Prognostic and Therapeutic Implications of TP53 Mutations ... · ii Prognostic and Therapeutic...

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Prognostic and Therapeutic Implications of TP53 Mutations in WNT and Sonic-Hedgehog Medulloblastomas by Nataliya Zhukova A thesis submitted in conformity with the requirements for the degree of Masters of Science Graduate Department of the Institute of Medical Science University of Toronto © Copyright by Nataliya Zhukova (2012)

Transcript of Prognostic and Therapeutic Implications of TP53 Mutations ... · ii Prognostic and Therapeutic...

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Prognostic and Therapeutic Implications of TP53 Mutations

in WNT and Sonic-Hedgehog Medulloblastomas

by

Nataliya Zhukova

A thesis submitted in conformity with the requirements

for the degree of Masters of Science

Graduate Department of the Institute of Medical Science

University of Toronto

© Copyright by Nataliya Zhukova (2012)

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Prognostic and Therapeutic Implications of TP53 Mutations in WNT and Sonic-Hedgehog

Medulloblastomas

by

Nataliya Zhukova

Masters of Science, 2012

Graduate Department of the Institute of Medical Science

University of Toronto

ABSTRACT

Recent discoveries enabled us to divide medulloblastoma into molecular sub-groups and

uncover novel mutations in these tumors. However, except for superior survival of the WNT

sub-group, the prognostic and therapeutic implications of these observations remain unclear.

TP53 mutations which confer radioresistance revealed conflicting clinical relevance in different

studies.

We hypothesized that the effect of TP53 mutations on survival is modulated through molecular

sub-grouping. This is especially important since therapeutic targeting of WNT can be achieved

with administration of lithium.

Here we first confirmed that TP53 mutant tumors confers unfavorable outcome only in SHH

subgroup, but not in WNT. We demonstrated that while TP53 mutations cause radioresistance,

activation of WNT/β-catenin signaling radiosensitizes medulloblastoma cells. We demonstrated

that lithium activates the WNT pathway and effectively sensitize medulloblastoma cells to

radiation. Furthermore, lithium did not sensitize normal neural stem cells to radiation,

suggesting its potential as an effective radiosensitizer for medulloblastoma.

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ACKNOWLEDGEMENTS

First and foremost, I would like to sincerely thank my supervisor Dr. Uri Tabori for accepting

me as his first graduate student and for providing me with guidance and support throughout my

entire academic training. Dr. Tabori provided many opportunities for my academic advancement

and was genuinely interested in and supportive of my pursuit of my personal goals in the field of

healthcare. His constant encouragement and advice greatly contributed to a pleasant and

productive graduate experience.

I would also like to thank the members of my program advisory committee, Dr. David Malkin,

Dr. Rob Bristow and Dr. Benjamin Alman for expertise provided to me throughout my training,

for their guidance and support during these two years.

A tremendous thank you extends to our SickKids and international collaborators and their

teams, Dr. Michael Taylor, SickKids, Dr. Vijay Ramaswamy, SickKids, Dr. Steve Clifford,

Northern Institute for Cancer Research, Newcastle upon Tyne, UK and Dr. Stefan Pfister,

German Cancer Research Center, University Hospital Heidelberg, Heidelberg and University

Medical Center, Hamburg-Eppendorf, Hamburg, Germany for their contributions to the

evolution of this project.

I also want to thank Dr. Berivan Baskin and the staff from the Division of Molecular Genetics,

Department of Paediatric Laboratory Medicine, SickKids, for their contribution to this project.

Many thanks are due to Dr. Eric Bouffet for his mentorship during my academic training.

I would also like to mention our lab technician, Cindy Zhang for her constant support through

my work in the lab. She provided an enormous amount of her time and her extensive expertise

in working with stem cells to perform normal neuronal stem cell experiments. In addition she

never fails to step forward and offer help to troubleshoot an experiment or to advise on a better

technique to use.

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Many thanks are due to the past and present members of the Tabori and Malkin Labs, who have

created a stimulating and challenging environment to work in and provided me with their

support and unique expertise both at the bench and in the office and made each day in the lab

enjoyable.

I am very grateful to my wonderful husband for his confidence in my abilities, constant

encouragement, sound advice, but moreover for his never-ending love and support for

everything I brave myself to.

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DATA ATRIBUTION

This thesis consists from two parts: clinical part and biological. This work represents a

collaborative effort on behalf of a number of individuals.

For the clinical part of the thesis, data collection, cohort assembly, statistical analysis and

interpretation of the results were performed by Nataliya Zhukova. Clinical and biological data

on individual patients used in this study were provided by Dr. Clifford, Northern Institute for

Cancer Research, Newcastle upon Tyne, UK; Dr. Pfister German Cancer Research Center,

University Hospital Heidelberg, Heidelberg, Germany; and Dr. Taylor, The Hospital for Sick

Children, Toronto, Canada. Medulloblastoma subgroup analysis for The Hospital for Sick

Children patients was performed by Dr. Ramaswamy, SickKids.

Conceptualization of the thesis, experimental design and execution of all biological

experiments, except normal neuronal stem cell experiments, and all data analysis and

interpretation were performed by Nataliya Zhukova.

Cindy Zhang, laboratory technician, performed normal neuronal stem cell work.

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TABLE OF CONTENTS

Abstract................................................................................................................................

ii

Acknowledgements...............................................................................................................

iii

Data Attribution...................................................................................................................

v

Table of contents..................................................................................................................

vi

List of figures.......................................................................................................................

x

List of abbreviations............................................................................................................

xii

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

1

1.0 Medulloblastoma: incidence, classification, clinical management and

Survival.........................................................................................................................

1

1.0.1. Risk stratification of medulloblastoma patients..................................................

1

1.0.2. Role of craniospinal radiation in treatment of medulloblastoma........................

1

1.0.3. Current management of medulloblastoma...........................................................

2

1.0.4. Treatment of infantile medulloblastoma..............................................................

2

1.1. Medulloblastoma: molecular biology of the tumor, molecular classification, clinical

implications..........................................................................................................................

3

1.1.1. Hereditary cancer syndromes and medulloblastoma...........................................

4

1.1.2. Early molecular studies.......................................................................................

4

1.1.3. Molecular subgroups of medulloblastoma..........................................................

5

1.2. Role of TP53 mutations in the progression and survival of

medulloblastoma..................................................................................................................

7

1.3. TP53 and its role in cellular radiation resistance..........................................................

10

1.4. Role of WNT pathway and CTNNB1 (β-catenin) mutations in the progression and

survival of medulloblastoma................................................................................................

12

1.5. WNT/β-catenin signalling pathway in development and cancer...................................

13

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1.5.1. Role of WNT signaling in embryonal development.............................................

13

1.5.2. WNT activation and stabilization of β-catenin in cancer......................................

14

1.5.3. Activation of WNT/β-catenin signaling as a prognostic marker in

cancer...................................................................................................................................

16

1.6. Role of WNT/β-catenin in radiation response...............................................................

18

1.7. Role of Lithium in mimicking WNT subtype of medulloblastoma..............................

19

1.8. Crosstalk between β-catenin and p53...........................................................................

20

PROJECT RATIONALE AND HYPOTHESIS............................................................

22

CHAPTER 2: MATERIALS AND METHODS............................................................

24

2.1. Patient cohort...............................................................................................................

24

2.1.1. Clinical data........................................................................................................

24

2.1.2. Statistical analysis..............................................................................................

24

2.2. Cell lines......................................................................................................................

24

2.3. TP53 and CTNNB1 sequencing...................................................................................

25

2.3 Chemicals.....................................................................................................................

26

2.4. Irradiation....................................................................................................................

26

2.5. Clonogenic experiments..............................................................................................

26

2.6. Transfection experiments............................................................................................

27

2.7. Combination of Lithium and radiation treatment........................................................

27

2.7.1. Lithium toxicity curves.......................................................................................

27

2.7.2. Clonogenic experiments with combination of lithium and radiation

exposure..............................................................................................................................

28

2.8. Western blot analysis...................................................................................................

28

2.9. β-catenin luciferase reporter assay...............................................................................

29

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2.10. Immunofluorescence – β-catenin nuclear translocation............................................ 29

2.10.1. β-catenin nuclear translocation.........................................................................

29

2.11. Normal neuronal stem cell viability experiments......................................................

30

2.12. Statistical Analysis..............................................................................................

30

CHAPTER 3: RESULTS................................................................................................

31

3.1. Survival of medulloblastoma patients with somatic TP53 mutations depends on

molecular subgroup...........................................................................................................

31

3.1.1. Distribution of TP53 mutations in subgroups....................................................

31

3.1.2. Patient characteristics and survival analysis......................................................

32

3.1.3. Validation of initial observations.......................................................................

36

3.2. TP53 mutant medulloblastoma cell lines are more resistant to radiation..................

38

3.3. CTNNB1 mutation (S33Y) sensitizes TP53 mutant medulloblastoma cells to

radiation............................................................................................................................

39

3.4. Lithium sensitises medulloblastoma cells to radiation..............................................

40

3.4.1. Medulloblastoma cells tolerate physiological doses of lithium.......................

45

3.4.2. Lithium radiosensitized medulloblastoma cells...............................................

45

3.4.3. Lithium phosphorylates GSK3β on Ser9..........................................................

47

3.4.4. Lithium results in nuclear translocation of β-catenin.......................................

47

3.4.5. Lithium constitutively activates canonical WNT signaling..............................

47

3.5. Lithium does not sensitize normal neuronal stem cells to radiation........................

50

3.5.1. Lithium does not decrease survival of normal neuronal stem cells.................

50

3.5.2. Normal neuronal stem cells treated with lithium do not demonstrate nuclear

translocation of β-catenin.................................................................................................

51

CHAPTER 4: DISCUSSION..........................................................................................

54

4.1. TP53 mutations are predictive of inferior survival in SHH, but not WNT

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medulloblastomas.............................................................................................................. 54

4.2. TP53 mutations in medulloblastomas confer resistance to radiation while activation

of canonical WNT signaling radiosensitizes medulloblastoma cells..................................

57

CHAPTER 5: CONCLUSIONS......................................................................................

61

CHAPTER 6: FUTURE DIRECTIONS.........................................................................

62

1) How does activation of WNT/β-catenin signalling offer a survival advantage?

............................................................................................................................................

62

2) Determining of mechanism of lithium action...........................................................

62

APPENDIX A: Phospho-histone H2AX (γH2AX) foci immunofluorescent microscopy

and imaging........................................................................................................................

68

CITATIONS.....................................................................................................................

69

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LIST OF FIGURES

CHAPTER 1: INTRODUCTION

Figure 1.1. Comparison of the various subgroups of medulloblastoma.................

8

Figure 1.2. Figure (a) OS analysis of molecular subgroups among all MB

patients...................................................................................................................

10

Figure 1.3. Canonical WNT/β-catenin signaling in normal and cancer cell............

16

Figure 1.4. Activation of canonical WNT/β-catenin signaling by lithium.................

23

CHAPTER 3: RESULTS

Figure 3.1.1. TP53 mutation spectrum...................................................................

36

Figure 3.1.2. Overall and progression-free survival for TP53 mutant and TP53 wild-type

patients...................................................................................................

39

Figure 3.1.3. Overall (OS) survival for TP53 mutant and TP53 wild-type patients,

MAGIC cohort........................................................................................................

41

Figure 3.2.1. TP53 mutation spectrum and distribution over functional domains in

medulloblastoma cell lines.................................................................................

44

Figure 3.2.2. TP53mut MB cells are more resistant to radiation............................

45

Figure 3.3.1. Nuclear translocation of β-catenin in CTNNB1-S33Y mutant cells...

47

Figure 3.3.2. Activation of WNT pathway signalling via S33Y-CTNNB1 mutation

radiosensitizes TP53 mutant cells..........................................................................

48

Figure 3.4.2. Lithium radiosensitizes both TP53wt and TP53mut medulloblastoma

cells............................................................................................

51

Figure 3.4.3. Lithium phosphorylates GSK3β on Ser9...........................................

52

Figure 3.4.4. Treatment with Lithium results in nuclear translocation of β-catenin in

both TP53 wt and TP53 mut cells.......................................................................

53

Figure 3.4.5. Lithium activates WNT/β-catenin transcriptional activity...................

55

Figure 3.5.1. Treatment with Lithium does not radiosensitize NNSC.....................

57

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CHAPTER 4: DISCUSSION

Figure 4.1. Clinicopathological flow chard for decision making in newly diagnosed MB

patients...........................................................................................

61

Figure 4.2. Relationship between β-catenin and p53 levels...................................

63

CHAPTER 6: FUTURE DIRECTIONS

Figure 6.1.A. Treatment with lithium increases number of γH2AX foci in TP53 wild-

type MB cells...................................................................................................

69

Figure 6.1.B. Treatment with lithium increases number of γH2AX foci in TP53 mutant

MB cells......................................................................................................

70

Figure 6.1.C. Treatment with lithium does not increase number of γH2AX foci in

NNSC.....................................................................................................................

71

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LIST OF ABREVIATIONS

APC Adematous polyposis coli

ATM Ataxia telangiectasia mutated

CHK2 Checkpoint kinase 2

CKIα Casein kinase Iα

CNS Central nervous system

CSC Cancer stem cell

CSI Craniospinal irradiation

CTNNB1 Cadherin-associated protein beta 1

DPLM Department of Paediatric Laboratory Medicine

DSB DNA double strand break

DSH Dishevelled protein

erbB-2 Erythroblastic leukemia viral oncogene homolog 2

FAP Familial adenomatous polyposis

FZ Frizzled receptor

GBM Glioblastoma multiforme

GFP Green fluorescent protein

GSK3β Glycogen synthase kinase 3β

HER2 Human Epidermal Growth Factor Receptor 2

HIC-1 Hypermethylated in cancer 1 protein

IARC International Agency for research of Cancer

IgG Immunoglobulin G

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JNK c-Jun N-terminal kinase

LFS Li-Fraumeni Syndrome

LiCl Lithium Chloride

MAGIC Medulloblastoma Advanced Genomics International Consortium

MB Medulloblastoma

mdm2 Murine double minute 2

miRNA microRNA

MLPA Multiple ligation-dependant probe amplification

MYC-N Myelocytomatosis viral related oncogene, neuroblastoma derived

NNSC Normal neuronal stem cells

OS Overall survival

PCP Planar cell polarity

PCR Polymerase chain reaction

PFS Progression free survival

PTCH Patched 2

SER Sensitizer enhancement ratio

SF2 Survival fraction at 2GY

SHH Sonic Hedgehog

siRNA Small interfering RNA

TCF/LEF T-cell factor / Lymphoid enhancer-binding factor

TCF4 Transcription factor 4

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TP53 Tumour protein 53

trkC Tyrosine kinase receptor

WNT WNT-Wingless pathway

yH2AX Phospho-histone H2AX

β-TrCP β-transducin repeat containing protein

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CHAPTER 1: INTRODUCTION

1.0 Medulloblastoma: incidence, classification, clinical management and survival

Medulloblastomas (MB) are the most common malignant central nervous system (CNS)

tumours of childhood. They occur at all ages; however, peak incidence is between 3 and 9 years

of age. In adults, medulloblastomas arise most commonly in the 30’s and 40’s and comprise 1-

2% of all brain tumours [1]. Overall males are 1.5 times more likely to be affected than females

[2-3].

Tumours which arise from the posterior fossa are called medulloblastomas; other histologically

similar embryonal tumours which originate from the pineal region or cerebrum are no longer

classified as medulloblastomas as they are molecularly distinct entities [1, 4]. Histologically

medulloblastoma appears as densely packed round cells with hyperchromatic nuclei and scanty

cytoplasm and as such belongs to the family of so-called “small round blue-cell tumours”.

