined by normalizing to -actin levels, and

comparing to the average from normal tissues. Solid line, mean fold change. *p0.05, tumor

vs. normal tissues. (E) Kaplan-Meier plot of DFS for the HNSCC patients from (D);

trichotomized based on interquartile range (low, medium, vs. high levels of UROD mRNA

expression). DFS was defined as absence of relapse or death, calculated from the time of

diagnosis. Median follow-up time was 6.9 years (range 2.310.8 yrs). (F) Cell viability

assessment of irradiated (2 Gy) primary normal human fibroblasts (MRC5, GM05757) and

untransformed fibroblasts from PCT patients (GM01482, GM00977, GM00961, GM01041) 96

h post-IR via MTS assay. *p0.05, MRC5 vs. PCT fibroblasts. (G) siCTRL- or siUROD-

transfected FaDu cells were treated with increasing doses of cisplatin (0.010.25 μM) or 5-FU

(125 μM) for 24 h, then assessed for cell viability 96 h later. ***p0.001 and **p0.01,

siCTRL drug vs. siUROD drug. Each datum represents the mean SEM from three

independent experiments.

4.5 Discussion

Intrinsic oxidative stress in cancer cells, due in part to oncogenic transformation, with

resultant increased metabolic activity and mitochondrial dysfunction, have long been

recognized to promote tumor genetic instability, cell growth, and proliferation [236]. Recently,

this distinct biochemical feature has been exploited for selective anti-cancer therapies [237,

238], wherein elevated basal levels of ROS-mediated signalling rendered neoplastic cells more

vulnerable to manipulations that enhanced oxidative stress. Thus, the addition of exogenous

ROS-inducing agents would increase intracellular ROS to toxic levels, triggering cell death in

cancer cells with already reduced antioxidant defence mechanisms [231-233]; whereas normal

cells have a greater capacity to contend with oxidative insults by virtue of their lower basal

ROS output, along with an intact anti-oxidant response system. These phenomena were

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recapitulated in this current study, wherein ROS levels were significantly augmented in tumor

cells after siUROD-plus-IR (Figures 4.4A and B), with the induction of cell cycle arrest and

apoptosis (Figure 4.3A). The compensatory anti-oxidant response (Figure 4.4G) was

inadequate compared to the capacity of normal cells, which demonstrated only a modest

elevation in ROS, with minimal consequences on viability (Figures 4.4C-E).

The novelty of our UROD discovery relates to the opportunity to perturb iron

homeostasis as the initiator of oxidative stress in tumor cells. Several lines of evidence support

a Fe-mediated mechanism of radiosensitization for UROD down-regulation, although we

cannot preclude Fe-independent mechanisms that might also contribute to this process. Firstly,

mitochondrial ferritin was significantly up-regulated by siUROD, even at early times post-

transfection (Figure 4.4G and data not shown), which was associated with a concomitant

increase in intracellular Fe (Figures 4.5A and B). Secondly, siUROD-plus-IR-induced

apoptosis was significantly suppressed by the Fe-chelator, deferoxamine (Figure 4.5C). Iron,

which exists in two oxidative states (Fe2+

and Fe3+

), is an essential element required for many

critical biological processes, including respiration, DNA synthesis, and O2 transport [234].

Transition metals such as Fe however, can also be powerful catalysts for ROS formation. In

the presence of H2O2, Fe2+

can be oxidized to Fe3+

via the Fenton reaction, producing highly

toxic hydroxyl radicals. When heme synthesis is disrupted, large quantities of iron, which

would normally be incorporated into PPIX to form heme, continue to be imported into the

mitochondria, causing an elevation in FTMT levels to sequester the excess Fe2+

and minimize

oxidative damage [239]. In cancers, the accretion of cellular Fe is further exacerbated by the

over-expression of transferrin receptor-1, a major mechanism of Fe-uptake to sustain the high

requirements of cellular and protein turn-over, plus DNA synthesis [240]. Our data

demonstrate the elevated Fe and consequential ROS formation due to siUROD alone to be

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non-lethal (Figure 4.3A); significant cytotoxicity was only observed when combined with IR,

which is clinically advantageous since RT is anatomically-targeted. Presumably, with siUROD

alone, the excess free Fe2+

led to an increase in the ambient concentration of free radicals with

which the cells can cope; however, the additional ROS insults induced by IR overwhelmed the

cell’s anti-oxidant capacity, resulting in the observed enhanced cell death.

Excess Fe2+

might also increase the effective range of radicals produced by -radiation.