Currently three histopathological variants of the tumour are recognised: 1) classic; 2)

anaplastic/large cell variant; and 3) desmoplastic with extensive nodularity [5].

1.0.1. Risk stratification of medulloblastoma patients

Risk stratification of patients has been developed as a result of multiple prospective, multicenter

randomised trials from Europe and North America and currently encompasses two groups: high-

risk patients who have disseminated disease at presentation or post-operative residual disease

more than 1.5 cm, and average-risk patients who have non-disseminated disease with total or

near-total resection of the primary tumour [1-2, 6]. Tumour histology is not universally included

in the risk stratification, however it is recognized that desmoplastic tumours of infancy have

better outcomes [1, 7]. Anaplastic histology is also widely accepted as a poor prognostic factor;

currently, in the US and Canada, all patients with anaplastic tumours regardless of the

dissemination of the disease or extent of the resection are assigned to a high-risk group [1, 8].

1.0.2. Role of craniospinal irradiation in treatment of medulloblastoma

Surgical resection alone of medulloblastoma is not curative. As was described by Cushing in the

1930s only 1 out of 63 patients survived with surgical resection alone in his series [9].

Recognition that medulloblastoma cells are sensitive to radiation, with 18 to 44% of cells

1

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surviving a dose of 2Gy [10], led to incorporation of radiotherapy into treatment protocols.

However, local radiation to the tumour site did not improve outcome [11]. Poor outcome for

children treated without cranio-spinal radiation (CSI) was related to a high proportion of

patients developing disseminated disease at recurrence [12]. The introduction of CSI allowed for

a dramatic increase in survival of medulloblastoma patients up to 50% of those treated. Edith

Paterson first demonstrated survival in 5 out of 12 children with medulloblastoma treated with

CSI in 1932 – 1947, followed by survival of 8 out of 15 patients treated with CSI in Toronto in

the 1950s; all 16 patients who did not receive CSI died [13]. Later, consistent survival rates

ranging from 27 to 40% have been demonstrated by multiple groups [9]. For many years the

standard dose of radiation was 36 Gy to the whole neural axis with a boost to the posterior fossa

to a total dose of 55 Gy to improve local control [14]. Detrimental long-term effects of radiation

to the developing brain resulted in attempts to reduce CSI to 24 Gy with the retention of the

posterior fossa boost at 55 Gy. Decrease of CSI radiation dose without addition of

chemotherapy resulted in both higher local relapse rate and higher rate of disseminated disease

at relapse [6, 15-16]. Addition of cis-platinum based chemotherapy to the treatment protocol

allowed for decrease of the CSI dose to 24 Gy in average-risk patients without compromising

survival [12, 17]. It is also recognised that inferior survival in infants and children less than

three years old is greatly related to avoidance / delay in CSI in order to minimize radiation

toxicity to the developing brain [18].

1.0.3. Current management of medulloblastoma

Treatment of medulloblastoma currently involves surgical tumour removal, radiation therapy

and adjuvant chemotherapy. As discussed above, treatment with radiation alone yields inferior

5-year and overall survival (OS) as well as progression free survival (PFS) (60%) in multiple

studies in average risk patients. Implementation of risk adapted therapy with high-dose adjuvant

chemotherapy and hematopoietic stem cell rescue in addition to radiation allowed for improved

tumour control with 5 year OS of 85% and 70% for average- and high-risk patients, respectively

[19-21].

1.0.4. Treatment of infantile medulloblastoma

Treatment of infantile medulloblastoma is highly problematic as use of cranio-spinal radiation

therapy is limited by significant neuro-cognitive impairment following irradiation of the

2

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immature brain. Thus, treatment in patients less than 3 years of age and in some studies less than

5 years of age is limited to chemotherapy-only regimens to avoid or delay cranio-spinal

radiation. The results of such trials are mixed [2, 6-7, 22-24]. Duffner et.al. showed that patients

younger than 3 years can be treated with chemotherapy only until they reach 3 years of age and

then receive cranio-spinal radiation with survival rates of 40% or less. However survival was

limited predominantly to patients with non-disseminated disease and was accompanied by a

significant neurocognitive decline secondary to radiation [23]. In subsequent studies, attempts

were made to intensify chemotherapy and avoid radiation, which resulted in 5-year PFS of 32%

and OS of 43% with almost 58% of children being able to avoid radiation [22]. Addition of

intravenous and intraventricular Methotrexate has significantly improved PFS and OS in non-

disseminated completely resected disease (82% and 93%, respectively). However, although

improved slightly for patients with residual disease (50% and 56%, respectively), outcomes

remained inferior for infants with disseminated disease (33% and 38%, respectively). In all

patients in this series IQ was lower than in healthy peers, however, markedly higher compared

to children received cranio-spinal radiation. Desmoplastic histology was determined to be an

independent prognostic factor for better survival in those series [7]. In addition, improved

outcomes could be partially attributed to better differential diagnostics. In particular, in recent

studies, 15-20% of infants who would have been diagnosed with medulloblastomas are now

diagnosed using immunohistochemical and molecular/genetic techniques with atypical

teratoid/rhabdoid tumours associated with extremely poor survival and are removed from the

analysis of the medulloblastoma study population [1]. Furthermore, most of the patients with

favorable outcome in infant studies have desmoplastic pathology. Together, these clinical

observations of inferior outcome for children treated with radiation sparing protocols further

support the central role of radiation therapy in survival of childhood medulloblastoma.

1.1. Medulloblastoma: molecular biology of the tumour, molecular classification,

clinical implications

The increase in understanding of molecular biology of cancer, and of medulloblastoma in

particular, led to multiple studies to improve diagnostics, risk stratification and better tailoring

of treatment based on tumour molecular and genetic alterations.

3

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1.1.1. Hereditary cancer syndromes and medulloblastoma

Several hereditary cancer syndromes have higher frequencies of medulloblastoma. These

include Turcot (WNT pathway and APC alterations) [25], Gorlin (Sonic Hedgehog pathway and

PTCH mutations) [26-27] Li-Fraumeni (TP53 germline mutations) and several DNA damage

repair syndromes. These genetic predisposition syndromes have pointed to the importance of

these pathways and gene alterations in the origin, development and clinical course of

medulloblastomas. Approximately 4% of Gorlin syndrome (nevoid basal cell carcinoma)

patients develop medulloblastomas; 8-10% of sporadic medulloblastomas, primarily of

desmoplastic variant, have been shown to have a somatic PTCH mutation, 9q22.3 alterations

and Sonic Hedgehog pathway activation as in Gorlin syndrome patients. Interestingly, PTCH

mutations in medulloblastoma have been correlated to favorable outcome [2, 26]. As detailed

below, these pathways, specifically WNT, SHH and TP53 will play a major role in the

classification and prognosis of medulloblastomas.

1.1.2. Early molecular studies

Early studies have shown overexpression of neurotropin-3 receptor trkC which is involved in

cell differentiation, growth and apoptotic response being a favorable prognostic marker [2, 28-

29]. trkC was found in approximately 48% of tumours; patients with high levels of trkC

demonstrated higher 5-year survival rates with longer PFS time compared to those with low

levels, 89% vs. 46% respectively [29].

In contrast, high expression of the erbB-2 oncogene correlated with poor prognosis with 10-year

OS of 10% vs. 48% in non-overexpressing tumours [30]; HER2-HER4 receptor heterodimer

overexpression was associated with poor survival, despite individual overexpression of either

HER2 or HER4 being not prognostic [31].

Prognostic value of MYC remains controversial. Grotzer et.al. has shown that expression levels

of MYC do not always correlate with MYC gene amplification, however low MYC expression

levels are a powerful independent predictor of better survival [32]. MYC-C amplifications have

been found in 5-10% of medulloblastomas [5, 33]. High MYC-C amplification was reported to

correlate with clinically aggressive tumours with short progression free survival times and

tendency to disseminate [33]. A recent study done by Pfister et.al. has demonstrated that both

MYC-C and MYC-N are associated with poor prognosis [5]. Interestingly, Northcott et.al. has

4

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shown differential distribution of MYC-C and MYC-N within molecular subgroups with MYC-C

being found exclusively in Group C (Group 3) medulloblastomas with the most aggressive

behavior and MYC-N being equally distributed between SHH, Group C (Group 3) and Group D

(Group 4) tumours [34]. A recent study has demonstrated that MYC-C amplifications define an

aggressive subgroup of medulloblastoma which is consistent with previous findings; in turn,

MYC-N amplifications are clustered within two subgroups: SHH – 38% of cases where half of

MYC-N tumours also have TP53 mutations and Group D (Group 4) – 62% of cases and

associated with worse prognosis [35].

Loss of 17p with isochromosome 17q has been described as the most common genetic

aberration in medulloblastoma, which is present in 35-50% of tumours, and has been associated

with poor prognosis in multiple studies [36-40]. HIC-1 which encodes a zinc finger

transcriptional repressor is most often disrupted by the 17p deletion. Under normal conditions,

HIC-1 is upregulated by TP53 and silenced by hypermethylation [2]; Hypermethylation of HIC-

1 is frequent in medulloblastomas with intact 17p and is predictive of poor outcome [41].

In addition to frequent 17p deletion and iso-17q, unbalanced translocations or deletions of

chromosomes 8, 10q, 11p and 16q are common in medulloblastomas [42], whereas deletions of

chromosomes 1, 2, 9q, 6q are less prevalent [5, 42]. Medulloblastomas typically have gains of

chromosomes 4, 7, 8 and 18, with less frequent gains of 6q. The prognostic value of these

genomic alterations is uncertain, except for the 6q aberrations: deletion of 6q is strongly

correlated with excellent survival, while 6q gain is associated with poor outcome [5]. Together,

these exciting molecular and genetic determinants fail to produce reliable results in larger

studies and therefore are not currently used in clinical stratification of medulloblastoma.

1.1.3. Molecular subgroups of medulloblastoma

Multiple studies describing roles of molecular markers and genetic aberrations, discussed in

detail above, suggest the existence of several subtypes within the single diagnosis of

medulloblastoma. Development of computational biology and high-throughput methods of

genomic data processing and analysis coupled with access to large patient cohorts through

establishment of international collaborations and multicenter studies, have made it possible to

study transcriptional and copy number changes across whole genome, allowing for the

publications of multiple studies describing molecular subtypes in both paediatric and adult

5

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medulloblastoma. In 2006 Thomson and colleagues demonstrated among 46 patient samples the

existence of five medulloblastoma subgroups, named A through E, with unique gene expression

signatures. As part of this study, he identified WNT activating/6q deleted tumours as group B

and SHH tumours as group D [43]. These findings were supported by Kool et.al. who, in a study

of 62 patients, identified five molecular variants within medulloblastoma including WNT and

SHH subtypes along with subtypes expressing neuronal differentiation genes (groups C and D)

and photoreceptor genes (group D and E). He has also demonstrated that identified groups are

different in demographics, presence of metastatic disease, age at presentation and histology [44].

Later, Fattet and colleagues correlated CTNNB1 mutations and nuclear translocation of β-

catenin in 72 paediatric medulloblastomas with strong expression profile of WNT activation and

demonstrated that this group of patients have superior survival [45]. In 2010 Northcott and

colleagues, using 103 samples, demonstrated that four medulloblastoma subgroups, named

WNT, SHH, group C and group D, distinguished by their demographics, clinical presentation

and survival outcomes, can be identified using their expression signatures. They subsequently

demonstrated that immunohistochemical techniques can be successfully implemented to identify

them robustly [34]. Six molecular subgroups were identified by Cho and colleagues using a

correlation between clinical characteristics and a unique combination of structural chromosomal

aberrations together with copy number variations and miRNA profiles in 194 patients [46].

Similar work was done in adult medulloblastoma where three groups, WNT, SHH and group D,

were identified on 28 patients using transcriptome analysis and then validated on an independent

cohort of 103 patients using tissue microarray. These subgroups demonstrated distinct

differences in demographics, clinical behaviour, histology as well as survival [47]. At the same

time Al-Halabi and colleagues demonstrated that although a SHH subset of tumours was

identified within adult medulloblastomas those tumours bore some notable differences in

molecular profile compared to paediatric SHH medulloblastomas. In addition, they were not

associated with favorable prognosis [48].

In summary, multiple research groups have demonstrated that medulloblastoma indeed is not a

homogeneous disease, but rather is comprised of several molecular subgroups. However, there

is a lack of uniformity in the number of subgroups reported and the criteria by which those

subgroups were characterized. In order to rectify this problem a consensus meeting, involving

all major medulloblastoma study groups was held in Boston in 2010. Recommendations were

adopted for universal use for clinical and research purposes of four subgroups of

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medulloblastoma: WNT and SHH, named after major pathways affected in those tumours, and

Group 3 and Group 4, in which no specific pathway alterations were yet identified based on

expression profiling. The major differences in clinical, molecular and genetic characteristics

between those groups are described in Figure 1.1. [49].

In addition, meta-analysis performed by Kool and colleagues in 2012 on 550 pooled samples

from 7 previous studies clearly demonstrated that medulloblastoma consists of the four

subgroups identified previously. These subgroups exhibit major differences in demographics,

clinical representation, genetic alterations and have unique transcription profiles [3].

Importantly, despite striking differences allowing reliable subgroup segregation, molecular

profiling fails to provide a robust instrument for patient risk-stratification. As it is evident from

Figure 1.2. only WNT tumours were identified as a favorable prognostic group, while the rest of

the subgroups demonstrated inferior survival without significant intergroup differences [3].

Therefore, the need still exists for development of better more robust prognostic markers that

use universally available techniques.

1.2. Role of TP53 mutations in the progression and survival of medulloblastoma

Medulloblastomas are among three brain tumours commonly found in patients who harbour

TP53 germline mutations (the other two are high grade gliomas and choroid plexus carcinomas)

and are defined as a major component tumour of the Li-Fraumeni syndrome (LFS) [50-51]. In

addition, as discussed above, multiple cytogenetic studies have demonstrated somatic non-

random loss of chromosome 17p, suggesting that TP53 (located on chromosome17p.13) may

have a role in medulloblastoma development and progression. The prognostic role of TP53

mutations in medulloblastoma remains unclear. The prevalence of somatic TP53 mutations in

medulloblastoma patients is less than 10% [52]. Despite frequent 17p monosomy, genetic events

specific to TP53 function, such as inactivating mutations or small deletions of TP53 itself or

alterations in mdm2 are rare events with unknown prognostic implications [40, 53-55]. On the

other hand, loss of TP53 function has been attributed to an increased incidence and earlier

development of medulloblastoma in mice heterozygously deficient for PTCH [56] suggesting

that it plays an important role in the aggressiveness of the disease. Strong association of nuclear

p53 immunopositivity in medulloblastoma samples with poor survival has been demonstrated by

several more recent studies [57-58].

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Taylor et.al. Acta Neuropathol 2012

Figure 1.1. Comparison of the various subgroups of medulloblastoma including their affiliations with previously published papers on medulloblastoma molecular subgroup. (LCA – large cell anaplastic, M+ -

metastatic disease, CTNNB1- β-catenin)

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Figure 1.2. Figure (a) OS analysis of molecular subgroups among

all patients from Kool et.al. Acta Neuropathol 2012. Overall survival

(OS) analyses of molecular, clinical, and histological subgroups

within the gene expression profiling cohort using Kaplan – Meier

plots and log-rank tests.

Kool et.al. Acta Neuropathol 2012

Months

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Clinical data utilizing TP53 mutations as a prognostic marker for recurrence and survival of

medulloblastoma patients revealed contradictory results. Correlation of clinical and survival

data with tumour biology obtained for 119 medulloblastoma patients who underwent treatment

at The Hospital for Sick Children between 1985 and 2001 demonstrated that tissue immuno-

positivity for p53 protein is among four of the strongest predictors of survival, with p53

immuno-positive patients having worse survival compared to their immuno-negative

counterparts [57]. Tabori et.al. has reported rapid recurrence and death in all (8/52) of the TP53

mutant patients in their series [59]. This was supported by Pietsch and colleagues who

correlated inferior survival with p53 immuno-positive tumours [60] in metastatic patients. In

contrast, Pfaff et.al. has demonstrated no difference in 5-year overall survival between mutant

and non-mutant TP53 patients in their cohort [61]. Similarly, no adverse survival in TP53

mutant patients was demonstrated by Clifford and colleagues. Specifically three out of four

patients in the WNT subgroup had durable remission. The only patient who succumbed to

disease in this cohort had a germline TP53 mutation [62]. Interestingly, both Pfaff and Clifford

observed cohort enrichment of the TP53 mutant population in the WNT subgroup tumours [61-

62], while the SickKids cohort had a higher proportion of the SHH subtype (personal

communication, Dr. M. Taylor, The Hospital for Sick Children, Toronto).

In summary, conflicting reports regarding the role of TP53 mutations in disease progression and

patient survival coupled with variation in TP53 mutation distribution in WNT and SHH

molecular subgroups between cohorts suggest the presence of possible biological modulating

factors and warrant further investigation.

1.3. TP53 and its role in cellular radiation resistance

TP53 is a tumour suppressor gene whose biological function and role in tissue radiation

response has been intensively studied for the last three decades. TP53 is mutated or deleted in

over 50% human cancers [63]; in cancers which retain wild type TP53 the function of p53

protein is often silenced by other mechanisms which include complex formations with viral

(large T antigen of SV40 virus) or cellular proteins or alterations of genes up or downstream of

TP53 to abrogate p53 associated apoptosis, cell cycle arrest or DNA brake/repair response [64].

Missense mutations which result in altered amino acid sequence, protein structure and function

account for more than 85% of genetic alterations in TP53. The majority of mutations are located

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in the DNA binding domain and affect p53 transcriptional function. Codons 175, 245, 248, 273

and 283 are hot-spots for mutations and are overrepresented in all tumours due to their

susceptibility to spontaneous mutagenesis and slower repair rates in the core domain [65]. In

addition, these particular codons are part of promoter-specific structures essential for p53

transcriptional function [66]. The role of p53 in mediation of apoptosis, cell cycle arrest and

DNA damage response to radiation is well described in the literature. Apoptosis and cell cycle

arrest are tissue and tumour specific and are not entirely mutually exclusive. In haematopoietic

cells (thymocytes, lymphocytes) and other cells sensitive to radiation (germ cells) p53 induce

apoptosis, preventing potentially oncogenic cells from proliferation. In fibroblasts and epithelial

cells, p53 induces cell cycle arrest in G1 and G2/M phases of the cell cycle. Cell cycle arrest

can be transient or permanent depending on the extent of DNA damage [67]. Wild-type p53 is

phosphorylated directly by ataxia telagiectasia mutated (ATM) and indirectly by ATM activated

Chk2 in response to DNA damage. Phosphorylation of p53 leads to disruption of its interaction

with mdm2, resulting in stable p53 protein, and also facilitates nuclear accumulation of

transcriptionally active protein by inhibiting nuclear export. Stable p53 becomes actively

engaged in transcription of downstream targets modulating tissue specific cellular responses to

radiation: cell cycle arrest, apoptosis and facilitating DNA damage repair [67-70].

Multiple studies have demonstrated that loss of p53 wild-type function confers resistance to

ionizing radiation. The role of p53 in resistance to radiation has been discussed in numerous

reviews; loss of G1 check point control, associated with TP53 mutations, was shown to result in

resistance to radiation in vitro [65, 71]. Williams and colleagues have shown on an extensive

panel of cancerous cell lines that mutant p53 cells are more resistant to radiation than wild-type

p53 cells at both low and high radiation doses and argue that TP53 status can be used as a

marker of tumour radiosensitivity [72-73]. Interestingly, there have been several reports

correlating TP53 mutations with radiosensitive phenotype. Matsui and colleagues demonstrated

that both murine and human fibroblasts containing engineered TP53 mutations on a p53 null

background were more sensitive to radiation, despite retention of impaired G1 check point, than

the TP53 wild-type or TP53 null counterparts [74]. Similarly, Servomaa et al. has shown that

p53 wild-type squamous cell carcinoma cell lines are more radiosensitive than ones bearing

TP53 mutations [75]. The existing controversy is partially explained by tissue specific cellular

response to radiation, modulated through other cross-talking pathways as well as multiplicity of

p53 functions in the cell.

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The role of TP53 mutations in radioresistance of medulloblastoma cells has not been extensively

studied. Salaroli et. al. have demonstrated increased resistance to ionizing radiation in TP53

mutant medulloblastoma cells [76]. Craniospinal radiation therapy remains the main medical

treatment for patients with medulloblastoma; in light of recent studies which demonstrate

association of clinical outcomes with the TP53 mutation status, greater understanding of p53-

mediated cellular response to radiation, specific to medulloblastoma, is crucial in order to

improve therapies.

1.4. Role of WNT pathway and CTNNB1 (β-catenin) mutations in the progression and

survival of medulloblastoma

Activation of the WNT/β-catenin pathway is present in 15-25% of medulloblastomas [45, 52,

77]. Activation of WNT signalling was first described as a germline mutation in the APC gene in

Turcot syndrome [25], which led to speculation about the role of the pathway in

medulloblastoma development. In sporadic medulloblastomas, APC is intact, however somatic

pathway-activating mutations in the CTNNB1 (β-catenin) gene were described by several groups

[45, 49, 78-79]. Currently the WNT subgroup is identified by expression profile demonstrating

WNT/β-catenin pathway activation; an overwhelming majority of these tumours have CTNNB1

mutations in exon 3 [34, 44, 49]. However, expression profiles do not provide an unequivocal

subgroup assignment. Overexpression of some of the WNT signature genes was identified in

SHH and Group 3 tumours as well [34] in addition to a single case with both CTNNB1 and

PTCH mutations [49].

Multiple studies have demonstrated a strong correlation of widespread nuclear immunopositivity

for β-catenin with presence of exon 3 mutations in CTNNB1; at the same time none of the

immunonegative samples or samples having focal nuclear immunopositivity were identified as

having CTNNB1 (β-catenin) mutations [21, 45, 79-80]. This strong correlation of the nuclear

immunopositivity for β-catenin with presence of mutation in CTNNB1 in combination with

available reliable antibodies allows for use of the immunohistochemical staining in clinical

setting providing quick, inexpensive and reliable marker for CTNNB1 mutation.

Ellison and colleagues first correlated immunopositivity for β-catenin with significantly

improved survival. Activating mutations in β-catenin have been determined to be a universal

favorable prognostic marker for both average and high risk patients; notably, all metastatic

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patients within this subgroup are alive at least 5 years post-diagnosis [79]. Superior outcome for

this group has been confirmed by several other studies citing long-term survival for WNT

subgroup patients exceeding 90% [3, 21, 34, 44-45, 49, 77, 80-81]. However there is no

literature elucidating biological mechanism responsible for better survival in WNT

medulloblastomas.

1.5. WNT/β-catenin signalling pathway in development and cancer

1.5.1. Role of WNT signaling in embryonal development

The WNT pathway is highly conserved from Drosophila to humans. It plays important roles in

both embryogenesis and cancer. WNT proteins are key players in cell fate determination and

patterning during embryonic development [82]. Defects of this pathway result in severe

developmental abnormalities and defects of embryogenesis [82-84]. WNT signalling is involved

in regulation of cell proliferation, differentiation and epithelial-mesenchymal interactions in a

variety of tissues, from formation of body axes during the early stages of development to tissue

differentiation during organ development, including the brain [84]. The WNT pathway plays

important roles in nervous system development, including differentiation and axon growth,

synaptic formation and myelination. Mutations in WNT1 result in severe midbrain, hindbrain

and spinal cord abnormalities. Interestingly, prolonged activation of WNT and/or

overexpression of ectopic β-catenin results in expansion of the neuronal progenitor cell pool and

enlargement of the forebrain and spinal cord, and increased proliferation of the ventricular zone

accompanied by defects of glial and neuronal differentiation [85]. In adults, WNT signalling

plays a role in maintenance of the stem cell population in highly proliferative tissues [84].

Currently, more than 19 WNT proteins and more than 10 Frizzled receptors are described in

WNT pathway. Major effector branches of the pathway depend on a combination of a particular

WNT ligand and Frizzled receptor and are subdivided into three major streams: 1) canonical

WNT/β-catenin pathway which predominantly regulates cell proliferation and differentiation

through β-catenin TCF/LEF mediated transcriptional activation of target genes and is implicated

in cancer [82-84]; 2) WNT/Ca++

branch which regulates cell adhesion and cell motility [84]; and

3) planar cell polarity (PCP) pathway which is involved in cell morphogenesis and cell polarity

via JNK [84].

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The key event in canonical WNT/β-catenin signaling is accumulation of the active β-catenin in

the cytoplasm, its subsequent translocation to the nucleus and activation of the downstream

effectors. The WNT/β-catenin signalling in normal and tumour cells is outlined in Figure 1.3.

In normal cells, the absence of WNT ligand results in low β-catenin levels both in the cytoplasm

and nucleus. Endogenous β-catenin is found in cell-cell junctions forming complexes with

cadherin-associated proteins to execute adhesion function. Free β-catenin is constantly

destroyed by the protein complex consisting of glycogen synthase kinase 3β (GSK3β), Axin,

adematous polyposis coli (APC) protein and casein kinase Iα (CKIα). Phosphorylation of β-

catenin on serine-45 (Ser45) enables its subsequent phosphorylation on serine-33, 37 (Ser33,

Ser37) and threonine-41 (Thr41). Phosphorylated β-catenin is ubiquitinated by β-transducin

repeat containing protein (β-TrCP) and degraded in the proteosome [83-84, 86]. At the same

time in the nucleus WNT targets are silenced by T-cell factor (TCF) and lymphoid enhancer-

binding protein (LEF) transcription factors [83]. Activation of the pathway begins with the

binding of WNT ligand to the corresponding Frizzled (FZ) receptor. Frizzled recruits dishevelled

protein (DSH) to the plasma membrane; phosphorylated DSH through interaction with Axin

prevents GSK3β by its phosphorylation on serine-9 (Ser9) from phosphorylation of β-catenin.

This leads to stabilization of β-catenin, its cytoplasmic accumulation and subsequent nuclear

translocation. In the nucleus, β-catenin couples and interacts with TCF/LEF complexes allowing

for transcriptional activation of downstream targets such as Cyclin D1, MYC and others [84, 86].

1.5.2. WNT activation and stabilization of β-catenin in cancer

In tumour cells, WNT activation and β-catenin stabilization are commonly achieved by

deactivating mutations in APC and activating mutations in CTNNB1 (β-catenin) [25, 78, 82, 87].

In the case of medulloblastoma, CTNNB1 mutations are the predominant way to activate WNT

pathway. In this case in the continuous absence of WNT signal, mutations in Ser45, Ser33, Ser37

and Thr41 act to prevent GSK3β from phosphorylation of β-catenin; they result in accumulation

of stable β-catenin in the cytoplasm and its translocation to the nucleus with subsequent

transcriptional activation of downstream targets via interaction with TCF/LEF [82-84, 86, 88-

90].

Inappropriate activation of the WNT pathway is implicated in cancer development.

Overexpression of WNT1 has been linked to development of mouse mammary carcinomas.

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Figure 1.3. Canonical WNT/β-catenin signaling in normal andcancer cell. (A) Phosphorylation of β-catenin by GSK3β resulted in

proteosomal degradation of the β-catenin protein; Canonical WNT

activation (B) – binding of multiple WNT ligands to Fz receptor

subsequently inhibits GSK3β via DSH, this results in stabilization of β-

catenin, it’s nuclear translocation and activation of TCF/LEF mediated

signaling;

(C) Mutation in β-catenin prevents GSK3β from phosphorylation and

targeting β-catenin for degradation, resulting in nuclear translocation of

β-catenin and initiation of TCF/LEF mediated transcription; Presence of

mutation constitutively activates WNT/β-catenin signalling similar to

canonical WNT signaling in normal cell.

A B C

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WNT1, WNT2 and WNT3 proteins are known to have high transforming potential and conduct

their signal through the canonical β-catenin pathway. Transfection of normal epithelial cells

with these WNT family proteins results in high rates of transformation [82]. In addition,

mutations in downstream members of the WNT pathway are detected in multiple cancers. APC

mutations are a key genetic event defining familial adenomatous polyposis (FAP) and are also

present in 80% of sporadic colon cancers [91-92]. Mutations in CTNNB1 are rare in colon

cancer and are always mutually exclusive with APC mutations [82]. Nuclear translocation of β-

catenin is observed in a third of primary melanoma samples [93]. Despite the fact that activating

mutations in CTNNB1 or deactivating mutations of APC are frequently found in melanoma cell

lines [94], they are rare events in primary tumours, suggesting a different mode of WNT

activation [93]. In prostate cancer, β-catenin mutations are found only in 5% of cases [95],

however transcriptionally active β-catenin has been shown to interact with androgen receptors

contributing to hormone resistance during prostate cancer progression [82, 96-97]. WNT

pathway activation is also implicated in thyroid, liver, ovarian and endometrial cancers [82, 84,

86]. In gliomas, levels of β-catenin in the tumour tissue and its nuclear accumulation correlate

with World Health Organization (WHO) grading of these tumours with higher level of nuclear

β-catenin being the feature of high grade gliomas [98]. Aberrations in WNT signalling are

described in paediatric embryonal solid tumours: 75% of hepatoblastoma cases have activating

CTNNB1 alterations – mutations and small frame shift deletions; 15% nephroblastomas have

aberrant activation of WNT through mutations in CTNNB1, predominantly affecting codon 45;

62% of pancreatoblastomas have activating mutations in CTNNB1. However, no correlation

with tumour progression or survival has been studied. Approximately 15-25% of sporadic

medulloblastomas demonstrate activation of WNT signalling with presence of CTNNB1

mutations. While mutations in APC are extremely rare in sporadic tumours [99-100][78, 84], as

mentioned before, germline APC mutations are found in patients with Turcot syndrome, a form

of familial adenomatous polyposis (FAP) with high incidence of medulloblastomas [25].

1.5.3. Activation of WNT/β-catenin signaling as a prognostic marker in cancer

The effects of WNT/β-catenin pathway activation are tissue and cell type dependent. Nuclear

accumulation of transcriptionally active β-catenin can promote aggressive behaviour in some

cells and apoptosis / cell growth arrest in the others [76]. In colorectal carcinomas, specific

mutations in the APC gene which prevent proper APC – β-catenin interactions have been

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predictive of poor survival [101]. In both colorectal cancer and hepatocellular carcinoma, β-

catenin nuclear accumulation has been correlated with reduced survival associated with

metastatic disease and aggressive tumour behaviour; in hepatocellular carcinomas nuclear β-

catenin accumulation has been correlated with high levels of Ki67, suggestive of increased cell

proliferation and tumour progression [86, 102-103]. Similarly, in breast cancer transcriptionally

active β-catenin is associated with overexpression of Cyclin D1 and poor survival [104].