Upon IR, superoxide and hydroxyl radicals are formed [241], both of which can react with

themselves to form H2O2, initiating the Fenton reaction and ultimately, oxidative damage.

Thus, the same phenomenon (i.e. Fe-overload) that cancer cells rely on for rapid proliferation

and DNA synthesis could be exploited for the liberation of detrimental radicals with -

radiation, exposing the double-edged sword of iron in cancer cells.

Similar to PDT, our siUROD radiosensitizing strategy exploits the heme pathway to

harness its anti-cancer effects; however, siUROD is distinct and superior for several reasons.

Tumor hypoxia severely hampers PDT efficacy, since molecular O2 is a prerequisite for the

production of photo-induced singlet oxygen molecules [242, 243]. However, siUROD-plus-IR

retained radiosensitizing efficacy even under hypoxia (Figure 4.4F), likely due to its reliance

on the Fe2+

-catalyzed Fenton reaction to yield highly cytotoxic radicals. H2O2 can be generated

via recombination of free radicals formed from water radiolysis [241]; hence, there is less

reliance on the presence of O2. The applicability of PDT is further limited since the light

source used to excite porphyrins and its derivatives occupy the visible spectrum, which cannot

penetrate tissues 0.8 cm, restricting PDT to superficial lesions [244]. Moreover, porphyrins

cannot be excited by the high-energy photons of x-rays or -rays [245], thereby accounting for

the modest radiosensitizing efficacies of porphyrins [244, 246, 247]. Thus, siUROD provides a

clear therapeutic advantage with significant sensitization by -rays, a mainstay in the standard

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anti-cancer therapeutic armamentarium. The possibility of utilizing siUROD as an adjunct to

photosensitizers also warrants additional examination, further broadening its potential clinical

application.

There is a paucity of literature surrounding UROD and cancer. Only a few studies have

reported enhanced heme biosynthesis in human cancers, wherein increased UROD activity was

observed in breast tumors vs. normal tissues [248, 249]; the basis for which remained unclear.

Our current study is the first such report in HNC, whereby UROD was markedly over-

expressed in primary HNSCC vs. corresponding normal tissues (Figure 4.7D). A potential

predictive value for UROD was also revealed, wherein lower levels of pre-treatment UROD

expression appeared to correlate with improved DFS in HNSCC patients treated with RT

(Figure 4.7E). The power of this association may be underestimated due to the skewed

outcome, in that there were only 8 non-relapsed vs. 30 relapsed cases. Thus, additional

evaluation of more balanced HNC cohorts is strongly warranted. The possible role of UROD

over-expression in predicting radioresistance was strongly supported by the reversal of the

radiosensitive phenotype of UTSCC-42a cells (Figure 4.7C); thereby facilitating the selection

of cancer patients who would be amenable to UROD-mediated radiosensitization.

The potential therapeutic application of siUROD in human cancers appears to be quite

extensive. UROD down-regulation not only radiosensitized a wide range of solid cancers

while sparing normal cells (Figures 4.7A and F and 4.4E), but also surprisingly sensitized

tumor cells to standard chemotherapeutic agents (Figure 4.7G). In theory, an ideal “sensitizer”

should have no inherent cytotoxicity, and should exert its effect only when administered with

RT or chemotherapy. However, many of the so-called “radiosensitizers” commonly used today

(e.g. cisplatin) exhibit inherent systemic toxicities and cause damage to normal tissues [250].

Overall, siUROD alone induced a modest reduction in tumor survival, which was significantly

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enhanced by IR. A few cancer cell lines demonstrated sensitivity to siUROD alone;

nonetheless these siUROD-mediated effects IR were tumor-selective, underscoring a clear

therapeutic window and potential therapeutic advantage of utilizing siUROD as either a stand-

alone anti-cancer treatment, or a sensitizing strategy combined with either RT or

chemotherapy. Furthermore, a naturally occurring state of UROD deficiency causes PCT, a

chronic non-fatal disorder [226]; hence, a transient development of “PCT” during the weeks of

RT and/or chemotherapy should be well-tolerated. Evidence for minimal toxicity is provided

by the few case reports wherein no significant increase in toxicities was observed when PCT-

cancer patients underwent RT [251-253].

Thus, the novel identification of down-regulating UROD has significant implications in

the management of human cancers. The therapeutic application of this approach is broad and

effective in the tumor-selective enhancement of radiation and chemotherapy efficacy.