Interestingly, some studies have shown that accumulation of mutant β-catenin in the nucleus is

associated with non-invasive tumours and better prognosis, suggesting that accumulation of wild

type β-catenin due to upstream pathway alterations and mutant β-catenin may have different

roles in tumour progression [86]. Activation of WNT/β-catenin signalling with nuclear

accumulation of β-catenin has been shown to decrease tumour size in melanoma both in patients

and in animal models [105]. Several reports link overexpression / accumulation of

transcriptionally active β-catenin to apoptosis and /or growth arrest: in kidney cell lines (COS7

and 293) β-catenin appears to be a mediator of apoptosis [106]. In turn, Julling and colleagues

have demonstrated in an osteosarcoma model that overexpression of β-catenin induces apoptosis

through a p53-independent pathway [107] . In retinoblastoma, activation of the WNT pathway

significantly reduces cell viability via induction of cell cycle arrest in cell lines. In primary

tumour tissues and mouse models canonical WNT signalling is suppressed, suggesting that at

least in retinoblastoma WNT acts as tumour suppressor [108]. In medulloblastoma, multiple

studies have shown that WNT pathway activation is associated with accumulation of

transcriptionally active β-catenin in the nucleus and is associated with improved survival as

described above [45, 79-80].

In summary, the role of WNT/β-catenin activation in both development and cancer has been

extensively studied and multiple reports exist, associating WNT activation and presence of

nuclear β-catenin with cancer phenotype. It is clearly evident that in a large number of cancers,

notably mostly in adults, WNT activation and nuclear β-catenin are associated with an

aggressive tumour phenotype, presence of metastatic disease and poor prognosis. At the same

time, in melanoma and two major paediatric cancers, retinoblastoma and medulloblastoma,

activation of WNT/β-catenin signalling was clearly associated with favorable disease outcome.

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1.6. Role of WNT/β-catenin in radiation response

Involvement of β-catenin in response to radiation is poorly studied. In the limited published

literature it has been shown that WNT/β-catenin mediates radiation resistance in both mouse

mammary progenitor cells and human breast cancer cell lines. [109-110]. Recent studies suggest

that aberrant activation of WNT/β-catenin signalling may contribute to radiation resistance of the

cancer stem cell (CSC) population. Exact mechanisms of radioresistance in CSC are yet to be

elucidated, however speculations have been made that excessive WNT/β-catenin signalling

results in chromosomal instability via deregulation of mitotic spindles and high tolerance to

DNA damage [111]. Contribution of aberrantly activated WNT/β-catenin pathway to

radioresistance was also described in oesophageal and colorectal cancers [112-113].

Interestingly, in colorectal cancer WNT involvement was independent of active β-catenin

accumulation and limited to tumours with overexpression of TCF4 [113]. In glioblastomas

multiforme (GBM) WNT/β-catenin pathway is activated in post-radiated tissues. In both cell

lines and in vivo mouse models enrichment for stem cell like populations resistant to radiation

was observed after irradiation with cells positive for transcriptionally active β-catenin,

suggesting that WNT/β-catenin activation contributes to radiation resistance. In addition,

pharmacological and/or siRNA inhibition of WNT signalling resulted in radiosensitisation of

tumour cells and depletion of β-catenin positive subpopulations [114].

Salaroli and colleagues first showed that in medulloblastoma cell lines radiation triggers WNT

activation. Subsequent activation of cell cycle arrest and apoptosis in both TP53 wild type and

mutant cells suggests that in TP53 deficient medulloblastoma cells, growth arrest and apoptosis

may be mediated through WNT/β-catenin bypassing p53 pathway.

In summary, a significant body of literature demonstrates that WNT plays an important role in

cellular radiation response and activation of WNT/β-catenin signalling promotes resistance to

radiation in a number of cancers. Association of WNT activation with highly favorable patient

survival in radiation-sensitive tumours provide grounds to suggest that the role of WNT/β-

catenin radiation resistance is cell type dependent and that in medulloblastoma it may confer

sensitivity to radiation.

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1.7. Role of Lithium in mimicking WNT subtype of medulloblastoma

Lithium has been used to treat bipolar disorder for more than 50 years [115], however its

mechanism of action is largely unknown. Lithium has been shown to affect embryonic

development in a manner consistent with WNT pathway activation, affect metabolism through

stimulating glycogen synthesis by activating glycogen synthase, modulate neurotransmitters and

alter neuronal communication, increase cell proliferation and stimulate hematopoiesis [116].

Two main lithium sensitive pathways have been identified to date: 1) depletion of inositol via

inhibition of inositol monophosphatase which results in a decrease of neurotransmitter

signalling and is thought to be mainly involved in lithium antipsychotic effects; 2) inhibition of

GSK3β, a negative regulator of WNT pathway, mimicking canonical WNT signalling. GSK3β is

known to be involved in multiple complex cellular processes both as a part of canonical WNT

signalling pathway and outside it: embryonal development, cell cycle progression regulation,

apoptosis and survival mediation, cellular metabolism, transcription and cytokinetics. The role

of GSK3β and WNT signalling in tumourogenesis was discussed above. Inhibition of GSK3β by

lithium is non-competitive and specific, as it does not affect other protein kinases [117]. It has

been demonstrated that both radiation exposure and lithium inhibit GSK3β by phosphorylation

on Ser9 [118-119]. Multiple studies have demonstrated that, by inhibition of GSK3β, lithium

activates downstream components of the WNT pathway and results in constitutive activation of

the pathway marked by nuclear accumulation of β-catenin [116, 120].

The effects of lithium are cell type specific. Lithium stimulated cell proliferation and invasion in

mammary tumour cells [76] and contributes to radiation resistance in pancreatic cancer models

[121]. Lithium also inhibits cell proliferation in melanoma, hepatocellular carcinoma and

prostate cancer [76], and inhibits cell invasion and migration in glioma [122]. Lithium, in

physiological concentration, has been shown to induce apoptosis in rat immature cerebellar

granular cells but, interestingly, at the same time promotes survival in mature neurons in in vitro

models [123]. It has also been reported that lithium has neuroprotective effects on hypocampal

neurons against radiation in mouse models [124-125].

Activation of the WNT pathway marked by presence of a CTNNB1 mutation resulting in nuclear

accumulation of β-catenin is the hallmark of WNT subgroup of medulloblastoma, the most

prognostically favorable variant of the disease [3, 21, 34, 44, 49, 77, 79]. As discussed above,

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administration of lithium is consistent with GSK3β-mediated activation of the canonical WNT

pathway and can be defined by nuclear β-catenin translocation and transcriptional activation of

β-catenin downstream targets. Thus, lithium can be used to mimic WNT subgroup

medulloblastoma in vitro. Activation of the WNT pathway by lithium is shown in Figure 1.4.

This model has certain limitations due to possible undescribed off-target effects of lithium

occurring both through inhibition of GSK3β and/or other pathways.

1.8. Crosstalk between β-catenin and p53

Aberrant activation of WNT/β-catenin signalling with accumulation of excessive

transcriptionally active β-catenin is strongly implicated in development of multiple cancers,

suggesting that it has an oncogenic role. The TP53 tumour suppressor gene plays a critical role

in prevention of malignancies safeguarding propagation of cells with deregulated proliferative

properties; TP53 is a frequent target for inactivation in many cancers [126]. In addition to its

oncogenic properties β-catenin has been shown to increase transcriptionally active p53 through

both mdm2 dependent and independent pathways associated with senescence phenotype in colon

cancer cells and fibroblasts [126-128]. It was also found that not only β-catenin can affect levels

of p53, but p53 in turn can modulate levels of β-catenin – thus activation of p53 in response to

genotoxic stress can result in increased proteosomic degradation of β-catenin possibly through a

GSK3β mediated mechanism. In the absence of a functional p53 pathway, β-catenin is free to

exert its oncogenic functions; interestingly, wild type TP53 is not able to down regulate mutant

β-catenin [126, 129]. It is not known if similar cross talk between β-catenin and p53 exists in

medulloblastomas.

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Figure 1.4. Activation of canonical WNT/β-catenin signaling bylithium. Treatment with Lithium inhibits GSK3β activity by Ser9

phosphorylation GSK3β. Inactivation of GSK3β results in impaired

phosphorylation of β-catenin and failed β-catenin degradation.

As a result of increased cytoplasmic levels, β-catenin translocates

to the nucleus, where it activates TCF/LEF mediated transcription,

constitutively activating canonical WNT signaling.

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PROJECT RATIONALE AND HYPOTHESIS

Although survival of children with medulloblastoma has significantly improved over the last

years with introduction of radiation and adjuvant chemotherapy, up to 30% of children will

experience recurrence and ultimately die from their disease.

Currently, medulloblastomas are stratified based on clinical criteria alone, which include age of

onset, metastatic spread and residual tumour after surgery. These clinical risk criteria and the

establishment of large cooperative clinical trials resulted in improved survival for these

individuals. However, the high morbidity associated with aggressive multimodal treatment

protocols which may be unnecessary for some patients with favorable tumours and the lack of

ability to predict recurrence in each clinical risk group resulted in efforts for genetic and

molecular stratification of the disease. As of today, none of the known molecular markers are

routinely used in the clinic or as a part of clinical trials. We have reported previously, using the

Toronto cohort, that survival is poor for children with medulloblastoma harboring TP53

mutations. However, a similar analysis of patients from the Heidelberg cohort failed to

demonstrate unfavorable prognosis of TP53 mutated tumours particularly those harbouring

concomitant mutations in CTNNB1 (β-catenin). Moreover, reports from an English cohort and

an additional German cohort revealed mixed findings suggesting that the role of TP53

alterations in medulloblastoma remains uncertain. Recently, several groups were able to

demonstrate that although morphologically similar, medulloblastomas could be divided into four

subtypes, named WNT, SHH and Group 3 and Group 4 based on their expression profile, and

genetic and cytogenetic markers. However, except for the WNT subgroup which is consistently

associated with improved survival, SHH tumours, group 3 and group 4 show heterogeneous, but

consistently inferior outcomes. Recent meta-analysis revealed no statistical difference in the

overall survival (OS) between the latter 3 groups. Our research group was recently able to

observe a unique association of SHH/TP53 mutant tumours with poor survival, as well as

enrichment in the Heidelberg cohort for WNT/TP53 mutant tumours with favorable outcome, we

hypothesize that the adverse poor survival associated with TP53 mutations is modulated by

other genetic alterations, such as activation of WNT or SHH signalling. This is especially

important since WNT activation can be achieved with administration of lithium, which presents

a feasible opportunity for pathway targeted therapy. Knowing that radiation therapy is a key

medical treatment for medulloblastoma patients leading to cure in the majority of patients we

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further hypothesise that constitutive activation of WNT/β-catenin signaling pathway with lithium

will rescue radiation resistance of the TP53 mutation-associated phenotype and sensitize

medulloblastoma cells to radiation thus providing the basis for novel therapy for this disease.

Therefore, the objectives of this thesis were:

1) To determine whether adverse survival in patients with TP53 mutant medulloblastoma is

subgroup dependent.

2) To determine whether WNT/β-catenin activation with lithium can sensitize

medulloblastoma cells to radiation

In order to clarify the prognostic role of TP53 mutations, we assembled all new data from recent

studies focusing on clinico-pathological associations of TP53 mutations with molecular

subgroups (discovery cohort). In order to confirm our observations, we sequenced a large,

independent validation cohort for which both clinical and molecular subgroup information was

available.

In order to investigate effect of WNT activation and lithium treatment on medulloblastoma

radiation response, we performed a series of clonogenic assays to assess cell viability and

clonogenicity in both TP53 wild-type and mutant medulloblastoma cells. Functional studies

were performed to evaluate WNT/β-catenin pathway involvement in radiation response.

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CHAPTER 2: MATERIALS AND METHODS

2.1. Patient cohort

2.1.1. Clinical data

Patient data, both clinical and biological, were obtained through a collaborative effort of several

international groups in Germany, the United Kingdom and Canada. Subgroup assignment,

CTNNB1, and TP53 sequencing was performed in house separately for each study. Briefly, for

the Newcastle group, patients with the WNT subgroup were identified by presence of a CTNNB1

mutation in exon 3, patients with SHH subgroup were identified using transcriptional profile,

and TP53 mutation status was determined using standard PCR-based DNA sequence analysis of

exons 4 to 9 [62, 77, 130]; For the Heidelberg group, mutation status was determined using

standard PCR-based DNA sequence analysis of exon 3 for CTNNB1 and exons 2 to 11,

including exon-intron boundaries, for TP53 [61], and subgroups were identified using antibody

panel as previously described [34]. For The Hospital for Sick Children group and MAGIC

cohort, mutation status of the CTNNB1 gene was determined using standard PCR-based DNA

sequence analysis of exon 3, TP53 gene was sequenced exon 2 to 11 and through the spanning

intron-exon junctions as previously published [131]. Specific medulloblastoma subgroups were

determined by expression levels of the 25 subgroup specific signature genes (CodeSet) using

nanoString nCounter Technology as previously described [132].

2.1.2. Statistical analysis

Overall and progression free survival were estimated using the Kaplan-Meier method with

significance (α=0.05) based on long-rank test, using GraphPad Prism version 5.00 for Windows,

GraphPad Software, San Diego California USA, www.graphpad.com. Overall survival was

defined as date of diagnosis to death of any cause or to the date of the last follow-up visit.

Progression-free survival was defined as date of diagnosis to date of first progression, relapse,

death of any cause or last date of follow-up. For correlative studies, Fisher exact test was used

when appropriate.

2.2. Cell lines

Human MB wild-type TP53 cells lines: ONS76, D283, Med8a, D458 and TP53 mutant cell

lines: UW228, RES256, D425 and Daoy were obtained as a courtesy of Drs. Michael Taylor and

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Annie Huang, The Hospital for Sick Children, Toronto. ONS76, UW228 cells were cultured in a

monolayer in Dulbecco’s Modified Eagle’s medium (DMEM) (Wisent, 319-005-CL)

supplemented with 10% of Fetal Bovine Serum (FBS) (Wisent, 080450) and 1% of Penicillin /

Streptomycin / Amphotericine (Pen/Strep/Am) (Wisent, 450-115-EL); Daoy, D283 and Med8a

cells were cultured in a monolayer in Alpha Modification of Eagle’s medium (AMEM) (Wisent,

310-010-CL), supplemented with 10% FBS and 1% Pen/Strep/Am solution; RES256 was

cultured in a monolayer in DMEM/F12 medium (Wisent, 219-095-QK), supplemented with

10% FBS and 1% Pen/Strep/Am solution; D425 and D458 were cultured as a suspension culture

in DMEM supplemented with 10% FBS, 1% Pen/Strep/Am solution and non-essential amino

acids (Wisent, 321-012). All cells were grown in a humidified atmosphere containing 5% CO2 at

37°C. 0.25% Trypsin EDTA (Wisent, 325-042-EL) was used to detach cells for passaging.

Normal fetal neural stem cells Hf5205 were obtained as courtesy of Dr. Peter Dirks, the

Hospital for Sick Children, Toronto; cells were isolated by Dr. Dirks’ group (Unpublished data)

and cultured as previously described [133]. In brief, Hf5205 cells were cultured in NeuroCult

NS-A Basal Medium (StemCell Technologies, 05750), supplemented with Glutamax (Wisent,

2mM), N2, B27 (Life Technologies, Invitrogen), EGF, FGF (10ng/ml, PeproTech) and Heparin

(2 μg/ml, Sigma). All culture vessels were coated with Laminin in PBS (2 μg/ml, Sigma) for 3

hours at 37C prior to use. Cells were grown in a monolayer at 37°C in a humidified atmosphere

containing 5% CO2 and the medium was replaced every 3-4 days. Accutase (Sigma) was used

to detach or dissociate cells before passaging or seeding.

2.3. TP53 and CTNNB1 sequencing

The TP53 mutation status of the cell lines was determined in the Department of Paediatric

Laboratory Medicine (DPLM) at The Hospital for Sick Children by direct sequence analysis of

exons and intron/exon boundaries of TP53 gene and by multiple ligation-dependent probe

amplification (MLPA) methods to detect deletions of the TP53 gene.