Furthermore, the recent identification of an endogenous inhibitor against UROD [254], along

with the already described crystal structure of human UROD [255] provide important insights

to pave the path for the development of small molecule inhibitors targeting UROD. Finally,

our discovery uncovers the translational significance of iron homeostasis and dysregulation in

cancer, warranting further investigations into this important biological process.

4.6 Acknowledgments

We thank Thomas Sun and Frederick Vizeacoumar for assistance with the HTS; Nadine

Kolas and Yanina Eberhard for technical guidance; and Melania Pintilie for statistical advice.

This work was supported by the Canadian Institutes of Health Research (Grant 69023), the

Elia Chair in Head and Neck Cancer Research, and in part from the Ministry of Health and

Long-Term Planning.

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CHAPTER 5: DISCUSSION

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5.1 Research Summary

Despite the recent advances in therapeutic options for HNC, treatment-associated

toxicities and overall clinical outcomes have remained disappointing. Radiation therapy, which

remains the primary curative modality for HNC, is often administered concomitantly with

radiosensitizing agents; many of which are neither selective nor tumor-specific. Thus, this

thesis focused on the goal of improving outcome for HNC patients by searching for novel

therapeutic strategies that synergize with radiation to enhance tumor cell killing, with minimal

damage to the surrounding normal tissues. In order to expedite this discovery process, a high-

throughput screening approach was employed.

In our initial attempt to search for novel radiosensitizers, we conducted a cell-based

phenotype-driven HTS of ~2,000 commercially available natural products, utilizing the short-

term tetrazolium-based MTS assay as a read-out for cell viability (Chapter 2). Although this

work was successful in identifying cetrimonium bromide as a novel tumor-selective

apoptogenic agent with in vitro and in vivo efficacy against several HNC models, two major

limitations of our discovery were that CTAB did not synergize with IR, nor was its precise

cellular target(s) elucidated. Furthermore, the HTS itself did not incorporate radiation

treatment; hence, the screen parameters were not ideal for discovering radiosensitizers.

Subsequently, in the continued search for novel radiosensitizers, an alternative strategy

was proposed involving a target-driven siRNA-based HTS. Radiation treatment was also

integrated into the screen, allowing for the direct assessment of the effects of both gene

knockdown and radiation. In terms of read-outs, it is well established that the clonogenic assay

is the gold standard for measuring cellular effects of IR in vitro. However, the long-term

kinetics, difficulty in large-scale automation, and limited robustness (i.e. colony-forming

capacity) of the assay has restricted its appeal and amenability to high-throughput platforms.

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Although this thesis was successful in developing an automated, 96-well high-throughput CFA

as a powerful and time-effective tool in the discovery of potent anti-cancer cytotoxics (Chapter

3), a technical drawback was its limited dynamic range due to the smaller surface area of the

96-well format and fewer cells that could be plated. Thus, we utilized the BrdU incorporation

assay, a viable CFA alternative, which measures replicative DNA synthesis and detects all

modes of cell death with long-term kinetics that is reflective of the therapeutic response; the

BrdU assay is also more sensitive and has a wider dynamic range compared to the MTS assay.

From the siRNA-based HTS of ~7000 human genes, uroporphyrinogen decarboxylase was

identified as a novel, potent radiosensitizing target against HNC models in vitro and in vivo;

wherein radiosensitization appeared to be mediated via tumor-selective enhancement of

oxidative stress. Thus, employing a high-throughput screening approach, this thesis was

successful in identifying two novel therapeutic strategies with clinical potential in the

management of HNC.

5.2 Future Directions

5.2.1 Empirical to Target-Driven Cancer Drug Discovery

Advances in our understanding of the molecular mechanisms underpinning tumor

development and growth has instigated a paradigm shift in our approach to cancer-based drug

discovery; transitioning the focus from the development of non-specific cytotoxic

chemotherapy to the rational design of target-based anti-cancer therapeutics. Despite the

successful application of chemoradiotherapy over the past few decades, the main drawback of

chemotherapy lies in its exacerbation of normal tissue toxicities. Thus, improving the

therapeutic index is a fundamental objective to enhancing cure rates with RT. As revealed in

this thesis, one approach is through the use of molecularly-targeted radiosensitizers that exploit

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tumor-specific aberrations to enhance IR-mediated tumor cell killing. In this respect, their

relative tumor-specificity and generally broad therapeutic margin might offer a theoretical

advantage over classical cytotoxics, as overlapping toxicity with RT on normal tissues is

potentially minimized. Additionally, targeted therapies may offer the prospect of improved

patient selection, rational dosing, predictable side-effects, known mechanisms of resistance,

and the testing of rational combination regimens [256].