Sequencing of CTNNB1 gene was performed by in our lab. Primers were designed using

Primer3Plus Version 2.3.0 software [134] to sequence exon 3. Following primers were used:

5’TGATTTGATGGAGTTGGACA’ and 3’GCTACTTGTTCTTGAGTGAAG’ PCR

conditions: initiation of denaturation – 95°C, 15 min - 1 cycle, denaturation – 94°C, 30 sec,

annealing – 55°C, 30 sec, elongation – 72°C 50 sec and final elongation – 72°C, 7 min – 35

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cycles. Sanger sequencing was performed by The Centre for Applied Genomics, The Hospital

for Sick Children, Toronto, Canada. Sequence was analysed using FinchTV 4.1 software

(PerkinElmer, Inc. USA) Human Genome Assembly 2009 UCSC (Center for Biomolecular

Science and Engineering, Santa Cruz, USA).

2.3 Chemicals

Lithium Chloride (Sigma, 02685-1EA), was dissolved in sterile water to prepare a stock solution

of 500mM, sterilized by filtration. Working solution of 2mM used for cell treatment was

prepared by dissolving stock solution in regular growth medium at the time of treatment.

2.4. Irradiation

Radiation exposure was performed using a Nordion Gammacell 40 irradiator with the central

dose rate 1.24 Gy/min. Single discrete doses of 1 to 10 Gy were delivered according to

experimental design.

2.5. Clonogenic experiments

Radiation sensitivity curves: ONS76, Med8a, D283, Daoy and UW228 cells were maintained in

T-75 flasks until 80% confluent, trypsinized to generate a single-cell suspension, counted using

a haemocytometer and seeded at specific numbers in triplicates into 10 cm dishes to yield

between 50 and 150 colonies after. Cells were allowed to adhere overnight and then irradiated

with the range of doses from 1 to 5 Gy single doses. The medium was changed after irradiation

and cells were maintained at 37°C for 14 days to allow colonies of more than 50 cells per

colony to form. The colonies were then fixed and stained with Crystal violet (0.1%) and counted

manually. The surviving fractions were calculated after adjustment for plating efficiency of non-

irradiated controls as follows:

SF (survival fraction) = [(number of colonies observed) / (number of cells plated)] / PE (plating

efficiency of control)

PE (plating efficiency of control) = (number of colonies observed in non-irradiated sample) /

(number of cells plated)

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Data from 3 independent experiments with triplicates were each plotted as a function of

radiation dose on a semi-logarithmic plot.

2.6. Transfection experiments

For each cell line 3x105 cells were transiently transfected in parallel with 2µg of plasmid-of-

interest DNA or 2ug pcDNA3 plasmid as control by Nucleoporation, Lonza, as per the

manufacturer’s instructions using Amaxa Mouse Neural Stem Cell Kit (Nucleofector, VPG-

1004) and X001 program. After transfection, medium was replaced with normal growth medium

and cells were transferred to 35 mm dishes and left for 24 hr. Subsequently cells were

trypsinized, counted, and plated in 10 cm dishes for clonogenic assay as described above.

Plasmids used: pcDNA3-TP53R175H, pcDNA3 (courtesy of Dr. David Malkin, The Hospital for

Sick Children, Toronto), pcDNA3-S33Y β-catenin [135] obtained through Addgene web-site:

http://www.addgene.org/. The figure was plotted from 3 independent experiments with

triplicates each; data was plotted as a function of radiation dose on a semi-logarithmic plot.

Transfection efficiency was estimated for the pcDNA3-TP53R175H/pcDNA3 plasmid

combination by co-transfection with the pcDNA3-GFP plasmid. The proportion of GFP-positive

cells was estimated by manual cell counting under fluorescence microscopy.

Transfection efficiency for the pcDNA3-S33Y plasmid was estimated by immunofluorescent

microscopy for FLAG-tag. Transfected cells were plated on cover slips and further processed as

described in section 2.10 and proportion of FLAG-positive cells was obtained by manual

counting.

2.7. Combination of Lithium and radiation treatment

2.7.1. Lithium toxicity curves

ONS76 and UW228 cells were trypsinized and plated for clonogenics as described above. Cells

were treated with 0.1mM, 0.5mM, 1mM, 2mM and 10mM of lithium chloride for 24 hours.

Drug-containing medium was then replaced by regular culture medium and cells were

maintained as described above until colonies of 50 or more cells appear (approximately 14

days), then fixed, stained with Crystal Violet (0.1%) and manually counted. Survival curves

were generated as described above. Data from 3 independent experiments, each in triplicate,

were plotted on a linear plot.

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2.7.2. Clonogenic experiments with combination of Lithium and radiation exposure

ONS76 and UW228 cells were cultured, trypsinised, counted and plated at specific densities and

kept overnight to adhere as described above. Cells were exposed to lithium chloride 2mM for 24

hours, and then irradiated with the range of doses from 2 to 8 Gy single doses. Drug-containing

medium was aspirated and replaced with normal growth medium. Cells were maintained as

described above until colonies of 50 or more cells appear (approximately 14 days), then fixed,

stained with Crystal Violet (0.1%) and manually counted. The surviving fractions were

calculated as described above, and survival curves were generated after normalizing for the

amount of lithium chloride that induced death. The survival fraction at 2 Gy (SF2 value) was

calculated for each curve; SF2 value has been shown to be the best discriminator of the cellular

radiosensitivity in vitro and represents the clinically relevant low-dose portion of radiation

survival curve [136]. Survival fractions at higher doses of radiation, SF4, SF5, Sf6 and SF8

were calculated when appropriate. The sensitizer enhancement ratio (SER) at 10% survival for

lithium chloride was calculated as following, where D is the dose of radiation [137]:

SER10 = D10 (without drug) / D10 (lithium chloride)

Data from 3 independent experiments, each in triplicates, were plotted as a function of radiation

dose on a semi-logarithmic plot.

2.8. Western blot analysis

Protein expression levels were determined by western blot analysis. Cells were lysed after 24 hr

of exposure to 2mM of lithium chloride or after 4 hr post 5 Gy of single dose irradiation;

untreated cells were used as control. Protein extraction was performed using RIPA extraction

buffer [138]. Protein quantification was done using BioRad Protein Assay (BioRad, 500-0006).

30µg of protein were used for the assay. Whole cell lysates were separated on 12%

polyacrylomide gel and transferred to PVDF membrane. Membranes were incubated with

appropriate blocking buffer and primary/secondary antibodies; imaging was done using the

LiCor Odyssey imaging system, LICOR Bioscience. Primary antibodies were the following:

Rabbit anti-GSK3β-total (Cell Signalling, 9315L) 1:1000, Rabbit anti-Phospho-Ser9-GSK3β

(Cell Signalling, 5558S) 1:1000, Rabbit anti-β-actin (Abcam, ab8227) 1:2000 and mouse anti-β-

catenin (BD Transduction Labs, 6101153) 1:1000. Experiments were done at least 3 times.

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2.9. β-catenin luciferase reporter assay

For the reporter assay, 1x105 cells were plated in 24-well plates in triplicate per sample and left

to attach overnight. Cells were transiently transfected in parallel with either 0.5µg of the TCF-

reporter construct (M50 Super 8x TOPFlash) DNA or 0.5µg of the mutated reporter construct

(M51 Super 8x FOPFlash) DNA [139] together with 1ng of Renilla DNA. Plasmids were

obtained through the Addgene web-site: http://www.addgene.org/. Transfections were

performed using Lipofectamine 2000 (Invitrogen, 11668-027) according to manufacturer’s

protocol. After 4 hr post transfection medium was replaced with the regular growth medium and

cells were treated with 2mM of lithium chloride for 24 hr. TCF-mediated transcriptional activity

was determined by the ration of TOPFlash/FOPFlash luciferase activity, normalized to the

Renilla luciferase activity using Promega Dual Luciferase Reporter Assay System, (Promega,

E1960). 3 independent experiments were done.

2.10. Immunofluorescence – β-catenin nuclear translocation

Cells were cultured on cover slips in 6-well dishes, then fixed using 4% paraformaldehyde/0.2%

Triton X-100, 20 min at RT followed by 20 min in 0.5% NP40. Cells were exposed to 2%

Bovine Serum Albumin (BSA) / 1% donkey serum in PBS blocking solution for 1 hr at RT.

After this, cells were incubated with appropriate primary antibodies at 4°C overnight followed

by secondary antibodies at room temperature over 1 hour. Cover slips were mounted on slides

using “Vectashield” mounting medium for Fluorescence (Vector laboratories, H-1000). Slides

were stored at 4°C in the dark. Images and quantifications were performed using Quorum

Spinning Disk Confocal Microscope and Perkin Elmer Velocity 6.0.1 software (Perkin Elmer,

USA).

Primary antibodies: Mouse anti-β-catenin (BD Transduction Labs, 6101153) 1:500, Secondary

antibodies: Alexa Fluor 488 donkey-anti-mouse IgG (Invitrogen, A21202) 1:500.

2.10.1. β-catenin nuclear translocation

For β-catenin nuclear translocation, cells were pre-treated with 2mM of lithium chloride for 24

hr and then fixed according to the protocol above. Untreated cells were used as control.

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For β-catenin nuclear translocation, following introduction of the S33Y-CTNNB1 mutation cells

were transfected on cover slips and then fixed 48 hr post transfection. Both untransfected cells

and cells transfected with pcDNA3 were used as controls.

All experiments were repeated 3 times.

2.11. Normal neuronal stem cell viability experiments

Normal neuronal stem cells (Hf5205) were seeded at 4x104 cells per well in 6-well plates and

treated with lithium chloride (2mM, Sigma) for 24hrs before radiation; radiated with 2-8Gy

single dose. Cells were counted 5 days after radiation using Cell Viability Analyzer (Vi-Cell

XR, Beckman Coulter). Survival curves were generated as described above. The figure was

plotted from three independent experiments with triplicate each.

2.12. Statistical Analysis

Results were expressed as the mean ± SE of 3 or more independent experiments. Statistically

significant differences between samples were determined using Student 2-tailed t-test and two-

way ANOVA with Bonferroni correction method when appropriate using GraphPad Prism

version 5.00 for Windows, GraphPad Software, San Diego California USA,

www.graphpad.com; p value < 0.05 was considered significant. The error bars in the figures

represent SEs.

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CHAPTER 3: RESULTS

3.1. Survival of medulloblastoma patients with somatic TP53 mutations depends on

molecular subgroup

Clinical data, TP53 and CTNNB1 status and subgroup were available for 183 patients. This data

was obtained as a collaborative effort of 3 research groups and was originally published

separately as parts of three independent peer reviewed publications: 20 patients from Northern

Institute for Cancer Research, Newcastle upon Tyne, UK [62]; 95 patients from The Hospital for

Sick Children, Toronto, Canada [59] and 68 patients from Heidelberg Group, German Cancer

Research Center, University Hospital Heidelberg, Heidelberg and University Medical Center,

Hamburg-Eppendorf, Hamburg, Germany [61].

3.1.1. Distribution of TP53 mutations in subgroups

Mutation analysis was done on the pooled cohort of patients, which included 183 patients from

our discovery cohort and 156 patients from the validation cohort described below. For this

analysis patients of all ages were included. We did not have compete data on all of 339 patients,

therefore, patients who either did not have a group assigned or who had TP53 status determined

by immunohistochemical staining for p53 protein, but not by sequencing were excluded (n=40).

The total number of patients we analysed was 299; out of whom WNT were 93 patients (78 were

TP53 wild-type, and 15 were TP53 mutant), SHH were 170 patients (136 of those patients were

TP53 wild-type and 34 were TP53 mutant) Group 3 had 13 patients (all patients were TP53

wild-type) Group 4 had 23 patients (16 TP53 wild-type and 7 were TP53 mutant).

We did not attempt to estimate frequency of TP53 mutations in our population. This analysis

was not possible because data was collected with a bias towards WNT and SHH subgroups and

patients bearing TP53 mutations, and information on other subgroups was limited, because only

TP53 mutant data was available for WNT and SHH subgroups from our collaborators. However,

recognising limitations of our study we attempted to analyse distribution of TP53 mutations by

molecular subgroups. We found that TP53 mutations are present in higher proportion in Group 4

and in SHH subgroup and are completely absent in Group 3 patients (30.4%, 20% and 0%

respectively); the WNT subgroup had 16.1% of tumours mutated for TP53 (Figure 3.1.1.D).

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In both discovery and validation cohorts we found a total of 57 TP53 mutations: 26 mutations

were homozygous, 19 were heterozygous and 1 was a compound heterozygote; 11 patients with

TP53 mutations have missing information about the type of the mutation. The majority of

mutations were found in the DNA-binding domain (exons 5 to 8), with 2 mutations each in the

transactivation and proline-rich domains (exon 2 to 4), one mutation in the tetramerization

domain (exon 10), and one mutation was found in exon 11. No clustering of mutations with

respect to molecular subgroup was observed. Figure 3.1.1.A shows a schematic representation

of TP53 mutations. Interestingly, we found that the majority of the TP53 mutations in WNT

tumours are heterozygous: 8 (75.3%) vs. 4 (26.7%) out of 12 patients. In contrast, SHH tumours

bear predominantly homozygous alterations in TP53 (19 (55.9%) vs. 7 (20.6%)) out of 34

patients (p<0.05). In addition, 20.6% SHH tumours have other genetic alterations in the TP53

gene, such as insertions and deletions (n=5). The presence of more than 1 TP53 mutation per

tumour was also described (n=2) and only 1 WNT tumour has disruption of TP53 function by

homozygous duplication (6.7%) (Figure 3.1.1.B&C). Interestingly, this patient had metastatic

disease, but survived. Together, these observations suggest higher degree of genomic instability

and possible aggressive phenotype in SHH TP53 mutant medulloblastomas when compared to

WNT ones.

3.1.2. Patient characteristics and survival analysis

For survival analysis, we considered only patients over 3 years old who received the treatment

protocol, which included radiation therapy. Of the total 183 patients, 62 patients were in WNT

subgroup, 51 patient in the SHH subgroup, 18 patients in group 3 and 38 patients in group 4.

Fifteen patients had undetermined group due to technical difficulties or lack of available tissue

and were excluded from further analysis. Twenty-one patients younger than 3 years of age were

further excluded. These were: 10 from SHH subgroup, 6 from group 3, and 5 were missing

subgroup assignment. All the patients in WNT subgroup were older than 3 years of age. Out of

21 excluded patients, 1 had a TP53 mutation and 7 did not have TP53 mutation status other than

by immunohistochemical staining for p53 protein (5 tumours were p53-positive including 1

sample which also was found to have TP53 mutation). The discovery cohort for this specific

thesis therefore included n=139 patients. Characteristics by subgroups are presented below.

WNT subgroup had 62 patients (n=62), 48 patients were TP53 wild-type and 10 patients were

TP53 mutant. Median age was 10.6 years (range 5 to 30 years old). Median observed time to last

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follow up was 85 months (range from 16 to 192 months). The SHH subgroup had 41 patients

(n=41), 18 patients were TP53 wild-type, 21 patient was TP53 mutant. Median age was 10.1

years (range 3 to 45 years of age). Median observed time to last follow up was 35 months (range

from less than 1 month to 185 months). For Group 3, we had 13 patients, all of them were TP53

wild-type with median age of 4.99 years old (range from 2.1 to 11.5 years). Median observed

time to last follow up was 77.1 months (range from 5.1 to 164 months). Twenty-three patients

were group 4, with 16 being TP53 wild-type and 7 being TP53 mutant. Median age was 8 years

(range from 3.24 to 17 years). Median observed time to last follow up was 79.2 months (range

from 16.5 to 179 months).

Both overall (Figure 3.1.2.A) and progression-free survival (Figure 3.1.2.B) were significantly

worse for TP53 mutant patients comparing to patients with TP53 wild-type tumours (p<0.0001

for both analyses). Accordingly, overall survival was 48.7% for TP53 mutant patients. Five year

OS for TP53 wild-type patients exceeded 75%, with median survival time of 76 months for

TP53 mutant patients. Median time to progression was 54.7 and 179.0 months for TP53 mutant

and TP53 wild-type patients, respectively (p<0.001).

Interestingly, separate survival analysis of the WNT and SHH subgroups revealed marked

differences in the role of TP53 mutation on survival. In the WNT subgroup overall and

progression free survival (Figure 3.1.2.C&D) for patients with TP53 mutant tumours was not

significantly different than for patient with TP53 wild-type tumours. Overall survival was 76.4%

and 87.5% for TP53 mutant and TP53 wild-type patients, respectively (p=0.27 and p=0.22,

respectively). Progression-free survival for TP53 mutant patients was 72.9% and 76.2% for

TP53 wild-type patients.

In contrast, in the SHH subgroup we observed a significant decrease in overall survival in TP53

mutant patients – 16.7%, compared to TP53 wild-type patients 88.9 % (p<0.001) (Figure

3.1.2.E), with median survival time of 43 months for TP53 mutant patients; 14/21 TP53 mutant

patients are dead of their disease while only 2/18 in TP53 wild-type group. We observed a

significant difference in progression-free survival between TP53 mutant and TP53 wild-type

patients (p<0.001) (Figure 3.1.2.F); 16 out of 21 TP53 mutant patients progressed, with median

progression time of 19.1 months; only 4 patients progressed in the TP53 wild-type group.

Overall, in the discovery cohort, most deaths in the SHH group were attributed to TP53

mutations.

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0%

20%

40%

60%

80%

100%

WNT SHH

HOM

HET

0%

20%

40%

60%

80%

100%

WNT SHH

Other genetic

events

Point

mutation

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

WNT SHH Group 3 Group 4

TP53wt

TP53mut

Figure 3.1.1. TP53 mutation spectrum. (A) Distribution of TP53

mutations in functional domains according to subgroups: WNT – red,

SHH – blue, Group 4 - green; (B) Percentage of homozygous (HOM)

and heterozygous (HET) TP53 mutations in WNT and SHH subgroups;

(C) Percentage of other than point mutations genetic events

(deletions/insertions and complex mutations) disrupting TP53 function

in WNT and SHH medulloblastomas; (D) Percentage of TP53

mutations in medulloblastoma subgroups in discovery and validation

cohort together.

D

CB

A

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Figure 3.1.2. Overall and progression-free survival for TP53 mutant and

TP53 wild-type patients. Kaplan-Meier estimates of survival for patients with

TP53 mutant and TP53 wild-type medulloblastomas; Overall (OS) (A) and

progression-free (PFS) (B) survival by TP53 mutation status for all patients

age 3+ years, p<0.0001 for both analyses; OS (C) and PFS (D) by TP53

status for WNT patients, p=0.27 and p=0.22 respectively; OS (E) and PFS (F)

by TP53 status for SHH patients, p<0.001 for both analyses.

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3.1.3. Validation of initial observations

To validate our findings we used a separate non-overlapping cohort of 156 patients from

MAGIC (Medulloblastoma Advanced Genomics International Consortium) study which

comprises data on MB patients collected as a part of an international collaboration in multiple

countries in Western and Eastern Europe, Asia, United States and Canada [140]. This validation

cohort consisted of WNT and SHH patients only. The access to this data was a courtesy of Dr.

Michael Taylor, The Hospital for Sick Children, Toronto. For the purpose of this thesis, we

excluded all patients younger than 3 years of age (n=44, among those all but 1 patients were

from SHH subgroup. Only 1 patient, also SHH, had TP53 mutation). The final analysis included

112 patients older than 3 years of age Ninety-three patients (83%) were TP53 wild-type and 16

(14.3%) were TP53 mutant. The WNT subgroup consisted of 34 patients: 29 were TP53 wild-

type and 5 were TP53 mutant. Their median age was 9.33 years (range: 3 – 56.3 years) and

median observed time to the last follow up was 60.5 months (range: 3 – 252 months). The SHH

cohort consisted of 78 patients, of whom 11 were TP53 mutant and 67 were TP53 wild-type.

Their median age was 11.5 years (range: 3 – 33 years), with a median observed time to last

follow up of 40.5 months (range: < 1 month – 300 months).

Overall survival for TP53 mutant patients was significantly worse than for TP53 wild-type

patients, being 27.3% and 62.7%, respectively (p=0.01), with a median survival of 81 months

for TP53 mutant patients. In the WNT subgroup, overall survival did not differ between TP53

mutant and TP53 wild-type patients, being 80% and 71.9%, respectively (p=0.17). However, in

the SHH subgroup TP53 mutant patients had marked reduced overall survival compared to TP53

wild-type patients (20.5% vs. 63.3%, respectively (p=0.01)) with a median survival for TP53

mutant patients of 45 months (Figure 3.1.3.A,B&C). These results were strikingly similar to

results obtained from our discovery cohort.

In conclusion of this part, these findings reveal a strong association of TP53 mutations with

inferior survival in SHH subgroup; in contrast WNT MB exhibit no difference in survival with

respect to TP53 mutation status, suggesting that adverse effects of TP53 mutations can be

modulated by a pathway, specific to WNT tumours which is yet to be determined.

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OS All patients

OS WNT

OS SHH

Figure 3.1.3. Overall (OS)survival for TP53 mutantand TP53 wild-type

patients, MAGIC cohort.

Kaplan-Meier estimates of

survival for patients with

TP53 mutant and TP53 wild-

type MB; OS (A) survival by

TP53 mutation status for all

patients age 3+ years

(p=0.01); (B) OS by TP53

status for WNT MB patients

(p=0.17); (C) OS by TP53

status for SHH MB patients,

(p=0.01).

A

B

C

37

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Since we hypothesized that these differences between subgroups are a result of difference in

radiosensitivity modulated by TP53 status in each molecular background, we performed the

following experiments to test our hypothesis:

3.2. TP53 mutant medulloblastoma cell lines are more resistant to radiation

To determine the role of TP53 in radiation resistance in MB cells we used two different

methods: 1) we performed a series of clonogenic assays using a panel of TP53 wild-type and

TP53 mutant cell lines; 2) we used TP53 wild-type cell line and transiently transfected it with a

plasmid containing R175H TP53 mutation; clonogenic assays were subsequently performed to

assess survival.

Sequencing analysis of 8 MB cell lines revealed 4 TP53 mutant cell lines, of which 3 had point

mutations and one had simultaneous deletion/insertion of two base pairs resulting in an amino

acid change at codon 274. All three mutations were missense, homozygous and were located in

the DNA-binding domain resulting in non-functional protein. All mutations were described as

somatic mutations in multiple cancers (IARC TP53 Database http://www-p53.iarc.fr). The list

and distribution of mutations over the TP53 functional domains is shown in Figure 3.2.1. All

cell lines were wild-type for the CTNNB1 gene as determined by sequencing of exon 3

performed in our laboratory as described above.

The following medulloblastoma cell lines: ONS76 (TP53 wild-type), Med8A (TP53 wild-type),

D283 (TP53 wild-type), UW228 (TP53 mutant) and Daoy (TP53 mutant) were further used in

this project. Three cell lines were excluded: D425 (TP53 mutant) and D458 (TP53 wild-type)

cells grow as a suspension culture and were not suitable for clonogenic assay; and RES256 did

not yield consistent numbers of colonies on multiple attempts. The clonogenic assays were

performed and clonogenic survival curves were generated to assess whether resistance to

radiation depends on TP53 status, with doses ranging from 2 to 5 Gy. Irradiated samples were

compared to non-irradiated controls. As expected, we observed significantly higher survival in

response to radiation treatment in TP53 mutant cell lines Daoy and UW228 compared to TP53

wild-type cell lines, ONS76, Med8a and D283 (p<0.01) (Fig.3.2.2A). Specifically, minimal

survival differences at physiologically relevant radiation doses of 2 Gy was 3% between the

Daoy and ONS76 cell lines and a maximum difference was 43% between the UW228 and

Med8A cells. Survival fraction was distributed as following: 76% +/-2.5% for UW228, 67% +/-

38

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4.7% for Daoy, 64% +/- 2.2% for ONS76, 54% +/-4.7% for D283 and 33% +/-9.9% for Med8A

cells. With increase of radiation dose TP53 mutant cells retain their survival advantage. At a

dose of 5 Gy the minimal difference in survival was 15% between Daoy and ONS76 and the

maximum difference was 43% between UW228 and Med8A cells with the following

distribution of survival fractions: 48% +/-8% for UW228, 35% +/-6.5% for Daoy, 19.8% +/-

1.2% for ONS76, 16.4% +/-3.9% and 5.4% +/-2.8% for Med8A cells.

Since cell lines in the panel were not isogenic, other genetic alterations, which were unknown to

us, may have contributed to the survival differences described above. In order to further confirm

the pro-survival effect of TP53 mutation under the radiation exposure, the wild-type TP53 cell

lines D283 and ONS76 were transiently transfected with known dominant-negative TP53

R175H mutation. Transfection efficiency was estimated based on co-transfection of a GFP-

expressing plasmid using immunofluorescent microscopy; percentage of GFP-positive cells was

obtained and transfection efficiency was calculated: 59.85% +/-12.3% (Figure 3.2.2.F).

Transfection of the ONS76 TP53 wild-type cells with TP53 R175H mutation resulted in a

significant increase in radiation resistance in transfected cells compared to the empty-vector

control (p<0.01) (Figure 3.2.2B). In order to assess the effect of TP53 mutation on cell survival

at lower, clinically relevant, radiation dose, survival fraction at 2 Gy (SF2) was calculated: 89%

+/- 2% and 57.4% +/- 1.8% for TP53 R175H and TP53 wild-type cells respectively; with a 32%

survival advantage for TP53 mutant cells (p< 0.0001). At a higher dose of radiation, (6 Gy),

TP53 mutant cells demonstrated 6.8% better survival compared to wild-type counterparts

(p<0.001) with survival fraction at 6Gy (SF6) calculated as following: 20% +/-0.9% and 13.2%

+/- 0.7%, respectively. (Figure 3.2.2.C&D). For D283, only survival at 5 Gy (SF5) was

estimated. SF5 was 24% +/-0.6% and 15.5% +/- 0.4% for TP53 R175H and TP53 wild-type

respectively, with 8.5% survival advantage of the mutant cells (p<0.05) (Figure 3.2.2.E).

From the above observations we conclude that TP53 mutations confer radiation resistance in

MB cell lines.

3.3. CTNNB1 mutation (S33Y) sensitizes TP53 mutant medulloblastoma cells to radiation.

Since we hypothesized that improved survival of patients with WNT tumours are a result of

higher radiosensitivity, we examined the role of activating mutations and subsequent WNT

pathway activation in sensitizing TP53 mutant MB cells to radiation. We transfected our wild-

39

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type CTNNB1 MB cells with the S33Y mutation. UW228 TP53 mutant cells were transfected as

described above. The CTNNB1-S33Y plasmid was FLAG-tagged. Transfection efficiency was

evaluated using immunofluorescence microscopy; percentage of the FLAG-positive cells was

estimated at 24, 48 and 72 hours post transfection: CTNNB1-S33Y transfected cells

demonstrated a maximum nuclear immunopositivity of close to 90% at 24 hr with almost

complete resolution of FLAG-expression by 72hr. Cells were irradiated between 24 and 48

hours post transfection.

Nuclear translocation of β-catenin was demonstrated in CTNNB1-S33Y cells compared to cells

bearing wild type CTNNB1 by immunofluorescent imaging. Indeed, β-catenin mutation

facilitates nuclear translocation of the protein (Figure 3.3.1.).

Clonogenic survival curves were generated to assess whether TP53 mutation-associated

resistance to radiation is counteracted by presence of the CTNNB1-S33Y mutation. Cells were

exposed to a range of radiation doses of 2 to 8 Gy.

We observed that presence of the CTNNB1-S33Y mutation radiosensitizes TP53 mutant MB

cells (p<0.05) compared to empty vector control. Although the difference in survival at 2 Gy

(SF2) did not achieve statistical significance and was calculated as 12% with 56% +/-5.9% and

44.4% +/-6.2% for UW228/pcDNA3 cells and UW228/S33Y cells respectively (p>0.05), this

radiosensitizing effect was more pronounced at higher radiation doses, with SF5 being 26.7%

+/-1.2% and 17% +/-1.6%, respectively, where a 10% survival difference between CTNNB1

wild type and mutant cells was achieved (p<0.001) (Figure 3.3.2.A&B).

We therefore conclude that introduction of a CTNNB1-S33Y mutation into TP53 mutant MB

cells results in both activation of WNT signaling and sensitization of the cells to radiation,

suggesting that WNT signalling activation in MB may abrogate resistance to radiation associated

with TP53 mutations.

3.4. Lithium sensitises medulloblastoma cells to radiation:

Lithium is a known inhibitor of GSK3β. We took advantage of this property of lithium to mimic

constitutive activation of the WNT pathway by CTNNB1 mutation in order to test its ability to

reverse the radioresistance caused by TP53 mutations in MB.

40

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Figure 3.2.1. TP53 mutation spectrum and distribution overfunctional domains in medulloblastoma cell lines. (A) List of

TP53 mutations obtained through sequencing of MB cell lines; all

the mutations are homozygous and all except one result in non-

functional protein. All, except one, mutations are described in

multiple sporadic cancers; two of the mutations are also described

in LFS families. (B) All mutations are in the DNA-binding domain of

TP53 gene.

A

B

41

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Figure 3.2.2. TP53mut MB cells are more resistant to radiation. (A)

Survival curves for MB cell lines given different radiation doses. Red circles

represent UW228 TP53mut cell line, red triangles – Daoy TP53mut, blue

triangles – D283 TP53wt, blue squares – ONS76 TP53wt and blue circles –

Med8a TP53wt. (B) Transfection of ONS76 TP53wt cells with dominant-

negative R175H TP53 mutation resulted in increased radiation resistance of

cells. Blue circles – empty vector control, red circles – cells transfected with

R175H plasmid. (C-D) Bar graph of ONS76 TP53wt cells transfected with

R175H mutation showing the quantification of the surviving fraction

following 2Gy (SF2) and 6Gy (SF6) irradiation (**p<0.01). (E) Bar graph of

D283 TP53wt cells transfected with R175H mutation showing the

quantification of the surviving fraction following 5Gy (SF5) irradiation

(**p<0.01). (F) GFP-positive cells 24 hr post transfection, right – bright field

microscopy, left – immunofluorescent microscopy of the same field,

representative image.

IR Dose (Gy)IR Dose (Gy)** **

**

42

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Figure 3.3.1. Nuclear translocation of β-catenin in CTNNB1-

S33Y mutant cells.Transfection of TP53 mutant MB cells with mutant β-catenin

construct results in nuclear translocation of β-catenin (green),

nucleus (red), co-localization of β-catenin to nucleus (yellow).

43

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Figure 3.3.2. Activation of WNT pathway signalling via S33Y-CTNNB1

mutation radiosensitizes TP53 mutant cells. (A) Transfection of UW228

TP53mut cells with S33Y-CTNNB1 mutation resulted in moderate sensitization of

cells to radiation (*p<0.05). Survival curves for cells given increased dose of

radiation; empty vector control (pcDNA3) – blue circles, cells transfected with

pcDNA3-S33Y-CTNNB1 plasmid – red circles. (B) Bar graph of UW228 TP53mut

cells transfected with S33Y-CTNNB1 mutation showing the quantification of the

surviving fraction following 2Gy (SF2) (p=0.5) and 5Gy (SF5) irradiation

(**p<0.01).