Fittingly, this paradigm shift in drug discovery has also prompted a transition from

empirical compound-orientated preclinical screening to target-focused drug screening.

However, contrary to initial expectations, target-driven screens are now faced with challenges,

mainly due to the fundamental and theoretical limits of this approach. First, target-based

screening frequently involves ex cellulo assays that do not fully recapitulate in cellulo

complexities. The pharmacologic effect resulting from the inhibition of a specific molecular

target may be influenced by interactions with other proteins within pathways or networks in

the cell. Thus, target-based screening assays may not be predictive of drug effects within the

context of the whole cell, resulting in unexpectedly lower efficacy and/or unforeseeable

adverse effects [256]. Second, only a minority of potential cancer targets are considered

pharmacologically tractable by the pharmaceutical industry. Amongst the four basic types of

cellular macromolecules, proteins, nucleic acids, lipids, and polysaccharides, the latter three

are not readily amenable to drug-targeting. Thus, the majority of clinically successful

chemotherapeutic drugs exert their effects by targeting proteins [257]. An analysis of known

Lipinski rule-of-five compliant drugs suggests there are only ~399 non-redundant molecular

targets from ~130 protein families, which bind these drugs. More globally, assuming that the

sequence and functional similarities within a gene family are indicative of a conserved drug

binding-site architecture, then only ~3,051 of the predicted 30,000 genes in the human genome

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code for a “druggable” protein [257]. At present, ~50% of the proteins expressed by the

genome are functionally unclassified; some of which might prove to be druggable [257].

Moreover, alternative therapeutic strategies including those based on antibody, protein, and

oligonucleotide (e.g. anti-sense oligonucleotides, RNAi) technologies, may further expand the

scope of potential druggable targets to those not amenable to rule-of-five compliant therapies.

5.2.2 RNAi in Drug Discovery and Therapeutics

The use of RNAi technology has become not only a dynamic tool for expediting the

cancer drug discovery process, but has also been making strides to serve as a powerful

therapeutic; thus, tackling the aforementioned limitations of target-oriented drug screening.

The large-scale identification of novel cancer targets via genome-wide RNAi screens can be

conducted both at the cellular and whole-animal level, enabling the direct assessment of loss-

of-function phenotypes for specific targets in cellulo and in vivo [258]. RNAi has also become

an important part of the target validation process, wherein tumor xenograft models of shRNA-

transfected cells may be used to confirm target knockdown-mediated suppression of tumor

development. In particular, inducible shRNA systems, which allow tumor xenografts to

become established before silencing target expression, reflects to an extent, the

pharmacological administration of therapies to established tumors [258]. As demonstrated in

this thesis, systemic or localized intra-tumoral siRNA delivery in mice xenograft models are

alternative methods for in vivo target validation; this approach, which simulates more or less

clinical practice, also allows for the assessment of treatment-associated systemic toxicities

[259]. In addition to target identification and validation, RNAi technology can be employed to

streamline the later stages of drug discovery. The combination of RNAi and in vitro

phenotype-based small-molecule screens can be a powerful approach to improve compound

identification and lead optimization. Specific cellular phenotypes associated with RNAi-screen

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target hits can be applied to secondary cell-based chemical screens to identify candidate small

molecules that can recapitulate the inhibitory effects of target RNAi [258]. Genomic

approaches, together with RNAi, can also be used to enhance lead optimization, wherein

molecular expression profiles of target RNAi and lead compounds identified from the in vitro

screen can be generated and compared. The compounds that most effectively reproduce the

RNAi-generated profile would be given priority for further development [258]. In fact, gene-

expression signatures can now be accurately compared, independent of the platform on which

they were created via the Connectivity Map (C-Map), a large compendium of gene-expression

profiles from cultured human cells treated with various chemicals and genetic reagents [260].

Thus, microarray signatures generated from RNAi-screen hits can also be compared to those

within C-Map to identify compounds with similar profiles. If the identified compounds are

already approved for another indication, this could further expedite clinical development.