* *

*

UW228

TP53mut/

S33Y

UW228

TP53mut/

pcDNA3

UW228

TP53mut/

S33Y

UW228

TP53mut/

pcDNA3

A B

Su

rviv

al

Fra

cti

on

, 2G

y

Su

rviv

al

Fra

cti

on

, 5G

y

Su

rviv

al

Fra

cti

on

44

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3.4.1. Medulloblastoma cells tolerate physiological doses of lithium

First, we estimated whether MB cells tolerate physiological dose of lithium (1-2mM) and also

the cytotoxic dose. Clonogenic assays were performed and survival curves were generated for

the ONS76 TP53 wild-type and UW228 TP53 mutant cells with lithium doses between 0.1 and

10 mM.

Our results indicate that 24 hour exposure to lithium does not result in the different toxicity in

MB cells with respect to their TP53 status; both TP53 wild-type and TP53 mutant cells were

responding to increasing doses of lithium in a similar pattern. We observed that in both ONS76

and UW228 24 hour treatment with 2mM of lithium does not cause marked cytotoxicity

compared to non-treated samples 100% ± 17% vs. 130% ± 30% (p=0.1) for ONS76 and 100% ±

44% vs. 130% ± 44% (p=0.35) for UW228, respectively. Up to 50% decreased colony

formation was observed at 10mM in both cell lines.

A concentration of 2mM was chosen as optimal to further investigate lithium as a radiosensitizer

in combination treatment, because it does not produce additional cell death. In addition 2mM, is

at the high end of the tolerable dose used in clinical practice without inflicting major toxicity.

3.4.2. Lithium radiosensitized medulloblastoma cells

To examine the effects of lithium on the radiosensitivity of MB cells, we performed clonogenic

assays using ONS76 and UW228 cell lines. In this combination protocol after 24 hours of

lithium treatment, cells were irradiated and colony forming efficiency was determined 14 days

later. Pre-treatment with lithium resulted in increased radiation sensitivity in both wild-type and

mutant TP53 cell lines (p<0.01) (Figure 3.4.1.A&B).

Survival fraction for lithium treated cells at 2 Gy (SF2) was calculated for ONS76 and UW228

cell lines as 33.5% ±3.5% and 43.5% ±1.5% respectively, which was significantly different

from untreated samples, 55.1% ±6.5% and 56.6% ±3% respectively (p<0.01) with 21.6%

decrease in survival for TP53 wild-type cells and 13.1% for TP53 mutant cells (Figure

3.4.1.C&E). The difference in survival was consistent at higher doses of radiation, 6 Gy (SF6),

as well: 1.7% +/-0.3% and 3.9% +/- 0.8% for lithium treated cells compared to 10% +/- 2% and

8.9% +/- 0.7% in untreated controls for ONS76 and UW228, respectively (Figure 3.4.1.D&F)

with an 8.3% decrease in survival for ONS76 and 5% for UW228.

45

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ONS76 Lithium

ONS76 No Lithium

ONS76 Lithium

Surv

ival

Fra

ctio

n, 2

Gy

Surv

ival

Fra

ctio

n, 6

Gy

** *

ONS76 No Lithium

Surv

ival

Fra

ctio

n, 2

Gy

UW228 No Lithium

UW228 Lithium

Surv

ival

Fra

ctio

n, 6

Gy

UW228 No Lithium

UW228 Lithium

**

C D E F

B A

Figure 3.4.1. Lithium radiosensitizes both TP53wt and TP53mut medulloblastoma cells. Treatment with Lithium sensitizes MB cells to radiation (**p<0.01). Survival curves for ONS76 TP53wt (A) and UW228 TP53mut (B) cells given increasing radiation doses following 24 hr of treatment with Lithium (red circles) and untreated control (blue circles). Bar graph of ONS76 (C) and UW228 (E) cells showing the quantification of the surviving fraction of Lithium treated (red) and untreated (blue) cells following 2Gy irradiation; 21% for ONS76 (*p<0.05) and 13.1% for UW228 decrease in survival (**p<0.01). Bar graph of ONS76 (D) and UW228 (F) cells showing the quantification of the surviving fraction following higher radiation dose (6Gy); 8.3% for ONS76 (**p<0.01) and 5% for UW228 decrease in survival (**p<0.01).

**

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Treatment with lithium resulted in sensitizer enhancement ratios of 1.5 for ONS76 and 1.3 for

UW228 at a surviving fraction of 0.10.

In order to evaluate the role of lithium in the activation of WNT/β-catenin signaling and

determine whether Lithium exerts its radiosensetisation effects through activation of this

pathway we performed a series of functional assays.

3.4.3. Lithium phosphorylates GSK3β on Ser9

We analysed protein expression by western blotting to determine whether treatment with lithium

resulted in phosphorylation of GSK3β on Ser9 and therefore inhibition of GSK3β as described in

the literature [119]. Indeed we observed phosphorylation of GSK3β by lithium. Interestingly, we

were not able to demonstrate Ser9 phosphorylation of GSK3β with a 2 Gy dose of radiation

(Figure 3.4.2.).

3.4.4. Lithium results in nuclear translocation of β-catenin

To confirm activation of WNT signalling by lithium treatment we performed

immunofluorescence microscopy to assess nuclear translocation of β-catenin (which is a sign of

WNT activation). After 24 hour pre-treatment of both TP53 wild-type (ONS76) and TP53

mutant (UW228) cells with lithium we observed translocation of β-catenin to the nucleus.

Interestingly, TP53 mutant cells demonstrated a significantly greater increase in translocation of

β-catenin to the nucleus than TP53 wild-type cells (Figure 3.4.3.A&B).

3.4.5. Lithium constitutively activates canonical WNT signaling

In order to assess whether treatment with lithium results in activation of WNT signalling through

β-catenin activation we examined the transcriptional activation of β-catenin downstream

signalling by performing a dual luciferase reporter assay using the Super 8x TOPFlash / Super

8x FOPFlash β-catenin reporter system. TP53 wild-type ONS76 and TP53 mutant UW228 cells

were transfected with Super 8x TOPFlash β-catenin reporter plasmid. Transfection with Super

8x FOPFlash bearing mutant TCF/LEF, unable to activate luciferase transcription, was used to

control for transfection effect. After normalization for transfection with Renilla luciferase both

TP53 wild-type and mutant cells transfected with Super 8x TOPFlash reporter gene

47

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Figure 3.4.2. Lithium phosphorylates GSK3β on Ser9. Western blot protein analysis; marked increase of the amount of the phospo-Ser9-GSK3β protein after treatment with Lithium comparing to untreated control, irradiation along does not result in increase in the amount of phospho-GSK3β protein. Neither treatment with Lithium nor irradiation affects quantities of the total GSK3β protein. Loading control: β-actin.

48

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Figure 3.4.3. Treatment with Lithium results in nuclear translocation of β-catenin in both TP53 wt and TP53 mut cells. (A) Immunofluorescent imaging of the nuclear translocation of β-catenin (green), nucleus (DAPI-red), co-localization (yellow) following 24 hr exposure to Lithium. (B) Bar graph showing percentage of cell positive for nuclear β-catenin (n=1).

49

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demonstrated activation of β-catenin-TCF/LEF transcription upon treatment with lithium

compared to cells transfected with mutant TCF/LEF control plasmid. TP53 wild-type cells

demonstrated 144-fold increase in luciferase activity (p<0.01). At the same time, lithium

treatment resulted in only 43-fold increase of luciferase activity in TP53 mutant cells (p<0.01)

(Figure 3.4.4.A&B).

Each experiment was performed 3 times. Since data from one experiment contained a high

proportion of outliers (likely resulting from poor transfection efficiency) these values were

excluded from the analysis. Subsequent analysis was performed on pooled data containing 6-9

technical replicates.

In summary, we were able to confirm that treatment with lithium phosphorylates GSK3β on

Ser9, which is consistent with what is described in literature. This is accompanied by nuclear

translocation of β-catenin and activation of transcription. Taken together these findings support

the premise that lithium acts through activation of WNT/β-catenin signalling.

3.5. Lithium does not sensitize normal neuronal stem cells to radiation

To address potential detrimental effects of lithium as a radiosensitizer on normal developing

brain tissue we used normal neuronal stem cells (NNSC) as a model to study cell survival and

DNA damage / repair response in treated and non-treated cells.

3.5.1. Lithium does not decrease survival of normal neuronal stem cells

Hf5205 NNSC are not able to form well defined colonies due to their extreme proliferative and

migratory capabilities. Therefore, to investigate the radiosensitizing effect of lithium on NNSC

we performed a cell survival assay as described above. In the combination protocol, cells were

pre-treated with 2mM of lithium for 24 hours, irradiated, and then cell number was counted and

survival curves generated 5 days after radiation exposure. Interestingly, pre-treatment with

lithium did not result in decreased survival or proliferation of NNSC compared to the untreated

control. On the contrary, pre-treated cells demonstrated slight, though not statistically significant

(p=0.15), survival advantage over their untreated counterparts (Figure 3.5.1.A). Survival

fraction at 2 Gy was calculated for both treated and untreated cells: 33% +/- 8% vs. 27% +/- 3%,

respectively. The observed difference did not quite reach statistical significance (p=0.0465). We

50

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also examined cell survival at high radiation doses and observed that the survival fraction at 8

Gy was 1.9% +/- 6.1% vs. 1.4% +/- 3.4% (p=0.064).

3.5.2. Normal neuronal stem cells treated with lithium do not demonstrate nuclear

translocation of β-catenin

To investigate whether lithium is able to activate canonical WNT signalling in NNSC we

performed immunofluorescent microscopy to assess nuclear translocation of β-catenin.

Surprisingly, our preliminary data demonstrates that in both pre-treated and untreated samples

β-catenin is located in the cytoplasm and not the nucleus (Figure 3.5.1.B).

51

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ONS76 No Lithium

ONS76 Lithium

UW228 No Lithium

UW222 Lithium

Fold

Indu

ctio

n

Fold

Indu

ctio

n

Figure 3.4.4. Lithium activates WNT/β-catenin transcriptional activity. Increase in luciferase activity (pTA-Luc/Super8xTOPFlash) over control (pGL3/Super8xFOPFlash) in both ONS76 TP53wt (A) and UW228 TP53mut (B) cells in response to treatment with Lithium (red); untreated cells have minimal endogenous WNT/β-catenin signalling (blue) (**p<0.01).

52

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Figure 3.5.1. Treatment with Lithium does not radiosensitizeNNSC. (A) Survival curves for Hf5205 normal neuronal stem cells

given increasing radiation doses following 24 hr of pre-treatment

with Lithium (red circles) and untreated control (blue circles)

(p=0.054). (B) Immunofluorescent imaging of Hf5205 cells;

absence of the nuclear translocation of β-catenin (green), nucleus

(DAPI-red), is observed in both control cells and following 24 hr

exposure to Lithium.

53

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CHAPTER 4: DISCUSSION

4.1. TP53 mutations are predictive of inferior survival in SHH, but not WNT

medulloblastomas

Medulloblastoma are the most common brain tumour in children. Although currently

medulloblastoma is considered one of the most curable malignant brain tumours and a prototype

of success in paediatric neurooncology, disease related mortality still ranges between 20 – 40%

depending on the clinical risk group at diagnosis. Although addition of radiation and adjuvant

high-dose chemotherapy can cure the majority of patients, they also result in devastating long-

term side effects and morbidity. Exposure to radiation can result in skeletal growth retardation,

pan-endocrine deficiencies, cognitive impairment with progressive decline in intellectual

function, psychiatric disorders and neurological deficits. In addition, high-dose chemotherapy

can impact cardiac function and fertility. With recent increased understanding of the genetics

and genomics of medulloblastoma, numerous attempts have been made to improve risk group

stratification and allow for a more personalized approach for treatment of these patients with the

hope to decrease the intensity of therapies, in particular radiation dose, in prognostically

favorable groups and improve survival in high risk patients.

International collaborations have resulted in novel subgroup classification of medulloblastoma

and the potential for targeted therapies for patients with specific molecular subgroups. WNT

signature tumours are recognized as a clinically favorable subgroup [34, 49]. However, even

with implementation of molecular profiling prognostication is still challenging for the SHH,

Group 3 and Group 4 patients, because the expression signature does not allow for survival

prediction [3]. There is a clear need for reliable, easy-to-use markers to predict survival and risk

stratification in the field of medulloblastoma. In addition, there is a lack of target specific

treatment options for known pathway driven tumours.

In this thesis we propose a novel approach to risk stratification of medulloblastoma patients

using a combination of molecular profiling with TP53 status. We also explore lithium as a

potential novel targeted therapeutic agent to improve survival in high-risk medulloblastoma

patients and possibly decrease radiation dose in the favorable subgroup.

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In this study examining correlation of OS and PFS outcome with subgroup and TP53 status in

139 primary medulloblastoma patients older than 3 years of age we have demonstrated that

presence of somatic TP53 mutation remains a strong predictor of inferior survival which is

consistent with previous reports [59-60]. Importantly, OS and PFS with respect to TP53 status

are dramatically different between WNT and SHH subgroups. In the WNT subgroup, TP53

mutation seems not to be a predictor of poor survival or risk of relapse; those patients

demonstrate superior survival regardless of TP53 status, comparing to all other subgroups. In

the SHH subgroup, in contrast, patients with TP53 mutations have significantly worse survival

compared to their TP53 wild-type counterparts; in addition, TP53 mutant patients are also more

likely to experience relapse. In fact, all our TP53 mutant SHH patients relapsed within 8 years

from diagnosis. We have validated our findings using an independent cohort comprised of the

medulloblastoma patients from different countries who received radiation therapy. In the

validation cohort we demonstrated OS survival outcomes similar to our initial observations for

patients in the WNT and SHH subgroups, with TP53 mutations resulting in marked decreased

survival in SHH subgroup and no significant negative effect in the WNT subgroup. Although the

validation cohort was collected over a long time span and was comprised from patients who

were treated on different protocols, the validation set still demonstrates similar findings to our

discovery cohort and allows us to suggest that there is lack of inherent bias and combination of

molecular group and TP53 status can be confidently used as a predictor of survival. Further

support to our findings is available by a recent independent study of whole genome sequencing

by Robinson and colleagues, who demonstrated that in a cohort of 14 SHH patients 3 deaths

occurred and these were exclusively in TP53 mutant patients [141]. Considering that the

outcome of TP53 mutant patients largely depends on the molecular subgroup in one hand and

the outcome of specific MB subgroups is significantly modulated by presence or absence of a

TP53 mutation on the other, we propose the following decision-making model for newly

diagnosed medulloblastoma patients (Figure 4.1.). In this model we propose upfront testing of

all patients for either TP53 mutation or to determine the molecular subgroup. In patients who

have wild-type TP53 no subsequent action will be needed and those patients will proceed to the

local standard-of-care protocols. Similarly if WNT, Group 3 or Group 4 is observed, those

patients will not require additional TP53 status determined. It will be increasingly important to

identify the combination of TP53 mutation with the SHH subgroup, as these patients have the

worst outcome and should be upgraded to high risk regardless of presence or absence of other

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Figure 4.1. Clinicopathological flow chard for decision makingin newly diagnosed MB patients. For accurate risk stratification it

is imperative to be able to either determine TP53 status or molecular

group of the MB patient. Presence of TP53 mutation warrants

subsequent molecular group assignment to identify SHH/TP53

mutation combination in order to upgrade patients too high risk

subgroup and offer the appropriate therapy; similarly, all SHH

patients have to be tested for TP53 mutation.

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high-risk criteria. Intensification of the therapy should be considered in this subgroup including

targeted therapy and SHH pathway inhibitors.

Although we were not able to observe any segregation by the type or domain localisation of

TP53 mutations according to subgroups, the high rate of LOH along with presence of deletions

and insertions in SHH tumours, but not WNT tumours, suggestive of higher genomic instability.