Clinically, RNAi-based therapeutics represent a fundamentally new way to treat cancer

by addressing targets that are otherwise “non-druggable” with existing medicines. In contrast

to Lipinski rule-of-five compliant drugs, siRNAs are too large (13 kDa) and negatively-

charged to cross cellular membranes. Furthermore, naked siRNAs are relatively unstable in

biological fluids and tissues due to degradation by nucleases, contributing to their short half-

lives in vivo [259]. Thus, the effective and non-toxic delivery of RNAi serves as perhaps the

most challenging and significant barrier to successfully translating RNAi technology to a

clinical application. Considerable progress has been made in optimizing siRNA delivery via

chemical modifications to its sugars, backbone, or bases of the oligoribonucleotides,

conferring improved potency and stability against nuclease degradation. Other strategies to

facilitate the delivery and therapeutic efficacy of siRNAs involve the use of liposomes and

lipid complexes, cationic polymers, or conjugates with targeting small molecules, proteins, and

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antibodies for cell type-specific delivery [259]. Numerous proof-of-concept studies with tumor

xenograft models in mice have demonstrated the efficacy of local and systemic RNAi using

various delivery strategies. For example, therapeutic siRNAs targeting PLK1, HER2, and

VEGF have been successful in suppressing tumor growth in prostate and ovarian carcinoma

xenograft models [261-263]. The application of RNAi therapeutics for cancer treatment is also

progressing in the clinic. The first non-human primate study on targeted, systemic delivery of

siRNA against ribonucleotide reductase subunit M2 (CALAA-01) was conducted by Calando

Pharmaceuticals in 2007, demonstrating multiple, systemic doses of siRNA administration to

be safe [264]. CALAA-01 is now in phase I clinical testing for systemic administration to

patients with relapsed or refractory solid tumors. Pre-clinical development of a second siRNA

therapeutic targeting HIF-2 (CALAA-02) is also underway.

If successful, RNAi-based cancer drugs would address some of the major limitations of

traditional pharmaceutical drugs, comprising small-molecules and protein- or antibody-based

therapeutics (Table 5.1). The main advantages of an RNAi strategy include its amenability to

all targets, even those classified as “non-druggable”. Furthermore, lead compounds can be

quickly identified and optimized, as synthetic siRNAs are relatively easy to mass-produce

[259]. Compared to other oligonucleotide-based strategies, such as anti-sense oligonucleotides

(ASO), RNAi is much more potent [265]. Thus, RNAi-based therapeutics demonstrate great

promise as a potential new class of pharmaceutical drugs with the capacity to fill a significant

gap in modern medicine.

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Table 5.1 Comparison of therapeutic modalities

Key features of two major classes of traditional pharmaceutical drugs (small molecules, and

antibodies proteins) compared to RNAi as a therapeutic approach. This table is adapted from

[259].

Small Molecules Antibodies and Proteins RNAi

• Antagonism or agonism of

target

• Antagonism or agonism of target • Antagonism only

• Extracellular and

intracellular targets

• Extracellular targets • All targets, including

“non-druggable”

targets

• Not all target classes can be

modulated selectively and

potently

• Highly selective and potent • Highly selective and

potent

• Lead ID and optimization

slow

• Lead ID and optimization slow • Rapid lead ID and

optimization

• Easy to synthesize • Difficult to produce • Easy to synthesize

5.2.3 Clinical Trials for Molecularly-Targeted Therapies

With the abundance of pre-clinical evidence supporting the role of molecularly-

targeted agents in enhancing tumor response to RT, combining these two modalities appears to

be a rational strategy for cancer treatment. However, translating this approach from promising

pre-clinical findings to clinical trials has been challenging. Despite the increasing number of

targeted agents accrued over the last decade, few have made it into the clinic. There is limited

clinical data available for assessing the true benefits of combining targeted agents with RT.

Moreover, it remains controversial whether these agents can rival cytotoxic chemotherapy in

terms of radiosensitization; thus, clinical trials comparing RT alone, RT-plus-chemotherapy,

vs. RT-plus-targeted agents should be performed to substantiate the widespread notion that

molecularly-targeted radiosensitizers are more efficacious and less toxic than cytotoxic drugs.

The impediment and lack of concordance between pre-clinical and clinical results may be a

result of various factors. Pre-clinical data are often based on a limited number of cell-line

models, which cannot truly reflect the molecular heterogeneity of patient tumors [266].