This is further supported by the finding that these tumours have high rate of chromosomal

shattering termed chromothripsis [142]. Together, these findings may provide plausible

explanation for poor survival in SHH/TP53 mutant patients.

4.2. TP53 mutations in medulloblastomas confer resistance to radiation while activation of

canonical WNT signaling radiosensitizes medulloblastoma cells.

Although the implication of TP53 mutations on cellular resistance to radiation and radiation

response in normal and cancer cells has been well-described in literature; the association of

TP53 mutations with resistance to radiation in medulloblastoma in particular has not been

addressed. In this study, using a heterogeneous panel of medulloblastoma cell lines, we have

demonstrated that TP53 mutant medulloblastomas are more resistant to radiation. We further

confirmed our results using an isogenic in vitro model, transfecting TP53 wild-type cells with a

known dominant-negative mutation. These findings are in agreement with previously published

data on other cancers, as discussed above.

Interestingly, introduction of a CTNNB1 activating mutation into medulloblastoma cells resulted

in sensitization of those cells to radiation, which contradicts published data of other cancers for

which there is an association between WNT signaling activation and increased resistance to

radiation [109-114]. Notably, all published reports on the association of radiation resistance with

either presence of CTNNB1 mutations or other pathway alterations, such as APC mutations, as

well as chemical inhibition of GSK3β, has been addressed in cancers other than

medulloblastoma.

Our finding that introduction of CTNNB1 mutations cause radiosensitivity in medulloblastoma

cells is in concordance with clinical observations that WNT subgroup patients, characterized by

the presence of CTNNB1 mutations, routinely treated with radiation, demonstrate superior

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survival [3, 21]. This has major implications on future protocols which may try to reduce/omit

radiation for patients with WNT medulloblastomas.

We further demonstrated that sensitization of medulloblastoma cells to radiation can be

achieved with administration of a clinically tolerable dose of lithium prior to radiation exposure.

In vitro experiments have shown that sensitization of both TP53 wild-type and TP53 mutant

medulloblastoma cells to radiation can be successfully achieved with 24 hours exposure to 2mM

of lithium. Decreased survival associated with treatment of medulloblastoma cells with lithium

was accompanied by inhibitory phosphorylation of GSK3β, nuclear translocation of β-catenin

and transcriptional activation of β-catenin TCF/LEF mediated signaling which are the hallmarks

of the WNT pathway activation. These findings are strongly suggestive of involvement of

WNT/β-catenin signaling in radiation response in medulloblastoma.

The fact that TP53 wild-type cells demonstrated greater decrease in clonogenic survival,

compared to TP53 mutant cells, supports the previously described model in which increased

levels of β-catenin stimulate accumulation of transcriptionally active p53 and provide negative

regulation for β-catenin. It is speculated that negative regulation of β-catenin by p53 is

accomplished via its GSK3β-mediated proteosomal degradation [128-129] (Figure 4.2.). We

think that presence of CTNNB1 mutation prevents GSK3β from executing its role in regulation

of β-catenin levels, which in turns results in diversion of the damaged cell to the death pathway

by stable p53. In cells treated with lithium, inhibition of GSK3β is achieved chemically, but

results in the same outcome.

Importantly, the fact that lithium sensitizes both TP53 wild-type and TP53 mutant cells to

radiation suggests that lithium may act through other p53-independent mechanisms yet to be

determined, and possibly through GSK3β. For example, decrease of tumour size in low grade

neuroendocrine tumours was achieved with lithium treatment and was associated with

previously described GSK3β inhibition [143]. Lithium was also described to decrease glioma

cell invasion through GSK3β inhibition [122]. The effect of other small metal molecules on

chromatin remodeling and reconstitution of wild-type configuration of mutant p53 protein have

recently been described [144-146]. These findings, together with recognition that lithium is

biologically necessary for healthy embryonal development and both physical and mental

functioning of mammals including humans [147] provide plausible grounds to examine role of

lithium in chromatin remodeling and/or reconstitution of wild-type p53 configuration as one of

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Figure 4.2. Relationship between β-catenin and p53 levels . Regulation

of β-catenin levels via GSK3β mediated negative feedback loop. (Adapted

from Damalas et.al. EMBO J 1999 & Sadot et.al. Mol Cell Biol 2001).

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the possible mechanisms of radiosensetisation. Understanding of the mechanisms behind lithium

radiosensetisation in medulloblastoma cells may also provide valuable insights into the

mechanisms behind favorable survival in WNT medulloblastomas and the basis for development

of novel therapeutics.

In addition, combination of lithium with small-molecule inhibitors of SHH pathway in SHH

subtype medulloblastomas may represent an attractive possibility of combine treatment where

the pathway is attacked from two different sides – inhibition of SHH pathway and enhancement

of WNT signalling.

The objective of any combination treatment is to achieve improvement in therapeutic outcomes

and minimize normal tissue damage; thus, no therapeutic improvement will be gained if a

proposed agent sensitizes both cancer and normal cells to radiation equally. Our preliminary

results from NNSC experiments demonstrate that lithium does not activate WNT signaling in

normal neuronal stem cells, nor does it sensitize them to radiation. Moreover, we were able to

observe marginal improvement in cell survival in lithium treated NNSC which is in agreement

with published reports demonstrating protective capacity of lithium against radiation in mouse

models [124-125]. To date we were not been able to demonstrate nuclear translocation of β-

catenin in response to lithium in NNSC; however these experiments require further development

and completion. The difference in response to lithium in normal and cancer tissues suggests that

it may act through different pathways. Alternatively, normal cells have strong tumour

suppressive mechanisms, including, but not limited to functional TP53, which are altered in

cancer cells. These pathways and mechanisms need to be investigated in further studies.

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CHAPTER 5: CONCLUSIONS

This study highlights how multicenter collaborative efforts can resolve discrepancies between

clinical reports on uncommon events in paediatric cancer, such as determining the role of TP53

mutations on survival in medulloblastomas.

In this study, we first demonstrated that successful combination of genetic and genomic

approaches in cancer can improve risk stratification in medulloblastoma patients. We were able

to demonstrate that although somatic TP53 mutations remain a prognostically unfavorable

marker, their effect is largely modulated by molecular subgroup and ranges from insignificant in

WNT to highly lethal in SHH driven tumours. We were able to identify a novel prognostically

unfavorable subgroup of medulloblastoma patients, namely SHH/TP53 mutant patients.

Moreover, we suggest a new clinicopathological approach for risk stratification of newly

diagnosed medulloblastoma patients which involves combination of testing for TP53 and

molecular group assignment.

Furthermore, we were able to replicate our clinical observation that WNT patients have

improved survival using an in vitro model and demonstrate that association of decreased tumour

survival with activation of WNT/β-catenin signaling is confirmed in our cell model. Our

findings of lithium significantly decreasing clonogenic capacity in both TP53 wild-type and

TP53 mutant medulloblastoma cell lines coupled with both lack of radiosensetisation in NNSC

and its known neuroprotective capacity suggests that lithium may be a safe and clinically

effective drug to use. We recognize that despite being safely used in humans for treatment of

psychiatric disorders, lithium needs further pre-clinical evaluation prior to administration to

humans for the purpose of medulloblastoma treatment. Indeed, a phase 1 trial of radiation

concomitant with lithium for children with medulloblastoma is currently planned based on our

observations.

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CHAPTER 6: FUTURE DIRECTIONS

Based on our findings further studies are required to investigate the biological mechanisms that

explain favorable survival of WNT subgroup patients and lithium radiosensitizing capacity.

1) How does activation of WNT/β-catenin signalling offer a survival advantage?

It is important to assess how activation of WNT/β-catenin signaling via presence of CTNNB1

mutation translates to decreased cell survival under conditions of radiation treatment.

Construction of a stable conditionally expressing mutant CTNNB1 protein cell line with TP53

wild-type and TP53 mutant background will allow performing a range of functional experiments

to investigate possible effects of mutation on cell cycle, DNA damage response and interaction

with other proteins including p53. Expression arrays comparing isogenic wild-type and

genetically engineered CTNNB1 mutant cell lines could identify altered pathways involved in

radiation response of WNT tumours.

2) Determining of mechanism of lithium action

As discussed above, the mechanism of lithium action is not completely understood. There are

several studies describing lithium as a GSK3β inhibitor [117, 120, 122]; however, the

downstream effects of this inhibition are not well known. It will be valuable to determine the

pathway involved in lithium mediated effects on cell survival which involve and do not involve

GSK3β. Expression arrays comparing lithium-treated and untreated medulloblastoma cells will

be an efficient and useful means to determine pathways involved in lithium action in

comprehensive and time-efficient manner. Alternatively, systematic knockdown of members of

the known WNT signaling pathway, together with some members of the other pathways, such as

p53 pathway, using siRNA can be used to investigate the role of each member of the pathway

with relation to lithium treatment and combination exposure to lithium and radiation therapy.

One of the most significant biological effects of radiation therapy is induction of DNA DSBs.

Unrepaired DNA DSBs can lead to cell death or chromosomal aberrations subsequently

incompatible with cell survival. A vast body of literature strongly correlate persistence of

unrepaired DNA DSBs with radiosensetisation of cancer cells [148-149]. To provide insight into

mechanism by which lithium may contribute to sensitization of medulloblastoma cells to

radiation we performed a series of DNA DSBs/repair immunofluorescence assays (Appendix A

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for Materials & Methods) which demonstrated a correlation of increased number of γH2AX foci

with lithium exposure prior to radiation. Our preliminary data has demonstrated that lithium

treatment combined with radiation results in increase DNA damage and decrease in DNA repair,

as assessed by development and persistence of γH2AX foci, in both TP53 wild-type and TP53

mutant medulloblastoma cells suggesting that lithium may be involved in modulation of the

DNA damage/repair pathway. Here we demonstrated that treatment with lithium alone did not

result in significant change in the number of foci compared to untreated samples in both TP53

wild-type and TP53 mutant cells. Importantly, in both TP53 mutant and TP53 wild-type cells we

observed a marked increase in the formation of γH2AX foci in samples pre-treated with lithium

(p<0.001) and presence of persistent residual foci beyond 24 hours post-radiation (Figure

6.1.A&B). Interestingly, we did not observe neither complete nor approaching baseline

resolution of γH2AX foci by 24 hours in any cell lines.

Surprisingly, treatment of NNSCs with lithium did not result in a significant difference in the

number of foci between pre-treated and non-treated cells (p=0.68). In addition, NNSC

demonstrated a classical pattern of formation and resolution of γH2AX foci as described in

literature with a rapid peak in foci shortly after radiation and close to complete resolution to the

baseline at 24 hours in both lithium-treated and untreated samples (Figure 6.1.C).

Our experiments allow us to suggest that in the cancer cell, lithium, in combination with

radiation, both enhances DNA damage and significantly hinder the ability of the cell to process

DNA DSBs, resulting in accumulation of intolerable DNA damage and subsequent cell death.

Presence of the residual γH2AX foci in lithium treated cells beyond 24 hours post radiation is

suggestive of impaired non-homologous end-joining (NHEJ) mechanism inflicted by lithium

[150-152]. The fact that lithium increases the number of γH2AX foci in both TP53 wild-type and

mutant cells supports the suggestion that it acts at least partially independent of the TP53

pathway.

Striking differences which we have observed in response to combined lithium and radiation

treatment between medulloblastoma and NNSC cells are intriguing and warrant further

investigation. The comet assay will be useful to confirm our findings with respect to the increase

in DNA DSBs and to assess the kinetics of DNA damage repair. A role for ATM and MRN

complex as targets for radiosensetisation therapy was recently described [153], as they play

pivotal roles in the DNA damage/repair response to radiation. Thus, it will be important to

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investigate the other members of DNA damage/repair pathway, such as ATM, Rad50, Mre11,

NBS1 and checkpoint proteins employing functional studies. In addition, to elucidate

mechanisms of radiosensetisation through lithium we plan to study whether lithium has an effect

on cell cycle and apoptosis in medulloblastoma and NNSC. Annexin apoptosis assay and cell

cycle analysis using flow cytometry will be valuable techniques to address these questions.

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Figure 6.1.A. Treatment with lithium increases number of γH2AX foci inTP53 wild-type MB cells. (A) Bar graph – quantification of the number of γH2AX

foci expressed per nucleus at 30 min, 4 and 24 hr after radiation compared to

non-treated control and cells treated only with lithium (**p<0.0001). (B)

Immunofluorescence of ONS76 TP53wt cells at 30 min, 4 and 24 hr after

exposure to 5 Gy single dose of radiation. Untreated control (top panel) has

significantly less γH2AX foci per cell compared to cells pre-treated for 24 hr with

lithium (bottom panel). Nucleus – DAPI-blue, γH2AX foci - green.

A

B

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A

B

Figure 6.1.B. Treatment with lithium increases number of γH2AX foci inTP53 mutant MB cells. (A) Bar graph – quantification of the number of γH2AX

foci expressed per nucleus at 30 min, 4 and 24 hr after radiation compared to

non-treated control and cells treated only with lithium (**p<0.0001).

(B) Immunofluorescence of UW228 TP53mut cells at 30 min, 4 and 24 hr after

exposure to 5 Gy single dose of radiation. Untreated control (top panel) has

significantly less γH2AX foci per cell compared to cells pre-treated for 24 hr

with lithium (bottom panel). Nucleus – DAPI-blue, γH2AX foci - green.

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Figure 6.1.C. Treatment with lithium does not increases number of

γH2AX foci in NNSC. (A) Bar graph – quantification of the number of γH2AX

foci expressed per nucleus at 30 min (p=0.53), 4 hr (p=0.84) and 24 hr

(p=0.48) after radiation compared to non-treated control and cells treated only

with lithium. (B) Immunofluorescence of Hf5205 cells at 30 min, 4 and 24 hr

after exposure to 5 Gy single dose of radiation. Both untreated control (top

panel) and cells pre-treated for 24 hr with lithium (bottom panel) have similar

number of γH2AX foci per nucleus. Nucleus – DAPI-blue, γH2AX foci -

green.

A

B

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APPENDIX A

Phospho-histone H2AX (γH2AX) foci immunofluorescent microscopy and imaging

Medulloblastoma and NNSC were cells were cultured on cover slips in 6-well dishes and fixed

as described above.

Primary antibodies: mouse anti-phospho-histone-H2AX (Ser139) (Millipore, 05-636) 1:800.

Secondary antibodies: Alexa Fluor 488 donkey-anti-mouse IgG (Invitrogen, A21202) 1:500.

For phospho-histone H2AX (γH2AX) foci formation, cells were pre-treated with 2mM lithium

Chloride for 24 hr and then exposed to 5 Gy single dose of radiation. Untreated and non-

irradiated cells were used as control. Specimens were collected at 30 min, 4 hr and 4 hr post

radiation. At appropriate time points, cells were fixed and processed as described above.

For quantification of γH2AX foci 3 independent experiments were performed. Between 100 and

250 nuclei were examined and foci were counted using Perkin Elmer Velocity 6.0.1 software

(Perkin Elmer, USA) with 0.2um cut off for foci size. The following procedure was applied to

decrease inter-experimental variability: 1) all 3 experiments were pooled together and individual

values were used for analysis [154]; 2) average number of foci per nucleus was calculated; 3)

outliers, which were estimated as nuclei containing > 2 standard deviation (2 SD) from the

average for the treatment subgroup, were eliminated; 4) baseline number of foci per nucleus was

estimated in the untreated sample as average plus 2 SD; 5) to remove background noise nuclei

with baseline or less number of foci were eliminated from experimental samples and the

remaining nuclei were considered γH2AX-positive for further calculations; 6) number of foci in

γH2AX-positive nuclei was calculated and expressed as an average number of foci per nuclei; 7)

comparison was done between treatments using average number of foci per nucleus and number

of γH2AX-positive cells as defined above.

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