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Furthermore, there have been limited efforts put towards optimizing combinatorial treatment

regimens (e.g. concentration, exposure, sequence) pre-clinically and clinically. There have also

been limited attempts to determine the predictors of treatment response, and to apply these

biomarkers for patient selection [266]. More recently, it has been recognized that traditional

clinical designs for evaluating cytotoxic chemotherapy may be inappropriate for targeted

agents. For instance, the conventional phase I trial design to establish maximum tolerated

doses may be unsuitable for agents that are intrinsically less toxic to normal tissues or are used

at sub-cytotoxic doses to elicit radiosensitization [266]. A move towards combined phase I/II

studies aimed at defining an optimum dose with simultaneous consideration of tumor effect

and toxicity has been proposed; thus, novel trial designs and surrogate endpoints may be

needed. Given that RT is a curative therapeutic modality for many cancers, it will be a

challenge for clinical researchers to optimally apply this combined approach in further

improving clinical outcome and minimizing toxic side-effects. As such, the National Cancer

Institute Radiation Modifier Working Group is currently working to propose appropriate

clinical trial designs for assessing the combination of molecularly-targeted agents and RT

[267].

5.2.4 Developing UROD as a Therapeutic Radiosensitizing Target

From the two novel therapeutic strategies identified in this thesis, UROD demonstrated

significant potential as a tumor-selective radiosensitizing target, warranting priority for further

pre-clinical assessment. Drug development pipelines often contain compounds in which the

exact cellular mechanisms of action remain poorly understood, slowing their development.

Thus, to further expand our current knowledge and breadth of application of this therapeutic

target, future directions include determining the applicability of our siUROD radiosensitization

strategy to other human cancer models. In addition to the 10 cancer cell lines studied to date

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(Figure 4.7A), a panel of additional cell lines in which IR plays a curative or adjuvant role

(e.g. colon, stomach) should be examined for in vitro and in vivo efficacy. With the

identification of a range of sensitivities to siUROD-plus-IR from the cell lines tested above,

global gene-expression analyses should provide insights into the determinants of siUROD-

mediated radiosensitization. Microarray profiling of pairs of sensitive vs. resistant cells within

one cancer model may reveal a putative “siUROD radiosensitizing signature”, which could be

used as a clinical biomarker when selecting suitable candidates for treatment. In an effort to

acquire additional biological insights as to whether major oncogenic aberrations influence

siUROD radiosensitization, isogenic cell lines with specific common mutations (e.g. p53,

EGFR, ras) should also be tested. Lastly, follow-up studies on the clinical translational

observations reported in this thesis (Figures 4.7D and E) should be conducted. These data were

based on a skewed cohort wherein only 8 of the 38 HNSCC patients did not relapse. Hence,

this translational study should be repeated by performing UROD immunohistochemistry on an

independent larger-sized cohort of HNSCC patients who have been treated with RT to confirm

whether UROD expression would indeed provide a predictive value for clinical outcome.

In addition to the promising pre-clinical findings reported in this thesis, the recent

identification of an endogenous inhibitor against UROD [254], along with the already

described crystal structure of human UROD [255] advance the development of small molecule

inhibitors against the target. Thus, in silico virtual screening or secondary cell-based chemical

screens to identify potential lead candidates that can mimic the inhibitory effects of siUROD

are warranted. Given the evolving power of RNAi technology in drug discovery, and our

expertise with in vitro and in vivo siRNA delivery, it is only fitting that we exploit its use to

enhance our lead identification and optimization efforts for UROD inhibitors. In combination

with other genomic tools (e.g. microarray profiling, C-Map) as described in the previous

136

sections, it is anticipated that a lead compound can be identified. Furthermore, with the

substantial progress being made in terms of RNAi-based therapeutics, it is also plausible to

pursue siUROD as a drug in itself. Any lead UROD inhibitors that are identified will be

subjected to rigorous in vitro and in vivo assessments, as described above to ensure a smooth

transition from pre-clinical to clinical testing.

5.3 Conclusions

In conclusion, this thesis has laid the foundation for the future discovery and

development of novel HNC therapeutics via RNAi and high-throughput screening, with both

target- and phenotype-based screens continuing to play important roles as we further shift into

an era of molecular-targeting. The use of RNAi, and in particular high-throughput RNAi

approaches, has the potential to streamline many of the stages of drug discovery, ranging from

initial target identification to drug development. As the focus continues to progress towards

molecular-targeting, there is great potential to develop rational, hypothesis-driven, mechanism-

based molecular therapeutics for HNC. As such, UROD and the remaining 66 radiosensitizing

targets identified from our RNAi screen provide promising, therapeutically exploitable

avenues for advancing the quality and effectiveness of RT in HNC management. Although

many challenges exist, the powerful and constantly evolving techniques of genomics,

molecular biology, and chemical biology provide an unprecedented opportunity to address

these issues, ensuring excellent prospects for improving clinical outcome for HNC.

137

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